U.S. patent application number 13/866790 was filed with the patent office on 2013-12-12 for cellulases, nucleic acids encoding them and methods for making and using them.
This patent application is currently assigned to BP Corporation North America Inc.. The applicant listed for this patent is David Blum, Joslin Gemsch Cuenca, Mark Dycaico. Invention is credited to David Blum, Joslin Gemsch Cuenca, Mark Dycaico.
Application Number | 20130330783 13/866790 |
Document ID | / |
Family ID | 37024271 |
Filed Date | 2013-12-12 |
United States Patent
Application |
20130330783 |
Kind Code |
A1 |
Blum; David ; et
al. |
December 12, 2013 |
CELLULASES, NUCLEIC ACIDS ENCODING THEM AND METHODS FOR MAKING AND
USING THEM
Abstract
This invention relates to molecular and cellular biology and
biochemistry. In one aspect, the invention provides polypeptides
having cellulase activity, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or .beta.-glucosidase activity, polynucleotides
encoding these polypeptides, and methods of making and using these
polynucleotides and polypeptides. In one aspect, the invention is
directed to polypeptides cellulase activity, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or .beta.-glucosidase activity,
including thermostable and thermotolerant activity, and
polynucleotides encoding these enzymes, and making and using these
polynucleotides and polypeptides. The polypeptides of the invention
can be used in a variety of pharmaceutical, agricultural, food and
feed processing and industrial contexts.
Inventors: |
Blum; David; (Brentwood,
TN) ; Cuenca; Joslin Gemsch; (San Diego, CA) ;
Dycaico; Mark; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Blum; David
Cuenca; Joslin Gemsch
Dycaico; Mark |
Brentwood
San Diego
San Diego |
TN
CA
CA |
US
US
US |
|
|
Assignee: |
BP Corporation North America
Inc.
Houston
TX
|
Family ID: |
37024271 |
Appl. No.: |
13/866790 |
Filed: |
April 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11908672 |
Sep 21, 2009 |
8426184 |
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PCT/US2006/002516 |
Jan 13, 2006 |
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13866790 |
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60662224 |
Mar 15, 2005 |
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Current U.S.
Class: |
435/99 ; 435/165;
435/209; 435/254.11; 435/254.2; 435/320.1; 435/325; 435/348;
435/419; 536/23.2 |
Current CPC
Class: |
C10L 1/02 20130101; C12Y
302/01004 20130101; A61P 1/14 20180101; C10L 2290/26 20130101; C12Y
302/01091 20130101; A61P 43/00 20180101; C10L 1/023 20130101; Y02E
50/17 20130101; C12N 9/2437 20130101; Y02E 50/10 20130101; Y02E
50/16 20130101; C10L 2200/0423 20130101; C10L 2200/0469 20130101;
C12P 7/10 20130101 |
Class at
Publication: |
435/99 ;
536/23.2; 435/320.1; 435/325; 435/254.11; 435/254.2; 435/348;
435/419; 435/209; 435/165 |
International
Class: |
C12P 7/10 20060101
C12P007/10; C12N 9/42 20060101 C12N009/42 |
Goverment Interests
GOVERNMENT SUPPORT
[0001] This invention was made with United States Government
support under Contract Nos. DE-FG03-02ER83395 and
DE-FG02-03ER83865, awarded by the Department of Energy. The
Government has certain rights in this invention.
Claims
1. An isolated or recombinant nucleic acid comprising (a) a nucleic
acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more or complete sequence identity to SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57,
SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID
NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ
ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,
SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103,
SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID
NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,
SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID
NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139,
SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID
NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157,
SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163 or SEQ ID NO:165, over
a region of at least about 20, 30, 40, 50, 75, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1050, 1100, 1150 or more residues, wherein the nucleic
acid encodes at least one polypeptide having a cellulase activity,
and optionally the sequence identities are determined by analysis
with a sequence comparison algorithm or by a visual inspection; or
(b) a nucleic acid sequence that hybridizes under stringent
conditions to a nucleic acid comprising SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID
NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ
ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,
SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID
NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ
ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69,
SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID
NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ
ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97,
SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID
NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115,
SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID
NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133,
SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID
NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151,
SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID
NO:161, SEQ ID NO:163 or SEQ ID NO:165, wherein the nucleic acid
encodes a polypeptide having a cellulase activity, and the
stringent conditions include a wash step comprising a wash in
0.2.times.SSC at a temperature of about 65.degree. C. for about 15
minutes, and optionally the nucleic acid is at least about 20, 30,
40, 50, 60, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900,
1000 or more residues in length or the full length of the gene or
transcript; (c) a nucleic acid sequence encoding a polypeptide
having a sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ
ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID
NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ
ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,
SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID
NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ
ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90,
SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID
NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108,
SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID
NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126,
SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID
NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143,
SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID
NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162,
SEQ ID NO:164 or SEQ ID NO:166; or (d) a nucleic acid sequence
complementary to (a), (b) or (c).
2. The isolated or recombinant nucleic acid of claim 1, wherein the
nucleic acid sequence comprises a sequence as set forth in SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57,
SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID
NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ
ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,
SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID
NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103,
SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID
NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121,
SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID
NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139,
SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID
NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157,
SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163 or SEQ ID NO:165.
3. The isolated or recombinant nucleic acid of claim 1, wherein the
sequence comparison algorithm is a BLAST version 2.2.2 algorithm
where a filtering setting is set to blastall-p blastp-d "nr
pataa"-F F, and all other options are set to default.
4-21. (canceled)
22. The isolated or recombinant nucleic acid of claim 1, wherein
the cellulase activity comprises hydrolyzing a cellulose, a
cellulose derivative or a hemicellulose.
23. The isolated or recombinant nucleic acid of claim 22 wherein
the cellulase activity comprises hydrolyzing a cellulose or a
hemicellulose in a wood or paper pulp or a wood or paper
product.
24-25. (canceled)
26. The isolated or recombinant nucleic acid of claim 1, wherein
the cellulase activity comprises catalyzing hydrolysis of a glucan
in a microbial cell, a fungal cell, a mammalian cell, a plant cell
or any plant material comprising a cellulosic part.
27. The isolated or recombinant nucleic acid of claim 1, wherein
the cellulase activity is thermostable.
28. The isolated or recombinant nucleic acid of claim 27, wherein
the polypeptide retains a cellulase activity under conditions
comprising a temperature range of between about 37.degree. C. to
about 95.degree. C., or between about 55.degree. C. to about
85.degree. C., or between about 70.degree. C. to about 75.degree.
C., or between about 70.degree. C. to about 95.degree. C., or
between about 90.degree. C. to about 95.degree. C., or retains a
cellulase activity in a temperature in the range between about
1.degree. C. to about 5.degree. C., between about 5.degree. C. to
about 15.degree. C., between about 15.degree. C. to about
25.degree. C., between about 25.degree. C. to about 37.degree. C.,
or between about 37.degree. C. to about 95.degree. C., 96.degree.
C., 97.degree. C., 98.degree. C. or 99.degree. C.
29. The isolated or recombinant nucleic acid of claim 1, wherein
the cellulase activity is thermotolerant.
30. The isolated or recombinant nucleic acid of claim 29, wherein
the polypeptide retains a cellulase activity after exposure to a
temperature in the range from greater than 37.degree. C. to about
95.degree. C., from greater than 55.degree. C. to about 85.degree.
C., or between about 70.degree. C. to about 75.degree. C., or from
greater than 90.degree. C. to about 95.degree. C., or after
exposure to a temperature in the range between about 1.degree. C.
to about 5.degree. C., between about 5.degree. C. to about
15.degree. C., between about 15.degree. C. to about 25.degree. C.,
between about 25.degree. C. to about 37.degree. C., or between
about 37.degree. C. to about 95.degree. C., 96.degree. C.,
97.degree. C., 98.degree. C. or 99.degree. C.
31-36. (canceled)
37. An expression cassette comprising a nucleic acid comprising a
sequence as set forth in claim 1.
38. A vector comprising a nucleic acid comprising a sequence as set
forth in claim 1, wherein optionally the vehicle comprises an
expression vector.
39. A cloning vehicle comprising a nucleic acid comprising a
sequence as set forth in claim 1, wherein optionally the cloning
vehicle comprises a viral vector, a plasmid, a phage, a phagemid, a
cosmid, a fosmid, a bacteriophage or an artificial chromosome, and
optionally the viral vector comprises an adenovirus vector, a
retroviral vector or an adeno-associated viral vector, and
optionally the cloning vehicle comprises a bacterial artificial
chromosome (BAC), a plasmid, a bacteriophage P1-derived vector
(PAC), a yeast artificial chromosome (YAC), or a mammalian
artificial chromosome (MAC).
40. A transformed cell comprising a nucleic acid comprising a
sequence as set forth in claim 1, or an expression cassette as set
forth in claim 37, the vector of claim 38, or a cloning vehicle as
set forth in claim 39, wherein optionally the cell is a bacterial
cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell
or a plant cell.
41-47. (canceled)
48. An isolated or recombinant polypeptide (i) having an amino acid
sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or more, or 100% sequence identity to SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40,
SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ
ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,
SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID
NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ
ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96,
SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID
NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114,
SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID
NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132,
SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID
NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150,
SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID
NO:160, SEQ ID NO:162, SEQ ID NO:164 or SEQ ID NO:166, over a
region of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 75,
100, 150, 200, 250, 300 or more residues, wherein optionally the
sequence identities are determined by analysis with a sequence
comparison algorithm or by a visual inspection, and optionally the
sequence comparison algorithm is a BLAST version 2.2.2 algorithm
where a filtering setting is set to blastall-p blastp-d "nr
pataa"-F F, and all other options are set to default; (ii) having
an amino acid sequence encoded by a nucleic acid as set forth in
claim 1, wherein the polypeptide has a cellulase activity or has
immunogenic activity in that it is capable of generating an
antibody that specifically binds to a polypeptide having a sequence
as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ
ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36,
SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID
NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ
ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64,
SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID
NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ
ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,
SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID
NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110,
SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID
NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128,
SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID
NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146,
SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID
NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164
or SEQ ID NO:166; or (iii) having an amino acid sequence as set
forth in (i) or (ii), or a polypeptide encoded by a nucleic acid as
set forth in claim 1, and comprising at least one amino acid
residue conservative substitution, wherein optionally conservative
substitution comprises replacement of an aliphatic amino acid with
another aliphatic amino acid; replacement of a serine with a
threonine or vice versa; replacement of an acidic residue with
another acidic residue; replacement of a residue bearing an amide
group with another residue bearing an amide group; exchange of a
basic residue with another basic residue; or, replacement of an
aromatic residue with another aromatic residue, or a combination
thereof, and optionally the aliphatic residue comprises Alanine,
Valine, Leucine, Isoleucine or a synthetic equivalent thereof the
acidic residue comprises Aspartic acid, Glutamic acid or a
synthetic equivalent thereof; the residue comprising an amide group
comprises Aspartic acid, Glutamic acid or a synthetic equivalent
thereof the basic residue comprises Lysine, Arginine or a synthetic
equivalent thereof or, the aromatic residue comprises
Phenylalanine, Tyrosine or a synthetic equivalent thereof.
49-127. (canceled)
128. A method for hydrolyzing, breaking up or disrupting a glucan-
or cellulose-comprising composition comprising the following steps:
(a) providing a polypeptide having a cellulase activity as set
forth in claim 48, or a polypeptide encoded by a nucleic acid as
set forth in claim 1; (b) providing a composition comprising a
cellulose or a glucan; and (c) contacting the polypeptide of step
(a) with the composition of step (b) under conditions wherein the
cellulase hydrolyzes, breaks up or disrupts the glucan- or
cellulose-comprising composition, wherein optionally the
composition comprises a plant cell, a bacterial cell, a yeast cell,
an insect cell, or an animal cell, and optionally the polypeptide
has endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity.
129-143. (canceled)
144. A method for making a fuel comprising contacting a composition
comprising a cellulose or a fermentable sugar with a polypeptide as
set forth in claim 48, or a polypeptide encoded by a nucleic acid
as set forth in claim 1, wherein optionally the composition
comprising a cellulose or a fermentable sugar comprises a plant,
plant product or plant derivative, and optionally the plant or
plant product comprises cane sugar plants or plant products, beets
or sugarbeets, wheat, corn, soybeans, potato, rice or barley, and
optionally the polypeptide has activity comprising cellulase,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
activity, and optionally the fuel comprises a bioethanol or a
gasoline-ethanol mix.
145. A method for making bioethanol comprising contacting a
composition comprising a cellulose or a fermentable sugar with a
polypeptide as set forth in claim 48, or a polypeptide encoded by a
nucleic acid as set forth in claim 1, wherein optionally the
composition comprising a cellulose or a fermentable sugar comprises
a plant, plant product or plant derivative, and optionally the
plant or plant product comprises cane sugar plants or plant
products, beets or sugarbeets, wheat, corn, soybeans, potato, rice
or barley, and optionally the polypeptide has activity comprising
cellulase, endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity.
146-161. (canceled)
Description
FIELD OF THE INVENTION
[0002] This invention relates to molecular and cellular biology and
biochemistry. In one aspect, the invention provides polypeptides
having cellulase activity, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or .beta.-glucosidase activity, polynucleotides
encoding these polypeptides, and methods of making and using these
polynucleotides and polypeptides. In one aspect, the invention is
directed to polypeptides having cellulase activity, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or
.beta.-glucosidase activity, including thermostable and
thermotolerant activity, and polynucleotides encoding these
enzymes, and making and using these polynucleotides and
polypeptides. The polypeptides of the invention can be used in a
variety of pharmaceutical, agricultural and industrial
contexts.
BACKGROUND
[0003] Cellulose is the most abundant renewable resource on earth.
It is composed of a linear chain of .beta. 1-4 glucose units with
the repeating unit being cellobiose, which is a glucose dimer
having a structure as shown in FIG. 5. The polymer is degraded by a
suite of enzymes which include endoglucanases (EG) which randomly
hydrolyze the cellulose polymer, and cellobiohydrolases (CBH) which
remove terminal cellobiose residues from cellulose. Cellobiose and
cello-oligosaccharides are hydrolyzed to glucose by
.beta.-glucosidases (BG). All three of these enzymes are necessary
for the complete breakdown of cellulose to glucose. For each of
these three enzymes different structural variants exist that
perform the same function. In addition, fungi and bacteria are
known to produce multiple forms of the same structural variants in
addition to different structural variants.
[0004] Further complicating this system is the fact that some
anaerobic bacteria and fungi are known to produce these enzymes in
multi-enzyme complexes which contain multiple enzymes all attached
to an enzyme scaffold with molecular weights above 2 million
daltons. Why is such a complex system of enzymes necessary for such
a simple molecule? Some researchers believe that this complexity is
due to the recalcitrant nature of the substrate. The cellulose
chains form microfibrils that pack into a crystalline matrix via
hydrogen bonding of adjacent chains. This structure is highly
resistant to chemical or enzymatic degradation.
[0005] CBHs are thought to be the key enzyme in the degradation of
this crystalline cellulose because of the nature of their enzymatic
attack on cellulose. EGs unlike CBHs have an open cleft that
attacks the cellulose chain at a perpendicular angle. CBHs attack
the chain directly via a tunnel containing the active site. The
current thought is that the cellulose chains enter the tunnel and
at the same time, adjacent hydrogen bonding is disrupted. Once the
cellobiohydrolases have established this "foothold" on the
substrate, the EGs can then come in and more readily attack the
substrate.
[0006] A major deficiency of known CBHs is their low catalytic
activity. Some groups argue that the low activity stems from the
fact that energy from hydrolysis is transferred to kinetic energy
to disrupt hydrogen bonds and enable the enzyme to move along the
substrate. CBHs are exo-acting enzymes and are found in 6 of the 90
families of glycosyl hydrolases. They include families 5, 6, 7, 9,
10 and 48. Family 5 contains many different types of glycosyl
hydrolases including cellulases, mannanases and xylanases. Although
most cellulases in this family are endoglucanases, there are
examples of cellobiohydrolases, most notably CelO from Clostridium
thermocellum. Family 6 contains only endoglucanases or
cellobiohydrolases with more cellobiohydrolase members than
endoglucanases. The enzymes have an inverting mechanism and
crystallographic studies suggest that the enzyme has a distorted
.alpha./.beta. barrel structure containing seven, not eight
parallel .beta.-strands. Family 7 enzymes are also composed of both
endoglucanases and cellobiohydrolases with more cellobiohydrolases
and only known members are from fungi. The enzyme has a retaining
mechanism and the crystal structure suggests a .beta.-jellyroll
structure. Family 9 contains endoglucanases, cellobiohydrolases and
.beta.-glucosidases with a preponderance of endoglucanases.
However, Thermobifida fusca produces an endo/exo-1,4-glucanase, the
crystal structure of which suggests a (.alpha./.alpha.).sub.6
barrel fold. The enzyme has characteristics of both endo and
exo-glucanases CBHs. Family 10 contains only 2 members described as
cellobiohydrolases with mainly the rest described as xylanases.
Cellobiohydrolases and xylanases from family 10 have activity on
methyl-umbelliferyl cellobioside. Family 48 contains mainly
bacterial and anaerobic fungal cellobiohydrolases and
endoglucanases. The structure is a (.alpha./.alpha.).sub.6 barrel
fold similar to family 9.
[0007] There is a need for less expensive and renewable sources of
fuel for road vehicles. New fuel sources will be more attractive if
they produce nonharmful endproducts after combustion. Ethanol
offers an attractive alternative to petroleum based fuels and can
be obtained through the fermentation of monomeric sugars derived
from starch or lignocellulose. However, current economics do not
support the widespread use of ethanol due to the high cost of
generating it. One area of research aimed at decreasing costs is
enhancement of the technical efficacy of the enzymes that can be
used to generate fermentable sugars from lignocellulose. The
development of enzymes that more efficiently digest feedstock will
translate to decreased ethanol production costs. More efficient
processes will decrease the United State's reliance on foreign oil
and the price fluctuations that may be related to that reliance.
Using cleaner fuels for transportation like bioethanol also may
decrease net CO.sub.2 emissions that are believed to be partially
responsible for global warming.
SUMMARY
[0008] The invention provides cellulases, e.g., endoglucanases,
cellobiohydrolases and/or .beta.-glucosidase (beta-glucosidases),
and methods for making and using them. In one aspect, the enzymes
of the invention have an increased catalytic rate to improve the
process of substrate hydrolysis. This increased efficiency in
catalytic rate leads to an increased efficiency in producing
sugars, which can be useful in industrial applications, e.g., the
sugars so produced can be used by microorganisms for ethanol
production. In one aspect, the invention provides highly active
(e.g., having an increased catalytic rate) cellobiohydrolases,
endoglucanases and beta-glucosidase. The invention provides
industrial applications (e.g., biomass to ethanol) using enzymes of
the invention having decreased enzyme costs, e.g., decreased costs
in biomass to ethanol processes. Thus, the invention provides
efficient processes for producing bioethanol and
bioethanol-comprising compositions, including fuels comprising
bioethanol, from any biomass.
[0009] In one aspect, the enzymes of the invention have a
glucanase, e.g., an endoglucanase, activity, e.g., catalyzing
hydrolysis of internal endo-.beta.-1,4- and/or .beta.-1,3-glucanase
linkages. In one aspect, the endoglucanase activity (e.g.,
endo-1,4-beta-D-glucan 4-glucano hydrolase activity) comprises
hydrolysis of 1,4- and/or .beta.-1,3-beta-D-glycosidic linkages in
cellulose, cellulose derivatives (e.g., carboxy methyl cellulose
and hydroxy ethyl cellulose) lichenin, beta-1,4 bonds in mixed
beta-1,3 glucans, such as cereal beta-D-glucans or xyloglucans and
other plant material containing cellulosic parts.
[0010] In one aspect, the enzymes of the invention have
endoglucanase (e.g., endo-beta-1,4-glucanases, EC 3.2.1.4;
endo-beta-1,3(1)-glucanases, EC 3.2.1.6; endo-beta-1,3-glucanases,
EC 3.2.1.39) activity and can hydrolyze internal .beta.-1,4- and/or
.beta.-1,3-glucosidic linkages in cellulose and glucan to produce
smaller molecular weight glucose and glucose oligomers. The
invention provides methods for producing smaller molecular weight
glucose and glucose oligomers using these enzymes of the
invention.
[0011] In one aspect, the enzymes of the invention are used to
generate glucans, e.g., polysaccharides formed from 1,4-.beta.-
and/or 1,3-glycoside-linked D-glucopyranose. In one aspect, the
endoglucanases of the invention are used in the food industry,
e.g., for baking and fruit and vegetable processing, breakdown of
agricultural waste, in the manufacture of animal feed, in pulp and
paper production, textile manufacture and household and industrial
cleaning agents. In one aspect, the enzymes, e.g., endoglucanases,
of the invention are produced by a microorganism, e.g., by a fungi
and/or a bacteria.
[0012] In one aspect, the enzymes, e.g., endoglucanases, of the
invention are used to hydrolyze beta-glucans (.beta.-glucans) which
are major non-starch polysaccharides of cereals. The glucan content
of a polysaccharide can vary significantly depending on variety and
growth conditions. The physicochemical properties of this
polysaccharide are such that it gives rise to viscous solutions or
even gels under oxidative conditions. In addition glucans have high
water-binding capacity. All of these characteristics present
problems for several industries including brewing, baking, animal
nutrition. In brewing applications, the presence of glucan results
in wort filterability and haze formation issues. In baking
applications (especially for cookies and crackers), glucans can
create sticky doughs that are difficult to machine and reduce
biscuit size. Thus, the enzymes, e.g., endoglucanases, of the
invention are used to decrease the amount of .beta.-glucan in a
.beta.-glucan-comprising composition, e.g., enzymes of the
invention are used in processes to decrease the viscosity of
solutions or gels; to decrease the water-binding capacity of a
composition, e.g., a .beta.-glucan-comprising composition; in
brewing processes (e.g., to increase wort filterability and
decrease haze formation), to decrease the stickiness of doughs,
e.g., those for making cookies, breads, biscuits and the like.
[0013] In addition, carbohydrates (e.g., .beta.-glucan) are
implicated in rapid rehydration of baked products resulting in loss
of crispiness and reduced shelf-life. Thus, the enzymes, e.g.,
endoglucanases, of the invention are used to retain crispiness,
increase crispiness, or reduce the rate of loss of crispiness, and
to increase the shelf-life of any carbohydrate-comprising food,
feed or drink, e.g., a .beta.-glucan-comprising food, feed or
drink.
[0014] Enzymes, e.g., endoglucanases, of the invention are used to
decrease the viscosity of gut contents (e.g., in animals, such as
ruminant animals, or humans), e.g., those with cereal diets. Thus,
in alternative aspects, enzymes, e.g., endoglucanases, of the
invention are used to positively affect the digestibility of a food
or feed and animal (e.g., human or domestic animal) growth rate,
and in one aspect, are used to higher generate feed conversion
efficiencies. For monogastric animal feed applications with cereal
diets, beta-glucan is a contributing factor to viscosity of gut
contents and thereby adversely affects the digestibility of the
feed and animal growth rate. For ruminant animals, these
beta-glucans represent substantial components of fiber intake and
more complete digestion of glucans would facilitate higher feed
conversion efficiencies. Accordingly, the invention provides animal
feeds and foods comprising endoglucanases of the invention, and in
one aspect, these enzymes are active in an animal digestive tract,
e.g., in a stomach and/or intestine.
[0015] Enzymes, e.g., endoglucanases, of the invention are used to
digest cellulose or any beta-1,4-linked glucan-comprising synthetic
or natural material, including those found in any plant material.
Enzymes, e.g., endoglucanases, of the invention are used as
commercial enzymes to digest cellulose, e.g., in the wood
processing, pulp and/or paper industry, in textile manufacture and
in household and industrial cleaning agents, and/or in biomass
waste processing.
[0016] In one aspect the invention provides compositions (e.g.,
pharmaceutical compositions, foods, feeds, drugs, dietary
supplements) comprising the enzymes, polypeptides or
polynucleotides of the invention. These compositions can be
formulated in a variety of forms, e.g., as tablets, gels, pills,
implants, liquids, sprays, powders, food, feed pellets or as any
type of encapsulated form.
[0017] The invention provides isolated or recombinant nucleic acids
comprising a nucleic acid sequence having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to an exemplary nucleic acid of the
invention, including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID
NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ
ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,
SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID
NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ
ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,
SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID
NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109,
SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID
NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127,
SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID
NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145,
SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID
NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163
and SEQ ID NO:165; see also Tables 1, 2, and 3, Examples 1 and 4,
below, and Sequence Listing, over a region of at least about 10,
15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,
1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,
1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100,
2200, 2250, 2300, 2350, 2400, 2450, 2500, or more residues; and in
alternative aspects, these nucleic acids encode at least one
polypeptide having a cellulase activity, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity, or
encode a polypeptide capable of generating an antibody that can
specifically bind to a polypeptide of the invention, or, these
nucleic acids can be used as probes for identifying or isolating
cellulase-encoding nucleic acids, or to inhibit the expression of
cellulase-expressing nucleic acids (all these aspects referred to
as the "nucleic acids of the invention"). In one aspect, the
sequence identities are determined by analysis with a sequence
comparison algorithm or by a visual inspection.
[0018] Nucleic acids of the invention also include isolated or
recombinant nucleic acids encoding an exemplary enzyme of the
invention, including a polypeptide having a sequence as set forth
in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,
SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID
NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ
ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,
SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID
NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ
ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84,
SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID
NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,
SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID
NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120,
SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID
NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138,
SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID
NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156,
SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164 and SEQ
ID NO:166, see also Tables 1, 2, and 3, Examples 1 and 4, below,
and the Sequence Listing, and subsequences thereof and variants
thereof. In one aspect, the polypeptide has a cellulase activity,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity.
[0019] In one aspect, the invention provides cellulase-encoding,
e.g., endoglucanase-, cellobiohydrolase- and/or
beta-glucosidase-encoding nucleic acids having a common novelty in
that they are derived from mixed cultures. The invention provides
cellulose-degrading enzyme-encoding nucleic acids isolated from
mixed cultures comprising a polynucleotide of the invention, e.g.,
a sequence having at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more, or complete (100%) sequence identity to an exemplary nucleic
acid of the invention, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ
ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,
SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID
NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ
ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71,
SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID
NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ
ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99,
SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID
NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,
SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID
NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135,
SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID
NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,
SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID
NO:163 and SEQ ID NO:165, and see Tables 1, 2, and 3, Examples 1
and 4, below, and Sequence Listing, over a region of at least about
50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, or more.
[0020] In one aspect, the invention provides cellulase enzyme-,
e.g., endoglucanase enzyme-, cellobiohydrolase enzyme- and/or
beta-glucosidase enzyme-encoding nucleic acids, including exemplary
polynucleotide sequences of the invention, see also Tables 1, 2,
and 3, Examples 1 and 4, below, and Sequence Listing, and the
polypeptides encoded by them, including enzymes of the invention,
e.g., exemplary polypeptides of the invention, e.g., SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40,
SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ
ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,
SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID
NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ
ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96,
SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID
NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114,
SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID
NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132,
SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID
NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150,
SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID
NO:160, SEQ ID NO:162, SEQ ID NO:164 or SEQ ID NO:166, see also
Table 1 and Sequence Listing, having a common novelty in that they
are derived from a common source, e.g., an environmental source. In
one aspect, the invention also provides cellulase enzyme-, e.g.,
endoglucanase enzyme-, cellobiohydrolase enzyme- and/or
beta-glucosidase enzyme-encoding nucleic acids with a common
novelty in that they are derived from environmental sources, e.g.,
mixed environmental sources.
[0021] In one aspect, the sequence comparison algorithm is a BLAST
version 2.2.2 algorithm where a filtering setting is set to
blastall-p blastp-d "nr pataa"-F F, and all other options are set
to default.
[0022] Another aspect of the invention is an isolated or
recombinant nucleic acid including at least 10, 15, 20, 25, 30, 35,
40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,
600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,
1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700,
1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2200, 2250, 2300,
2350, 2400, 2450, 2500, or more consecutive bases of a nucleic acid
sequence of the invention, sequences substantially identical
thereto, and the sequences complementary thereto.
[0023] In one aspect, the isolated or recombinant nucleic acid
encodes a polypeptide having a cellulase activity, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
activity, which is thermostable. The polypeptide can retain a
cellulase activity under conditions comprising a temperature range
of between about 37.degree. C. to about 95.degree. C.; between
about 55.degree. C. to about 85.degree. C., between about
70.degree. C. to about 95.degree. C., or, between about 90.degree.
C. to about 95.degree. C. The polypeptide can retain a cellulase
activity in temperatures in the range between about 1.degree. C. to
about 5.degree. C., between about 5.degree. C. to about 15.degree.
C., between about 15.degree. C. to about 25.degree. C., between
about 25.degree. C. to about 37.degree. C., between about
37.degree. C. to about 95.degree. C., 96.degree. C., 97.degree. C.,
98.degree. C. or 99.degree. C., between about 55.degree. C. to
about 85.degree. C., between about 70.degree. C. to about
75.degree. C., or between about 90.degree. C. to about 99.degree.
C., or 95.degree. C., 96.degree. C., 97.degree. C., 98.degree. C.
or 99.degree. C., or more.
[0024] In another aspect, the isolated or recombinant nucleic acid
encodes a polypeptide having a cellulase activity, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
activity, which is thermotolerant. The polypeptide can retain a
cellulase activity after exposure to a temperature in the range
from greater than 37.degree. C. to about 95.degree. C. or anywhere
in the range from greater than 55.degree. C. to about 85.degree. C.
The polypeptide can retain a cellulase activity after exposure to a
temperature in the range between about 1.degree. C. to about
5.degree. C., between about 5.degree. C. to about 15.degree. C.,
between about 15.degree. C. to about 25.degree. C., between about
25.degree. C. to about 37.degree. C., between about 37.degree. C.
to about 95.degree. C., 96.degree. C., 97.degree. C., 98.degree. C.
or 99.degree. C., between about 55.degree. C. to about 85.degree.
C., between about 70.degree. C. to about 75.degree. C., or between
about 90.degree. C. to about 95.degree. C., or more. In one aspect,
the polypeptide retains a cellulase activity after exposure to a
temperature in the range from greater than 90.degree. C. to about
99.degree. C., or 95.degree. C., 96.degree. C., 97.degree. C.,
98.degree. C. or 99.degree. C., at about pH 4.5, or more.
[0025] The invention provides isolated or recombinant nucleic acids
comprising a sequence that hybridizes under stringent conditions to
a nucleic acid of the invention, including an exemplary sequence of
the invention, e.g., a sequence as set forth in SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ
ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,
SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID
NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ
ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59,
SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID
NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ
ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87,
SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID
NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105,
SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID
NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123,
SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID
NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141,
SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID
NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159,
SEQ ID NO:161, SEQ ID NO:163 or SEQ ID NO:165 (see also Tables 1,
2, and 3, Examples 1 and 4, below,), or fragments or subsequences
thereof. In one aspect, the nucleic acid encodes a polypeptide
having a cellulase activity, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity. The
nucleic acid can be at least about 10, 15, 20, 25, 30, 35, 40, 45,
50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200 or more
residues in length or the full length of the gene or transcript. In
one aspect, the stringent conditions comprise a wash step
comprising a wash in 0.2.times.SSC at a temperature of about
65.degree. C. for about 15 minutes.
[0026] The invention provides a nucleic acid probe for identifying
or isolating a nucleic acid encoding a polypeptide having a
cellulase activity, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase activity, wherein the probe
comprises at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,
60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or
more, consecutive bases of a sequence comprising a sequence of the
invention, or fragments or subsequences thereof, wherein the probe
identifies the nucleic acid by binding or hybridization. The probe
can comprise an oligonucleotide comprising at least about 10 to 50,
about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100
consecutive bases of a sequence comprising a sequence of the
invention, or fragments or subsequences thereof.
[0027] The invention provides a nucleic acid probe for identifying
or isolating a nucleic acid encoding a polypeptide having a
cellulase activity, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase activity, wherein the probe
comprises a nucleic acid comprising a sequence at least about 10,
15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or
more residues of a nucleic acid of the invention, e.g., a
polynucleotide having at least about 50%, 51%, 52%, 53%, 54%, 55%,
56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence
identity to an exemplary nucleic acid of the invention. In one
aspect, the sequence identities are determined by analysis with a
sequence comparison algorithm or by visual inspection. In
alternative aspects, the probe can comprise an oligonucleotide
comprising at least about 10 to 50, about 20 to 60, about 30 to 70,
about 40 to 80, or about 60 to 100 consecutive bases of a nucleic
acid sequence of the invention, or a subsequence thereof.
[0028] The invention provides an amplification primer pair for
amplifying (e.g., by PCR) a nucleic acid encoding a polypeptide
having a cellulase activity, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity,
wherein the primer pair is capable of amplifying a nucleic acid
comprising a sequence of the invention, or fragments or
subsequences thereof. One or each member of the amplification
primer sequence pair can comprise an oligonucleotide comprising at
least about 10 to 50, or more, consecutive bases of the sequence,
or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or more
consecutive bases of the sequence. The invention provides
amplification primer pairs, wherein the primer pair comprises a
first member having a sequence as set forth by about the first (the
5') 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36 or more residues of a nucleic
acid of the invention, and a second member having a sequence as set
forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36
or more residues of the complementary strand of the first
member.
[0029] The invention provides cellulase-encoding, e.g.,
endoglucanase-, cellobiohydrolase- and/or beta-glucosidase-encoding
nucleic acids generated by amplification, e.g., polymerase chain
reaction (PCR), using an amplification primer pair of the
invention. The invention provides cellulase-encoding, e.g.,
endoglucanase-, cellobiohydrolase- and/or beta-glucosidase-encoding
nucleic acids generated by amplification, e.g., polymerase chain
reaction (PCR), using an amplification primer pair of the
invention. The invention provides methods of making a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme by amplification, e.g., polymerase chain
reaction (PCR), using an amplification primer pair of the
invention. In one aspect, the amplification primer pair amplifies a
nucleic acid from a library, e.g., a gene library, such as an
environmental library.
[0030] The invention provides methods of amplifying a nucleic acid
encoding a polypeptide having a cellulase activity, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
activity comprising amplification of a template nucleic acid with
an amplification primer sequence pair capable of amplifying a
nucleic acid sequence of the invention, or fragments or
subsequences thereof.
[0031] The invention provides expression cassettes comprising a
nucleic acid of the invention or a subsequence thereof. In one
aspect, the expression cassette can comprise the nucleic acid that
is operably linked to a promoter. The promoter can be a viral,
bacterial, mammalian or plant promoter. In one aspect, the plant
promoter can be a potato, rice, corn, wheat, tobacco or barley
promoter. The promoter can be a constitutive promoter. The
constitutive promoter can comprise CaMV35S. In another aspect, the
promoter can be an inducible promoter. In one aspect, the promoter
can be a tissue-specific promoter or an environmentally regulated
or a developmentally regulated promoter. Thus, the promoter can be,
e.g., a seed-specific, a leaf-specific, a root-specific, a
stem-specific or an abscission-induced promoter. In one aspect, the
expression cassette can further comprise a plant or plant virus
expression vector.
[0032] The invention provides cloning vehicles comprising an
expression cassette (e.g., a vector) of the invention or a nucleic
acid of the invention. The cloning vehicle can be a viral vector, a
plasmid, a phage, a phagemid, a cosmid, a fosmid, a bacteriophage
or an artificial chromosome. The viral vector can comprise an
adenovirus vector, a retroviral vector or an adeno-associated viral
vector. The cloning vehicle can comprise a bacterial artificial
chromosome (BAC), a plasmid, a bacteriophage P1-derived vector
(PAC), a yeast artificial chromosome (YAC), or a mammalian
artificial chromosome (MAC).
[0033] The invention provides transformed cell comprising a nucleic
acid of the invention or an expression cassette (e.g., a vector) of
the invention, or a cloning vehicle of the invention. In one
aspect, the transformed cell can be a bacterial cell, a mammalian
cell, a fungal cell, a yeast cell, an insect cell or a plant cell.
In one aspect, the plant cell can be soybeans, rapeseed, oilseed,
tomato, cane sugar, a cereal, a potato, wheat, rice, corn, tobacco
or barley cell.
[0034] The invention provides transgenic non-human animals
comprising a nucleic acid of the invention or an expression
cassette (e.g., a vector) of the invention. In one aspect, the
animal is a mouse, a rat, a pig, a goat or a sheep.
[0035] The invention provides transgenic plants comprising a
nucleic acid of the invention or an expression cassette (e.g., a
vector) of the invention. The transgenic plant can be a cereal
plant, a corn plant, a potato plant, a tomato plant, a wheat plant,
an oilseed plant, a rapeseed plant, a soybean plant, a rice plant,
a barley plant or a tobacco plant.
[0036] The invention provides transgenic seeds comprising a nucleic
acid of the invention or an expression cassette (e.g., a vector) of
the invention. The transgenic seed can be a cereal plant, a corn
seed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a
palm kernel, a sunflower seed, a sesame seed, a peanut or a tobacco
plant seed.
[0037] The invention provides an antisense oligonucleotide
comprising a nucleic acid sequence complementary to or capable of
hybridizing under stringent conditions to a nucleic acid of the
invention. The invention provides methods of inhibiting the
translation of a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme message in a cell
comprising administering to the cell or expressing in the cell an
antisense oligonucleotide comprising a nucleic acid sequence
complementary to or capable of hybridizing under stringent
conditions to a nucleic acid of the invention. In one aspect, the
antisense oligonucleotide is between about 10 to 50, about 20 to
60, about 30 to 70, about 40 to 80, or about 60 to 100 bases in
length, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100 or more bases in length. The invention
provides methods of inhibiting the translation of a cellulase
enzyme, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme message in a cell comprising administering
to the cell or expressing in the cell an antisense oligonucleotide
comprising a nucleic acid sequence complementary to or capable of
hybridizing under stringent conditions to a nucleic acid of the
invention.
[0038] The invention provides double-stranded inhibitory RNA (RNAi,
or RNA interference) molecules (including small interfering RNA, or
siRNAs, for inhibiting transcription, and microRNAs, or miRNAs, for
inhibiting translation) comprising a subsequence of a sequence of
the invention. In one aspect, the siRNA is between about 21 to 24
residues, or, about at least 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60,
65, 70, 75, 80, 85, 90, 95, 100 or more duplex nucleotides in
length. The invention provides methods of inhibiting the expression
of a cellulase enzyme, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme in a cell comprising
administering to the cell or expressing in the cell a
double-stranded inhibitory RNA (siRNA or miRNA), wherein the RNA
comprises a subsequence of a sequence of the invention.
[0039] The invention provides isolated or recombinant polypeptides
comprising an amino acid sequence having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete
(100%) sequence identity to an exemplary polypeptide or peptide of
the invention over a region of at least about 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150,
175, 200, 225, 250, 275, 300, 325, 350 or more residues, or over
the full length of the polypeptide. In one aspect, the sequence
identities are determined by analysis with a sequence comparison
algorithm or by a visual inspection. Exemplary polypeptide or
peptide sequences of the invention include SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID
NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ
ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60,
SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID
NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ
ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88,
SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID
NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106,
SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID
NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,
SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID
NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142,
SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID
NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160,
SEQ ID NO:162, SEQ ID NO:164 and SEQ ID NO:166 (see also Tables 1,
2, and 3, Examples 1 and 4, below, and Sequence Listing), and
subsequences thereof and variants thereof. Exemplary polypeptides
also include fragments of at least about 10, 15, 20, 25, 30, 35,
40, 45, 50, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600 or more residues in length, or over the full
length of an enzyme. Polypeptide or peptide sequences of the
invention include sequence encoded by a nucleic acid of the
invention. Polypeptide or peptide sequences of the invention
include polypeptides or peptides specifically bound by an antibody
of the invention (e.g., epitopes), or polypeptides or peptides that
can generate an antibody of the invention (e.g., an immunogen).
[0040] In one aspect, a polypeptide of the invention has at least
one cellulase enzyme, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme activity. In alternative
aspects, a polynucleotide of the invention encodes a polypeptide
that has at least one cellulase enzyme, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity.
[0041] In one aspect, the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity is thermostable. The polypeptide can retain a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity under conditions comprising a
temperature range of between about 1.degree. C. to about 5.degree.
C., between about 5.degree. C. to about 15.degree. C., between
about 15.degree. C. to about 25.degree. C., between about
25.degree. C. to about 37.degree. C., between about 37.degree. C.
to about 95.degree. C., between about 55.degree. C. to about
85.degree. C., between about 70.degree. C. to about 75.degree. C.,
or between about 90.degree. C. to about 95.degree. C., or more. In
another aspect, the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity can be thermotolerant. The polypeptide can retain a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity after exposure to a temperature in
the range from greater than 37.degree. C. to about 95.degree. C.,
or in the range from greater than 55.degree. C. to about 85.degree.
C. In one aspect, the polypeptide can retain a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity after exposure to a temperature in the range from
greater than 90.degree. C. to about 95.degree. C. at pH 4.5.
[0042] Another aspect of the invention provides an isolated or
recombinant polypeptide or peptide comprising at least 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
125, 150 or more consecutive bases of a polypeptide or peptide
sequence of the invention, sequences substantially identical
thereto, and the sequences complementary thereto. The peptide can
be, e.g., an immunogenic fragment, a motif (e.g., a binding site),
a signal sequence, a prepro sequence or an active site.
[0043] The invention provides isolated or recombinant nucleic acids
comprising a sequence encoding a polypeptide having a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity and a signal sequence, wherein the
nucleic acid comprises a sequence of the invention. The signal
sequence can be derived from another cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme or a non-cellulase, e.g., non-endoglucanase,
non-cellobiohydrolase and/or non-beta-glucosidase enzyme (a
heterologous) enzyme. The invention provides isolated or
recombinant nucleic acids comprising a sequence encoding a
polypeptide having a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity, wherein the sequence does not contain a signal sequence
and the nucleic acid comprises a sequence of the invention. In one
aspect, the invention provides an isolated or recombinant
polypeptide comprising a polypeptide of the invention lacking all
or part of a signal sequence. In one aspect, the isolated or
recombinant polypeptide can comprise the polypeptide of the
invention comprising a heterologous signal sequence, such as a
heterologous cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme signal sequence or
non-cellulase, e.g., non-endoglucanase, non-cellobiohydrolase
and/or non-beta-glucosidase enzyme signal sequence.
[0044] In one aspect, the invention provides chimeric proteins
comprising a first domain comprising a signal sequence of the
invention and at least a second domain. The protein can be a fusion
protein. The second domain can comprise an enzyme. The enzyme can
be a non-enzyme.
[0045] The invention provides chimeric polypeptides comprising at
least a first domain comprising signal peptide (SP), a prepro
sequence and/or a catalytic domain (CD) of the invention and at
least a second domain comprising a heterologous polypeptide or
peptide, wherein the heterologous polypeptide or peptide is not
naturally associated with the signal peptide (SP), prepro sequence
and/or catalytic domain (CD). In one aspect, the heterologous
polypeptide or peptide is not a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme. The
heterologous polypeptide or peptide can be amino terminal to,
carboxy terminal to or on both ends of the signal peptide (SP),
prepro sequence and/or catalytic domain (CD).
[0046] The invention provides isolated or recombinant nucleic acids
encoding a chimeric polypeptide, wherein the chimeric polypeptide
comprises at least a first domain comprising signal peptide (SP), a
prepro domain and/or a catalytic domain (CD) of the invention and
at least a second domain comprising a heterologous polypeptide or
peptide, wherein the heterologous polypeptide or peptide is not
naturally associated with the signal peptide (SP), prepro domain
and/or catalytic domain (CD).
[0047] The invention provides isolated or recombinant signal
sequences (e.g., signal peptides) consisting of or comprising a
sequence as set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to
17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24,
1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to
32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40,
1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46 or 1 to 47, of
a polypeptide of the invention, e.g., the exemplary SEQ ID NO:2,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID
NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ
ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40,
SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID
NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ
ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68,
SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID
NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ
ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96,
SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID
NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114,
SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID
NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132,
SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID
NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150,
SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID
NO:160, SEQ ID NO:162, SEQ ID NO:164 or SEQ ID NO:166 (see Tables
1, 2, and 3, Examples 1 and 4, below, and Sequence Listing). In one
aspect, the invention provides signal sequences comprising the
first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 65, 66, 67, 68, 69, 70 or more amino terminal residues of a
polypeptide of the invention.
[0048] In one aspect, the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity comprises a specific activity at about 37.degree. C. in
the range from about 1 to about 1200 units per milligram of
protein, or, about 100 to about 1000 units per milligram of
protein. In another aspect, the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity comprises a specific activity from about 100 to about 1000
units per milligram of protein, or, from about 500 to about 750
units per milligram of protein. Alternatively, the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity comprises a specific activity at 37.degree. C. in
the range from about 1 to about 750 units per milligram of protein,
or, from about 500 to about 1200 units per milligram of protein. In
one aspect, the cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme activity comprises a
specific activity at 37.degree. C. in the range from about 1 to
about 500 units per milligram of protein, or, from about 750 to
about 1000 units per milligram of protein. In another aspect, the
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity comprises a specific activity at
37.degree. C. in the range from about 1 to about 250 units per
milligram of protein. Alternatively, the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity comprises a specific activity at 37.degree. C. in
the range from about 1 to about 100 units per milligram of
protein.
[0049] In another aspect, the thermotolerance comprises retention
of at least half of the specific activity of the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme at 37.degree. C. after being heated to the elevated
temperature. Alternatively, the thermotolerance can comprise
retention of specific activity at 37.degree. C. in the range from
about 1 to about 1200 units per milligram of protein, or, from
about 500 to about 1000 units per milligram of protein, after being
heated to the elevated temperature. In another aspect, the
thermotolerance can comprise retention of specific activity at
37.degree. C. in the range from about 1 to about 500 units per
milligram of protein after being heated to the elevated
temperature.
[0050] The invention provides the isolated or recombinant
polypeptide of the invention, wherein the polypeptide comprises at
least one glycosylation site. In one aspect, glycosylation can be
an N-linked glycosylation. In one aspect, the polypeptide can be
glycosylated after being expressed in a P. pastoris or a S.
pombe.
[0051] In one aspect, the polypeptide can retain cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity under conditions comprising about pH 6.5, pH 6, pH
5.5, pH 5, pH 4.5 or pH 4 or more acidic. In another aspect, the
polypeptide can retain a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity under conditions comprising about pH 7, pH 7.5 pH 8.0, pH
8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or more basic pH. In one
aspect, the polypeptide can retain a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity after exposure to conditions comprising about pH
6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4 or more acidic pH. In
another aspect, the polypeptide can retain a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity after exposure to conditions comprising about pH 7,
pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or
more basic pH.
[0052] In one aspect, the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme of the
invention has activity at under alkaline conditions, e.g., the
alkaline conditions of the gut, e.g., the small intestine. In one
aspect, the polypeptide can retains activity after exposure to the
acidic pH of the stomach.
[0053] The invention provides protein preparations comprising a
polypeptide (including peptides) of the invention, wherein the
protein preparation comprises a liquid, a solid or a gel. The
invention provides heterodimers comprising a polypeptide of the
invention and a second protein or domain. The second member of the
heterodimer can be a different cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme, a
different enzyme or another protein. In one aspect, the second
domain can be a polypeptide and the heterodimer can be a fusion
protein. In one aspect, the second domain can be an epitope or a
tag. In one aspect, the invention provides homodimers comprising a
polypeptide of the invention.
[0054] The invention provides immobilized polypeptides (including
peptides) having cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme activity, wherein the
immobilized polypeptide comprises a polypeptide of the invention, a
polypeptide encoded by a nucleic acid of the invention, or a
polypeptide comprising a polypeptide of the invention and a second
domain. In one aspect, the polypeptide can be immobilized on a
cell, a metal, a resin, a polymer, a ceramic, a glass, a
microelectrode, a graphitic particle, a bead, a gel, a plate, an
array or a capillary tube.
[0055] The invention also provides arrays comprising an immobilized
nucleic acid of the invention, including, e.g., probes of the
invention. The invention also provides arrays comprising an
antibody of the invention.
[0056] The invention provides isolated or recombinant antibodies
that specifically bind to a polypeptide of the invention or to a
polypeptide encoded by a nucleic acid of the invention. These
antibodies of the invention can be a monoclonal or a polyclonal
antibody. The invention provides hybridomas comprising an antibody
of the invention, e.g., an antibody that specifically binds to a
polypeptide of the invention or to a polypeptide encoded by a
nucleic acid of the invention. The invention provides nucleic acids
encoding these antibodies.
[0057] The invention provides method of isolating or identifying a
polypeptide having cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity comprising the steps of: (a) providing an antibody of the
invention; (b) providing a sample comprising polypeptides; and (c)
contacting the sample of step (b) with the antibody of step (a)
under conditions wherein the antibody can specifically bind to the
polypeptide, thereby isolating or identifying a polypeptide having
a cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme activity.
[0058] The invention provides methods of making an anti-cellulase,
e.g., anti-endoglucanase, anti-cellobiohydrolase and/or
anti-beta-glucosidase enzyme antibody comprising administering to a
non-human animal a nucleic acid of the invention or a polypeptide
of the invention or subsequences thereof in an amount sufficient to
generate a humoral immune response, thereby making an
anti-cellulase, e.g., anti-endoglucanase, anti-cellobiohydrolase
and/or anti-beta-glucosidase enzyme antibody. The invention
provides methods of making an anti-cellulase, e.g.,
anti-endoglucanase, anti-cellobiohydrolase and/or
anti-beta-glucosidase immune response (cellular or humoral)
comprising administering to a non-human animal a nucleic acid of
the invention or a polypeptide of the invention or subsequences
thereof in an amount sufficient to generate an immune response
(cellular or humoral).
[0059] The invention provides methods of producing a recombinant
polypeptide comprising the steps of: (a) providing a nucleic acid
of the invention operably linked to a promoter; and (b) expressing
the nucleic acid of step (a) under conditions that allow expression
of the polypeptide, thereby producing a recombinant polypeptide. In
one aspect, the method can further comprise transforming a host
cell with the nucleic acid of step (a) followed by expressing the
nucleic acid of step (a), thereby producing a recombinant
polypeptide in a transformed cell.
[0060] The invention provides methods for identifying a polypeptide
having cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme activity comprising the following
steps: (a) providing a polypeptide of the invention; or a
polypeptide encoded by a nucleic acid of the invention; (b)
providing cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme substrate; and (c)
contacting the polypeptide or a fragment or variant thereof of step
(a) with the substrate of step (b) and detecting a decrease in the
amount of substrate or an increase in the amount of a reaction
product, wherein a decrease in the amount of the substrate or an
increase in the amount of the reaction product detects a
polypeptide having a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity. In one aspect, the substrate is a cellulose-comprising
compound.
[0061] The invention provides methods for identifying cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme substrate comprising the following steps:
(a) providing a polypeptide of the invention; or a polypeptide
encoded by a nucleic acid of the invention; (b) providing a test
substrate; and (c) contacting the polypeptide of step (a) with the
test substrate of step (b) and detecting a decrease in the amount
of substrate or an increase in the amount of reaction product,
wherein a decrease in the amount of the substrate or an increase in
the amount of a reaction product identifies the test substrate as a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme substrate.
[0062] The invention provides methods of determining whether a test
compound specifically binds to a polypeptide comprising the
following steps: (a) expressing a nucleic acid or a vector
comprising the nucleic acid under conditions permissive for
translation of the nucleic acid to a polypeptide, wherein the
nucleic acid comprises a nucleic acid of the invention, or,
providing a polypeptide of the invention; (b) providing a test
compound; (c) contacting the polypeptide with the test compound;
and (d) determining whether the test compound of step (b)
specifically binds to the polypeptide.
[0063] The invention provides methods for identifying a modulator
of a cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme activity comprising the following
steps: (a) providing a polypeptide of the invention or a
polypeptide encoded by a nucleic acid of the invention; (b)
providing a test compound; (c) contacting the polypeptide of step
(a) with the test compound of step (b) and measuring an activity of
the cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme, wherein a change in the cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity measured in the presence of the
test compound compared to the activity in the absence of the test
compound provides a determination that the test compound modulates
the cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme activity. In one aspect, the
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity can be measured by providing a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme substrate and detecting a decrease in the
amount of the substrate or an increase in the amount of a reaction
product, or, an increase in the amount of the substrate or a
decrease in the amount of a reaction product. A decrease in the
amount of the substrate or an increase in the amount of the
reaction product with the test compound as compared to the amount
of substrate or reaction product without the test compound
identifies the test compound as an activator of cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity. An increase in the amount of the substrate or a
decrease in the amount of the reaction product with the test
compound as compared to the amount of substrate or reaction product
without the test compound identifies the test compound as an
inhibitor of cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme activity.
[0064] The invention provides computer systems comprising a
processor and a data storage device wherein said data storage
device has stored thereon a polypeptide sequence or a nucleic acid
sequence of the invention (e.g., a polypeptide or peptide encoded
by a nucleic acid of the invention). In one aspect, the computer
system can further comprise a sequence comparison algorithm and a
data storage device having at least one reference sequence stored
thereon. In another aspect, the sequence comparison algorithm
comprises a computer program that indicates polymorphisms. In one
aspect, the computer system can further comprise an identifier that
identifies one or more features in said sequence. The invention
provides computer readable media having stored thereon a
polypeptide sequence or a nucleic acid sequence of the invention.
The invention provides methods for identifying a feature in a
sequence comprising the steps of: (a) reading the sequence using a
computer program which identifies one or more features in a
sequence, wherein the sequence comprises a polypeptide sequence or
a nucleic acid sequence of the invention; and (b) identifying one
or more features in the sequence with the computer program. The
invention provides methods for comparing a first sequence to a
second sequence comprising the steps of: (a) reading the first
sequence and the second sequence through use of a computer program
which compares sequences, wherein the first sequence comprises a
polypeptide sequence or a nucleic acid sequence of the invention;
and (b) determining differences between the first sequence and the
second sequence with the computer program. The step of determining
differences between the first sequence and the second sequence can
further comprise the step of identifying polymorphisms. In one
aspect, the method can further comprise an identifier that
identifies one or more features in a sequence. In another aspect,
the method can comprise reading the first sequence using a computer
program and identifying one or more features in the sequence.
[0065] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity from an environmental sample comprising the steps
of: (a) providing an amplification primer sequence pair for
amplifying a nucleic acid encoding a polypeptide having a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity, wherein the primer pair is
capable of amplifying a nucleic acid of the invention; (b)
isolating a nucleic acid from the environmental sample or treating
the environmental sample such that nucleic acid in the sample is
accessible for hybridization to the amplification primer pair; and,
(c) combining the nucleic acid of step (b) with the amplification
primer pair of step (a) and amplifying nucleic acid from the
environmental sample, thereby isolating or recovering a nucleic
acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity from an environmental sample. One or each member of
the amplification primer sequence pair can comprise an
oligonucleotide comprising an amplification primer sequence pair of
the invention, e.g., having at least about 10 to 50 consecutive
bases of a sequence of the invention.
[0066] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity from an environmental sample comprising the steps
of: (a) providing a polynucleotide probe comprising a nucleic acid
of the invention or a subsequence thereof; (b) isolating a nucleic
acid from the environmental sample or treating the environmental
sample such that nucleic acid in the sample is accessible for
hybridization to a polynucleotide probe of step (a); (c) combining
the isolated nucleic acid or the treated environmental sample of
step (b) with the polynucleotide probe of step (a); and (d)
isolating a nucleic acid that specifically hybridizes with the
polynucleotide probe of step (a), thereby isolating or recovering a
nucleic acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity from an environmental sample. The environmental
sample can comprise a water sample, a liquid sample, a soil sample,
an air sample or a biological sample. In one aspect, the biological
sample can be derived from a bacterial cell, a protozoan cell, an
insect cell, a yeast cell, a plant cell, a fungal cell or a
mammalian cell.
[0067] The invention provides methods of generating a variant of a
nucleic acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity comprising the steps of: (a) providing a template
nucleic acid comprising a nucleic acid of the invention; and (b)
modifying, deleting or adding one or more nucleotides in the
template sequence, or a combination thereof, to generate a variant
of the template nucleic acid. In one aspect, the method can further
comprise expressing the variant nucleic acid to generate a variant
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme polypeptide. The modifications, additions
or deletions can be introduced by a method comprising error-prone
PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR,
sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis,
recursive ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, Gene Site Saturation
Mutagenesis (GSSM), synthetic ligation reassembly (SLR),
Chromosomal Saturation Mutagenesis (CSM) or a combination thereof.
In another aspect, the modifications, additions or deletions are
introduced by a method comprising recombination, recursive sequence
recombination, phosphothioate-modified DNA mutagenesis,
uracil-containing template mutagenesis, gapped duplex mutagenesis,
point mismatch repair mutagenesis, repair-deficient host strain
mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion
mutagenesis, restriction-selection mutagenesis,
restriction-purification mutagenesis, artificial gene synthesis,
ensemble mutagenesis, chimeric nucleic acid multimer creation and a
combination thereof.
[0068] In one aspect, the method can be iteratively repeated until
a cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme having an altered or different
activity or an altered or different stability from that of a
polypeptide encoded by the template nucleic acid is produced. In
one aspect, the variant cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
polypeptide is thermotolerant, and retains some activity after
being exposed to an elevated temperature. In another aspect, the
variant cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme polypeptide has increased
glycosylation as compared to the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme encoded
by a template nucleic acid. Alternatively, the variant cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase polypeptide has a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity under a high temperature, wherein the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme encoded by the template nucleic acid is not active under the
high temperature. In one aspect, the method can be iteratively
repeated until a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme coding sequence having an
altered codon usage from that of the template nucleic acid is
produced. In another aspect, the method can be iteratively repeated
until a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme gene having higher or
lower level of message expression or stability from that of the
template nucleic acid is produced.
[0069] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity to increase its expression in a host cell, the
method comprising the following steps: (a) providing a nucleic acid
of the invention encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity; and, (b) identifying a non-preferred or a less
preferred codon in the nucleic acid of step (a) and replacing it
with a preferred or neutrally used codon encoding the same amino
acid as the replaced codon, wherein a preferred codon is a codon
over-represented in coding sequences in genes in the host cell and
a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell,
thereby modifying the nucleic acid to increase its expression in a
host cell.
[0070] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity; the method comprising the following steps: (a)
providing a nucleic acid of the invention; and, (b) identifying a
codon in the nucleic acid of step (a) and replacing it with a
different codon encoding the same amino acid as the replaced codon,
thereby modifying codons in a nucleic acid encoding a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme.
[0071] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity to increase its expression in a host cell, the
method comprising the following steps: (a) providing a nucleic acid
of the invention encoding a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
polypeptide; and, (b) identifying a non-preferred or a less
preferred codon in the nucleic acid of step (a) and replacing it
with a preferred or neutrally used codon encoding the same amino
acid as the replaced codon, wherein a preferred codon is a codon
over-represented in coding sequences in genes in the host cell and
a non-preferred or less preferred codon is a codon
under-represented in coding sequences in genes in the host cell,
thereby modifying the nucleic acid to increase its expression in a
host cell.
[0072] The invention provides methods for modifying a codon in a
nucleic acid encoding a polypeptide having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity to decrease its expression in a host cell, the
method comprising the following steps: (a) providing a nucleic acid
of the invention; and (b) identifying at least one preferred codon
in the nucleic acid of step (a) and replacing it with a
non-preferred or less preferred codon encoding the same amino acid
as the replaced codon, wherein a preferred codon is a codon
over-represented in coding sequences in genes in a host cell and a
non-preferred or less preferred codon is a codon under-represented
in coding sequences in genes in the host cell, thereby modifying
the nucleic acid to decrease its expression in a host cell. In one
aspect, the host cell can be a bacterial cell, a fungal cell, an
insect cell, a yeast cell, a plant cell or a mammalian cell.
[0073] The invention provides methods for producing a library of
nucleic acids encoding a plurality of modified cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme active sites or substrate binding sites, wherein the
modified active sites or substrate binding sites are derived from a
first nucleic acid comprising a sequence encoding a first active
site or a first substrate binding site the method comprising the
following steps: (a) providing a first nucleic acid encoding a
first active site or first substrate binding site, wherein the
first nucleic acid sequence comprises a sequence that hybridizes
under stringent conditions to a nucleic acid of the invention, and
the nucleic acid encodes a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme active
site or a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme substrate binding site;
(b) providing a set of mutagenic oligonucleotides that encode
naturally-occurring amino acid variants at a plurality of targeted
codons in the first nucleic acid; and, (c) using the set of
mutagenic oligonucleotides to generate a set of active
site-encoding or substrate binding site-encoding variant nucleic
acids encoding a range of amino acid variations at each amino acid
codon that was mutagenized, thereby producing a library of nucleic
acids encoding a plurality of modified cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme active sites or substrate binding sites. In one aspect, the
method comprises mutagenizing the first nucleic acid of step (a) by
a method comprising an optimized directed evolution system, Gene
Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly
(SLR), error-prone PCR, shuffling, oligonucleotide-directed
mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo
mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis,
exponential ensemble mutagenesis, site-specific mutagenesis, gene
reassembly, and a combination thereof. In another aspect, the
method comprises mutagenizing the first nucleic acid of step (a) or
variants by a method comprising recombination, recursive sequence
recombination, phosphothioate-modified DNA mutagenesis,
uracil-containing template mutagenesis, gapped duplex mutagenesis,
point mismatch repair mutagenesis, repair-deficient host strain
mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion
mutagenesis, restriction-selection mutagenesis,
restriction-purification mutagenesis, artificial gene synthesis,
ensemble mutagenesis, chimeric nucleic acid multimer creation and a
combination thereof.
[0074] The invention provides methods for making a small molecule
comprising the following steps: (a) providing a plurality of
biosynthetic enzymes capable of synthesizing or modifying a small
molecule, wherein one of the enzymes comprises a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme encoded by a nucleic acid of the invention; (b) providing a
substrate for at least one of the enzymes of step (a); and (c)
reacting the substrate of step (b) with the enzymes under
conditions that facilitate a plurality of biocatalytic reactions to
generate a small molecule by a series of biocatalytic reactions.
The invention provides methods for modifying a small molecule
comprising the following steps: (a) providing a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme, wherein the enzyme comprises a polypeptide of the
invention, or, a polypeptide encoded by a nucleic acid of the
invention, or a subsequence thereof; (b) providing a small
molecule; and (c) reacting the enzyme of step (a) with the small
molecule of step (b) under conditions that facilitate an enzymatic
reaction catalyzed by the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme,
thereby modifying a small molecule by a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymatic reaction. In one aspect, the method can comprise a
plurality of small molecule substrates for the enzyme of step (a),
thereby generating a library of modified small molecules produced
by at least one enzymatic reaction catalyzed by the cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme. In one aspect, the method can comprise a
plurality of additional enzymes under conditions that facilitate a
plurality of biocatalytic reactions by the enzymes to form a
library of modified small molecules produced by the plurality of
enzymatic reactions. In another aspect, the method can further
comprise the step of testing the library to determine if a
particular modified small molecule that exhibits a desired activity
is present within the library. The step of testing the library can
further comprise the steps of systematically eliminating all but
one of the biocatalytic reactions used to produce a portion of the
plurality of the modified small molecules within the library by
testing the portion of the modified small molecule for the presence
or absence of the particular modified small molecule with a desired
activity, and identifying at least one specific biocatalytic
reaction that produces the particular modified small molecule of
desired activity.
[0075] The invention provides methods for determining a functional
fragment of a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme comprising the steps of:
(a) providing a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme, wherein the enzyme
comprises a polypeptide of the invention, or a polypeptide encoded
by a nucleic acid of the invention, or a subsequence thereof; and
(b) deleting a plurality of amino acid residues from the sequence
of step (a) and testing the remaining subsequence for a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity, thereby determining a functional
fragment of a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme. In one aspect, the
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity is measured by providing a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme substrate and detecting a decrease in the
amount of the substrate or an increase in the amount of a reaction
product.
[0076] The invention provides methods for whole cell engineering of
new or modified phenotypes by using real-time metabolic flux
analysis, the method comprising the following steps: (a) making a
modified cell by modifying the genetic composition of a cell,
wherein the genetic composition is modified by addition to the cell
of a nucleic acid of the invention; (b) culturing the modified cell
to generate a plurality of modified cells; (c) measuring at least
one metabolic parameter of the cell by monitoring the cell culture
of step (b) in real time; and, (d) analyzing the data of step (c)
to determine if the measured parameter differs from a comparable
measurement in an unmodified cell under similar conditions, thereby
identifying an engineered phenotype in the cell using real-time
metabolic flux analysis. In one aspect, the genetic composition of
the cell can be modified by a method comprising deletion of a
sequence or modification of a sequence in the cell, or, knocking
out the expression of a gene. In one aspect, the method can further
comprise selecting a cell comprising a newly engineered phenotype.
In another aspect, the method can comprise culturing the selected
cell, thereby generating a new cell strain comprising a newly
engineered phenotype.
[0077] The invention provides methods of increasing thermotolerance
or thermostability of a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
polypeptide, the method comprising glycosylating a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme polypeptide, wherein the polypeptide comprises at least
thirty contiguous amino acids of a polypeptide of the invention; or
a polypeptide encoded by a nucleic acid sequence of the invention,
thereby increasing the thermotolerance or thermostability of the
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase polypeptide. In one aspect, the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme specific activity can be thermostable or thermotolerant at a
temperature in the range from greater than about 37.degree. C. to
about 95.degree. C.
[0078] The invention provides methods for overexpressing a
recombinant cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase polypeptide in a cell comprising
expressing a vector comprising a nucleic acid comprising a nucleic
acid of the invention or a nucleic acid sequence of the invention,
wherein the sequence identities are determined by analysis with a
sequence comparison algorithm or by visual inspection, wherein
overexpression is effected by use of a high activity promoter, a
dicistronic vector or by gene amplification of the vector.
[0079] The invention provides methods of making a transgenic plant
comprising the following steps: (a) introducing a heterologous
nucleic acid sequence into the cell, wherein the heterologous
nucleic sequence comprises a nucleic acid sequence of the
invention, thereby producing a transformed plant cell; and (b)
producing a transgenic plant from the transformed cell. In one
aspect, the step (a) can further comprise introducing the
heterologous nucleic acid sequence by electroporation or
microinjection of plant cell protoplasts. In another aspect, the
step (a) can further comprise introducing the heterologous nucleic
acid sequence directly to plant tissue by DNA particle bombardment.
Alternatively, the step (a) can further comprise introducing the
heterologous nucleic acid sequence into the plant cell DNA using an
Agrobacterium tumefaciens host. In one aspect, the plant cell can
be a cane sugar, beet, soybean, tomato, potato, corn, rice, wheat,
tobacco or barley cell.
[0080] The invention provides methods of expressing a heterologous
nucleic acid sequence in a plant cell comprising the following
steps: (a) transforming the plant cell with a heterologous nucleic
acid sequence operably linked to a promoter, wherein the
heterologous nucleic sequence comprises a nucleic acid of the
invention; (b) growing the plant under conditions wherein the
heterologous nucleic acids sequence is expressed in the plant cell.
The invention provides methods of expressing a heterologous nucleic
acid sequence in a plant cell comprising the following steps: (a)
transforming the plant cell with a heterologous nucleic acid
sequence operably linked to a promoter, wherein the heterologous
nucleic sequence comprises a sequence of the invention; (b) growing
the plant under conditions wherein the heterologous nucleic acids
sequence is expressed in the plant cell.
[0081] The invention provides feeds or foods comprising a
polypeptide of the invention, or a polypeptide encoded by a nucleic
acid of the invention. In one aspect, the invention provides a
food, feed, a liquid, e.g., a beverage (such as a fruit juice or a
beer), a bread or a dough or a bread product, or a beverage
precursor (e.g., a wort), comprising a polypeptide of the
invention. The invention provides food or nutritional supplements
for an animal comprising a polypeptide of the invention, e.g., a
polypeptide encoded by the nucleic acid of the invention.
[0082] In one aspect, the polypeptide in the food or nutritional
supplement can be glycosylated. The invention provides edible
enzyme delivery matrices comprising a polypeptide of the invention,
e.g., a polypeptide encoded by the nucleic acid of the invention.
In one aspect, the delivery matrix comprises a pellet. In one
aspect, the polypeptide can be glycosylated. In one aspect, the
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity is thermotolerant.
[0083] In another aspect, the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity is thermostable.
[0084] The invention provides a food, a feed or a nutritional
supplement comprising a polypeptide of the invention. The invention
provides methods for utilizing a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme as a
nutritional supplement in an animal diet, the method comprising:
preparing a nutritional supplement containing a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme comprising at least thirty contiguous amino acids of a
polypeptide of the invention; and administering the nutritional
supplement to an animal. The animal can be a human, a ruminant or a
monogastric animal. The cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme can be
prepared by expression of a polynucleotide encoding the cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme in an organism selected from the group
consisting of a bacterium, a yeast, a plant, an insect, a fungus
and an animal. The organism can be selected from the group
consisting of an S. pombe, S. cerevisiae, Pichia pastoris, E. coli,
Streptomyces sp., Bacillus sp. and Lactobacillus sp.
[0085] The invention provides edible enzyme delivery matrix
comprising a thermostable recombinant cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme, e.g., a polypeptide of the invention. The invention
provides methods for delivering a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
supplement to an animal, the method comprising: preparing an edible
enzyme delivery matrix in the form of pellets comprising a
granulate edible carrier and a thermostable recombinant cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme, wherein the pellets readily disperse the
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme contained therein into aqueous media, and
administering the edible enzyme delivery matrix to the animal. The
recombinant cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme can comprise a polypeptide
of the invention. The cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme can be
glycosylated to provide thermostability at pelletizing conditions.
The delivery matrix can be formed by pelletizing a mixture
comprising a grain germ and a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme. The
pelletizing conditions can include application of steam. The
pelletizing conditions can comprise application of a temperature in
excess of about 80.degree. C. for about 5 minutes and the enzyme
retains a specific activity of at least 350 to about 900 units per
milligram of enzyme.
[0086] In one aspect, invention provides a pharmaceutical
composition comprising a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme of the
invention, or a polypeptide encoded by a nucleic acid of the
invention. In one aspect, the pharmaceutical composition acts as a
digestive aid.
[0087] In certain aspects, a cellulose-containing compound is
contacted a polypeptide of the invention having a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity at a pH in the range of between about pH 3.0 to
9.0, 10.0, 11.0 or more. In other aspects, a cellulose-containing
compound is contacted with the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme at a
temperature of about 55.degree. C., 60.degree. C., 65.degree. C.,
70.degree. C., 75.degree. C., 80.degree. C., 85.degree. C.,
90.degree. C., or more.
[0088] The details of one or more aspects of the invention are set
forth in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent
from the description and drawings, and from the claims.
[0089] All publications, patents, patent applications, GenBank
sequences and ATCC deposits, cited herein are hereby expressly
incorporated by reference for all purposes.
BRIEF DESCRIPTION OF DRAWINGS
[0090] The following drawings are illustrative of aspects of the
invention and are not meant to limit the scope of the invention as
encompassed by the claims.
[0091] FIG. 1 is a block diagram of a computer system.
[0092] FIG. 2 is a flow diagram illustrating one aspect of a
process for comparing a new nucleotide or protein sequence with a
database of sequences in order to determine the homology levels
between the new sequence and the sequences in the database.
[0093] FIG. 3 is a flow diagram illustrating one aspect of a
process in a computer for determining whether two sequences are
homologous.
[0094] FIG. 4 is a flow diagram illustrating one aspect of an
identifier process 300 for detecting the presence of a feature in a
sequence.
[0095] FIG. 5 is an illustration of the structure of
cellobiose.
[0096] FIGS. 6 and 7 illustrate the results of a TLC analysis of
reaction products from cellohexaose, as discussed in detail in
Example 1, below.
[0097] FIG. 8 illustrates in graph form data showing the release of
cellobiose from PASC by the exemplary enzyme 22/22a (a CBH) of the
invention, as discussed in detail in Example 2, below.
[0098] FIG. 9 illustrates in graph form data showing the release of
cellobiose from AVICEL.RTM. MCC by the exemplary enzyme 22/22a (a
CBH) of the invention, as discussed in detail in Example 2,
below.
[0099] FIG. 10 illustrates in graphic form data showing a typical
GIGAMATRIX.TM. breakout, where active clones expressing enzyme able
to hydrolyze methylumbelliferyl cellobioside are identified, as
discussed in detail in Example 4, below.
[0100] FIG. 11 illustrates in graph form data showing the activity
of selected enzymes against phosphoric acid-swollen cellulose
(PASC) by capillary electrophoresis (CE) analysis, as discussed in
detail in Example 4, below.
[0101] FIG. 12 illustrates in graph form data from assays of an
exemplary enzyme of the invention and subclone variants in
AVICEL.RTM. Microcrystalline Cellulose (MCC), where the reaction
products were analyzed by the BCA reducing sugar assay, as
discussed in detail in Example 4, below.
[0102] FIG. 13 illustrates in graph form data from primary GSSM
screening assays, as discussed in detail in Example 4, below.
[0103] FIG. 14 illustrates in graph form data from secondary GSSM
screening assays, as discussed in detail in Example 4, below.
[0104] FIG. 15 illustrates in graph form data from mixed, or
"blended", GSSM screening assays, as discussed in detail in Example
4, below.
[0105] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0106] The invention provides polypeptides with cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
activity, polynucleotides encoding them, and methods of making and
using these polynucleotides and polypeptides. The invention also
provides cellulase enzymes, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzymes, polynucleotides encoding
these enzymes, the use of such polynucleotides and
polypeptides.
[0107] In one aspect, the invention provides a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase, with an increased catalytic rate, improving the
process of substrate hydrolysis. This increased efficiency in
catalytic rate leads to an increased efficiency in producing sugars
that will subsequently be used by microorganisms for ethanol
production. In one aspect, microorganisms generating enzyme of the
invention are used with ethanol-producing microorganisms. Thus, the
invention provides methods for ethanol production and making "clean
fuels" based on ethanol, e.g., for transportation using
bioethanol.
[0108] In one aspect the invention provides compositions (e.g.,
enzyme preparations, feeds, drugs, dietary supplements) comprising
the enzymes, polypeptides or polynucleotides of the invention.
These compositions can be formulated in a variety of forms, e.g.,
as liquids, gels, pills, tablets, sprays, powders, food, feed
pellets or encapsulated forms, including nanoencapsulated
forms.
[0109] Assays for measuring cellulase activity, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
activity, e.g., for determining if a polypeptide has cellulase
activity, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity, are well known in the art and are within
the scope of the invention; see, e.g., Baker W L, Panow A,
Estimation of cellulase activity using a glucose-oxidase-Cu(II)
reducing assay for glucose, J Biochem Biophys Methods. 1991
December, 23(4):265-73; Sharrock K R, Cellulase assay methods: a
review, J Biochem Biophys Methods. 1988 October, 17(2):81-105;
Carder J H, Detection and quantitation of cellulase by Congo red
staining of substrates in a cup-plate diffusion assay, Anal
Biochem. 1986 Feb. 15, 153(1):75-9; Canevascini G., A cellulase
assay coupled to cellobiose dehydrogenase, Anal Biochem. 1985 June,
147(2):419-27; Huang J S, Tang J, Sensitive assay for cellulase and
dextranase. Anal Biochem. 1976 June, 73(2):369-77.
[0110] The pH of reaction conditions utilized by the invention is
another variable parameter for which the invention provides. In
certain aspects, the pH of the reaction is conducted in the range
of about 3.0 to about 9.0. In other aspects, the pH is about 4.5 or
the pH is about 7.5 or the pH is about 9. Reaction conditions
conducted under alkaline conditions also can be advantageous, e.g.,
in some industrial or pharmaceutical applications of enzymes of the
invention.
[0111] The invention provides cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase polypeptides
of the invention in a variety of forms and formulations. In the
methods of the invention, cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase polypeptides
of the invention are used in a variety of forms and formulations.
For example, purified cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase polypeptides
can be used in enzyme preparations deployed in bioethanol
production or in pharmaceutical or dietary aid applications.
Alternatively, the enzymes of the invention can be used directly in
processes to produce bioethanol, make clean fuels, process
biowastes, process foods, liquids or feeds, and the like.
[0112] Alternatively, the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase polypeptides
of the invention can be expressed in a microorganism using
procedures known in the art. In other aspects, the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
polypeptides of the invention can be immobilized on a solid support
prior to use in the methods of the invention. Methods for
immobilizing enzymes on solid supports are commonly known in the
art, for example J. Mol. Cat. B: Enzymatic 6 (1999) 29-39; Chivata
et al. Biocatalysis: Immobilized cells and enzymes, J. Mol. Cat. 37
(1986) 1-24: Sharma et al., Immobilized Biomaterials Techniques and
Applications, Angew. Chem. Int. Ed. Engl. 21 (1982) 837-54: Laskin
(Ed.), Enzymes and Immobilized Cells in Biotechnology.
Nucleic Acids, Probes and Inhibitory Molecules
[0113] The invention provides isolated and recombinant nucleic
acids, e.g., see Tables 1, 2, and 3, Examples 1 and 4, below, and
Sequence Listing; nucleic acids encoding polypeptides, including
the exemplary polynucleotide sequences of the invention, e.g., see
Table 1 and Sequence Listing; including expression cassettes such
as expression vectors and various cloning vehicles comprising
nucleic acids of the invention. The invention also includes methods
for discovering, identifying or isolated new cellulases, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
polypeptide sequences using the nucleic acids of the invention. The
invention also includes methods for inhibiting the expression of
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase encoding genes and transcripts using the nucleic
acids of the invention.
[0114] Also provided are methods for modifying the nucleic acids of
the invention, including making variants of nucleic acids of the
invention, by, e.g., synthetic ligation reassembly, optimized
directed evolution system and/or saturation mutagenesis such as
gene site saturation mutagenesis (GSSM). The term "saturation
mutagenesis", Gene Site Saturation Mutagenesis, or "GSSM" includes
a method that uses degenerate oligonucleotide primers to introduce
point mutations into a polynucleotide, as described in detail,
below. The term "optimized directed evolution system" or "optimized
directed evolution" includes a method for reassembling fragments of
related nucleic acid sequences, e.g., related genes, and explained
in detail, below. The term "synthetic ligation reassembly" or "SLR"
includes a method of ligating oligonucleotide fragments in a
non-stochastic fashion, and explained in detail, below. The term
"variant" refers to polynucleotides or polypeptides of the
invention modified at one or more base pairs, codons, introns,
exons, or amino acid residues (respectively) yet still retain the
biological activity of a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase of the
invention. Variants can be produced by any number of means included
methods such as, for example, error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, GSSM and any
combination thereof.
[0115] The nucleic acids of the invention can be made, isolated
and/or manipulated by, e.g., cloning and expression of cDNA
libraries, amplification of message or genomic DNA by PCR, and the
like. For example, exemplary sequences of the invention were
initially derived from environmental sources. Thus, in one aspect,
the invention provides cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme-encoding nucleic acids, and the polypeptides encoded by
them, having a common novelty in that they are derived from a
common source, e.g., an environmental, mixed culture, or a
bacterial source.
[0116] In practicing the methods of the invention, homologous genes
can be modified by manipulating a template nucleic acid, as
described herein. The invention can be practiced in conjunction
with any method or protocol or device known in the art, which are
well described in the scientific and patent literature.
[0117] The phrases "nucleic acid" or "nucleic acid sequence" as
used herein refer to an oligonucleotide, nucleotide,
polynucleotide, or to a fragment of any of these, to DNA or RNA of
genomic or synthetic origin which may be single-stranded or
double-stranded and may represent a sense or antisense
(complementary) strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like material, natural or synthetic in origin. The
phrases "nucleic acid" or "nucleic acid sequence" includes
oligonucleotide, nucleotide, polynucleotide, or to a fragment of
any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of
genomic or synthetic origin which may be single-stranded or
double-stranded and may represent a sense or antisense strand, to
peptide nucleic acid (PNA), or to any DNA-like or RNA-like
material, natural or synthetic in origin, including, e.g., iRNA,
ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g.,
iRNPs). The term encompasses nucleic acids, i.e., oligonucleotides,
containing known analogues of natural nucleotides. The term also
encompasses nucleic-acid-like structures with synthetic backbones,
see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;
Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)
Antisense Nucleic Acid Drug Dev 6:153-156. "Oligonucleotide"
includes either a single stranded polydeoxynucleotide or two
complementary polydeoxynucleotide strands which may be chemically
synthesized. Such synthetic oligonucleotides have no 5' phosphate
and thus will not ligate to another oligonucleotide without adding
a phosphate with an ATP in the presence of a kinase. A synthetic
oligonucleotide can ligate to a fragment that has not been
dephosphorylated.
[0118] A "coding sequence of" or a "nucleotide sequence encoding" a
particular polypeptide or protein, is a nucleic acid sequence which
is transcribed and translated into a polypeptide or protein when
placed under the control of appropriate regulatory sequences. The
term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the
coding region (leader and trailer) as well as, where applicable,
intervening sequences (introns) between individual coding segments
(exons). A promoter sequence is "operably linked to" a coding
sequence when RNA polymerase which initiates transcription at the
promoter will transcribe the coding sequence into mRNA. "Operably
linked" as used herein refers to a functional relationship between
two or more nucleic acid (e.g., DNA) segments. It can refer to the
functional relationship of transcriptional regulatory sequence to a
transcribed sequence. For example, a promoter is operably linked to
a coding sequence, such as a nucleic acid of the invention, if it
stimulates or modulates the transcription of the coding sequence in
an appropriate host cell or other expression system. Generally,
promoter transcriptional regulatory sequences that are operably
linked to a transcribed sequence are physically contiguous to the
transcribed sequence, i.e., they are cis-acting. However, some
transcriptional regulatory sequences, such as enhancers, need not
be physically contiguous or located in close proximity to the
coding sequences whose transcription they enhance.
[0119] The term "expression cassette" as used herein refers to a
nucleotide sequence which is capable of affecting expression of a
structural gene (i.e., a protein coding sequence, such as a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme of the invention) in a host compatible with
such sequences. Expression cassettes include at least a promoter
operably linked with the polypeptide coding sequence; and,
optionally, with other sequences, e.g., transcription termination
signals. Additional factors necessary or helpful in effecting
expression may also be used, e.g., enhancers, alpha-factors. Thus,
expression cassettes also include plasmids, expression vectors,
recombinant viruses, any form of recombinant "naked DNA" vector,
and the like. A "vector" comprises a nucleic acid which can infect,
transfect, transiently or permanently transduce a cell. It will be
recognized that a vector can be a naked nucleic acid, or a nucleic
acid complexed with protein or lipid. The vector optionally
comprises viral or bacterial nucleic acids and/or proteins, and/or
membranes (e.g., a cell membrane, a viral lipid envelope, etc.).
Vectors include, but are not limited to replicons (e.g., RNA
replicons, bacteriophages) to which fragments of DNA may be
attached and become replicated. Vectors thus include, but are not
limited to RNA, autonomous self-replicating circular or linear DNA
or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat.
No. 5,217,879), and include both the expression and non-expression
plasmids. Where a recombinant microorganism or cell culture is
described as hosting an "expression vector" this includes both
extra-chromosomal circular and linear DNA and DNA that has been
incorporated into the host chromosome(s). Where a vector is being
maintained by a host cell, the vector may either be stably
replicated by the cells during mitosis as an autonomous structure,
or is incorporated within the host's genome.
[0120] As used herein, the term "recombinant" encompasses nucleic
acids adjacent to a "backbone" nucleic acid to which it is not
adjacent in its natural environment. In one aspect, to be
"enriched" the nucleic acids will represent about 5% or more of the
number of nucleic acid inserts in a population of nucleic acid
backbone molecules. Backbone molecules according to the invention
include nucleic acids such as expression vectors, self-replicating
nucleic acids, viruses, integrating nucleic acids and other vectors
or nucleic acids used to maintain or manipulate a nucleic acid
insert of interest. In one aspect, the enriched nucleic acids
represent about 15% or more of the number of nucleic acid inserts
in the population of recombinant backbone molecules. In one aspect,
the enriched nucleic acids represent about 50% or more of the
number of nucleic acid inserts in the population of recombinant
backbone molecules. In a one aspect, the enriched nucleic acids
represent about 90% or more of the number of nucleic acid inserts
in the population of recombinant backbone molecules.
[0121] One aspect of the invention is an isolated or recombinant
nucleic acid comprising one of the sequences of the invention, or a
fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75,
100, 150, 200, 300, 400, or 500 or more consecutive bases of a
nucleic acid of the invention. The isolated or recombinant nucleic
acids may comprise DNA, including cDNA, genomic DNA and synthetic
DNA. The DNA may be double-stranded or single-stranded and if
single stranded may be the coding strand or non-coding (anti-sense)
strand. Alternatively, the isolated or recombinant nucleic acids
comprise RNA.
[0122] The isolated or recombinant nucleic acids of the invention
may be used to prepare one of the polypeptides of the invention, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, or 150 or more consecutive amino acids of one of the
polypeptides of the invention. Accordingly, another aspect of the
invention is an isolated or recombinant nucleic acid which encodes
one of the polypeptides of the invention, or fragments comprising
at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more
consecutive amino acids of one of the polypeptides of the
invention. The coding sequences of these nucleic acids may be
identical to one of the coding sequences of one of the nucleic
acids of the invention or may be different coding sequences which
encode one of the of the invention having at least 5, 10, 15, 20,
25, 30, 35, 40, 50, 75, 100, or 150 or more consecutive amino acids
of one of the polypeptides of the invention, as a result of the
redundancy or degeneracy of the genetic code. The genetic code is
well known to those of skill in the art and can be obtained, e.g.,
on page 214 of B. Lewin, Genes VI, Oxford University Press,
1997.
[0123] The nucleic acids encoding polypeptides of the invention
include but are not limited to: the coding sequence of a nucleic
acid of the invention and additional coding sequences, such as
leader sequences or proprotein sequences and non-coding sequences,
such as introns or non-coding sequences 5' and/or 3' of the coding
sequence. Thus, as used herein, the term "polynucleotide encoding a
polypeptide" encompasses a polynucleotide which includes the coding
sequence for the polypeptide as well as a polynucleotide which
includes additional coding and/or non-coding sequence.
[0124] In one aspect, the nucleic acid sequences of the invention
are mutagenized using conventional techniques, such as site
directed mutagenesis, or other techniques familiar to those skilled
in the art, to introduce silent changes into the polynucleotides o
of the invention. As used herein, "silent changes" include, for
example, changes which do not alter the amino acid sequence encoded
by the polynucleotide. Such changes may be desirable in order to
increase the level of the polypeptide produced by host cells
containing a vector encoding the polypeptide by introducing codons
or codon pairs which occur frequently in the host organism.
[0125] The invention also relates to polynucleotides which have
nucleotide changes which result in amino acid substitutions,
additions, deletions, fusions and truncations in the polypeptides
of the invention. Such nucleotide changes may be introduced using
techniques such as site directed mutagenesis, random chemical
mutagenesis, exonuclease III deletion and other recombinant DNA
techniques. Alternatively, such nucleotide changes may be naturally
occurring allelic variants which are isolated by identifying
nucleic acids which specifically hybridize to probes comprising at
least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,
or 500 consecutive bases of one of the sequences of the invention
(or the sequences complementary thereto) under conditions of high,
moderate, or low stringency as provided herein.
[0126] General Techniques
[0127] The nucleic acids used to practice this invention, whether
RNA, siRNA, miRNA, antisense nucleic acid, cDNA, genomic DNA,
vectors, viruses or hybrids thereof, may be isolated from a variety
of sources, genetically engineered, amplified, and/or
expressed/generated recombinantly. Recombinant polypeptides (e.g.,
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes) generated from these nucleic acids can be
individually isolated or cloned and tested for a desired activity.
Any recombinant expression system can be used, including bacterial,
mammalian, yeast, insect or plant cell expression systems.
[0128] Alternatively, these nucleic acids can be synthesized in
vitro by well-known chemical synthesis techniques, as described in,
e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997)
Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.
Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang
(1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;
Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
[0129] Techniques for the manipulation of nucleic acids, such as,
e.g., subcloning, labeling probes (e.g., random-primer labeling
using Klenow polymerase, nick translation, amplification),
sequencing, hybridization and the like are well described in the
scientific and patent literature, see, e.g., Sambrook, ed.,
MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold
Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997);
LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY:
HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic
Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
[0130] Another useful means of obtaining and manipulating nucleic
acids used to practice the methods of the invention is to clone
from genomic samples, and, if desired, screen and re-clone inserts
isolated or amplified from, e.g., genomic clones or cDNA clones.
Sources of nucleic acid used in the methods of the invention
include genomic or cDNA libraries contained in, e.g., mammalian
artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118;
6,025,155; human artificial chromosomes, see, e.g., Rosenfeld
(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);
bacterial artificial chromosomes (BAC); P1 artificial chromosomes,
see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors
(PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids,
recombinant viruses, phages or plasmids.
[0131] In one aspect, a nucleic acid encoding a polypeptide of the
invention is assembled in appropriate phase with a leader sequence
capable of directing secretion of the translated polypeptide or
fragment thereof.
[0132] The invention provides fusion proteins and nucleic acids
encoding them. A polypeptide of the invention can be fused to a
heterologous peptide or polypeptide, such as N-terminal
identification peptides which impart desired characteristics, such
as increased stability or simplified purification. Peptides and
polypeptides of the invention can also be synthesized and expressed
as fusion proteins with one or more additional domains linked
thereto for, e.g., producing a more immunogenic peptide, to more
readily isolate a recombinantly synthesized peptide, to identify
and isolate antibodies and antibody-expressing B cells, and the
like. Detection and purification facilitating domains include,
e.g., metal chelating peptides such as polyhistidine tracts and
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle
Wash.). The inclusion of a cleavable linker sequences such as
Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a
purification domain and the motif-comprising peptide or polypeptide
to facilitate purification. For example, an expression vector can
include an epitope-encoding nucleic acid sequence linked to six
histidine residues followed by a thioredoxin and an enterokinase
cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797;
Dobeli (1998) Protein Expr. Purif. 12:404-414). The histidine
residues facilitate detection and purification while the
enterokinase cleavage site provides a means for purifying the
epitope from the remainder of the fusion protein. Technology
pertaining to vectors encoding fusion proteins and application of
fusion proteins are well described in the scientific and patent
literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
[0133] Transcriptional and Translational Control Sequences
[0134] The invention provides nucleic acid (e.g., DNA) sequences of
the invention operatively linked to expression (e.g.,
transcriptional or translational) control sequence(s), e.g.,
promoters or enhancers, to direct or modulate RNA
synthesis/expression. The expression control sequence can be in an
expression vector. Exemplary bacterial promoters include lacI,
lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplary eukaryotic
promoters include CMV immediate early, HSV thymidine kinase, early
and late SV40, LTRs from retrovirus, and mouse metallothionein
I.
[0135] As used herein, the term "promoter" includes all sequences
capable of driving transcription of a coding sequence in a cell,
e.g., a plant or animal cell. Thus, promoters used in the
constructs of the invention include cis-acting transcriptional
control elements and regulatory sequences that are involved in
regulating or modulating the timing and/or rate of transcription of
a gene. For example, a promoter can be a cis-acting transcriptional
control element, including an enhancer, a promoter, a transcription
terminator, an origin of replication, a chromosomal integration
sequence, 5' and 3' untranslated regions, or an intronic sequence,
which are involved in transcriptional regulation. These cis-acting
sequences can interact with proteins or other biomolecules to carry
out (turn on/off, regulate, modulate, etc.) transcription.
"Constitutive" promoters are those that drive expression
continuously under most environmental conditions and states of
development or cell differentiation. "Inducible" or "regulatable"
promoters direct expression of the nucleic acid of the invention
under the influence of environmental conditions or developmental
conditions. Examples of environmental conditions that may affect
transcription by inducible promoters include anaerobic conditions,
elevated temperature, drought, or the presence of light.
[0136] "Tissue-specific" promoters are transcriptional control
elements that are only active in particular cells or tissues or
organs, e.g., in plants or animals. Tissue-specific regulation may
be achieved by certain intrinsic factors which ensure that genes
encoding proteins specific to a given tissue are expressed. Such
factors are known to exist in mammals and plants so as to allow for
specific tissues to develop.
[0137] Promoters suitable for expressing a polypeptide in bacteria
include the E. coli lac or trp promoters, the lad promoter, the
lacZ promoter, the T3 promoter, the T7 promoter, the gpt promoter,
the lambda PR promoter, the lambda PL promoter, promoters from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), and the acid phosphatase promoter. Eukaryotic
promoters include the CMV immediate early promoter, the HSV
thymidine kinase promoter, heat shock promoters, the early and late
SV40 promoter, LTRs from retroviruses, and the mouse
metallothionein-I promoter. Other promoters known to control
expression of genes in prokaryotic or eukaryotic cells or their
viruses may also be used. Promoters suitable for expressing the
polypeptide or fragment thereof in bacteria include the E. coli lac
or trp promoters, the lac/promoter, the lacZ promoter, the T3
promoter, the T7 promoter, the gpt promoter, the lambda P.sub.R
promoter, the lambda P.sub.L promoter, promoters from operons
encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK)
and the acid phosphatase promoter. Fungal promoters include the
.alpha.-factor promoter. Eukaryotic promoters include the CMV
immediate early promoter, the HSV thymidine kinase promoter, heat
shock promoters, the early and late SV40 promoter, LTRs from
retroviruses and the mouse metallothionein-I promoter. Other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses may also be used.
[0138] Tissue-Specific Plant Promoters
[0139] The invention provides expression cassettes that can be
expressed in a tissue-specific manner, e.g., that can express a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme of the invention in a tissue-specific
manner. The invention also provides plants or seeds that express a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme of the invention in a tissue-specific
manner. The tissue-specificity can be seed specific, stem specific,
leaf specific, root specific, fruit specific and the like.
[0140] The term "plant" includes whole plants, plant parts (e.g.,
leaves, stems, flowers, roots, etc.), plant protoplasts, seeds and
plant cells and progeny of same. The class of plants which can be
used in the method of the invention is generally as broad as the
class of higher plants amenable to transformation techniques,
including angiosperms (monocotyledonous and dicotyledonous plants),
as well as gymnosperms. It includes plants of a variety of ploidy
levels, including polyploid, diploid, haploid and hemizygous
states. As used herein, the term "transgenic plant" includes plants
or plant cells into which a heterologous nucleic acid sequence has
been inserted, e.g., the nucleic acids and various recombinant
constructs (e.g., expression cassettes) of the invention.
[0141] In one aspect, a constitutive promoter such as the CaMV 35S
promoter can be used for expression in specific parts of the plant
or seed or throughout the plant. For example, for overexpression, a
plant promoter fragment can be employed which will direct
expression of a nucleic acid in some or all tissues of a plant,
e.g., a regenerated plant. Such promoters are referred to herein as
"constitutive" promoters and are active under most environmental
conditions and states of development or cell differentiation.
Examples of constitutive promoters include the cauliflower mosaic
virus (CaMV) 35S transcription initiation region, the 1'- or
2'-promoter derived from T-DNA of Agrobacterium tumefaciens, and
other transcription initiation regions from various plant genes
known to those of skill. Such genes include, e.g., ACT11 from
Arabidopsis (Huang (1996) Plant Mol. Biol. 33:125-139); Cat3 from
Arabidopsis (GenBank No. U43147, Zhong (1996) Mol. Gen. Genet.
251:196-203); the gene encoding stearoyl-acyl carrier protein
desaturase from Brassica napus (Genbank No. X74782, Solocombe
(1994) Plant Physiol. 104:1167-1176); GPc1 from maize (GenBank No.
X15596; Martinez (1989) J. Mol. Biol. 208:551-565); the Gpc2 from
maize (GenBank No. U45855, Manjunath (1997) Plant Mol. Biol.
33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;
5,633,440.
[0142] The invention uses tissue-specific or constitutive promoters
derived from viruses which can include, e.g., the tobamovirus
subgenomic promoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA
92:1679-1683; the rice tungro bacilliform virus (RTBV), which
replicates only in phloem cells in infected rice plants, with its
promoter which drives strong phloem-specific reporter gene
expression; the cassava vein mosaic virus (CVMV) promoter, with
highest activity in vascular elements, in leaf mesophyll cells, and
in root tips (Verdaguer (1996) Plant Mol. Biol. 31:1129-1139).
[0143] In one aspect, the plant promoter directs expression of
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme-expressing nucleic acid in a specific
tissue, organ or cell type (i.e. tissue-specific promoters) or may
be otherwise under more precise environmental or developmental
control or under the control of an inducible promoter. Examples of
environmental conditions that may affect transcription include
anaerobic conditions, elevated temperature, the presence of light,
or sprayed with chemicals/hormones. For example, the invention
incorporates the drought-inducible promoter of maize (Busk (1997)
supra); the cold, drought, and high salt inducible promoter from
potato (Kirch (1997) Plant Mol. Biol. 33:897 909).
[0144] In one aspect, tissue-specific promoters promote
transcription only within a certain time frame of developmental
stage within that tissue. See, e.g., Blazquez (1998) Plant Cell
10:791-800, characterizing the Arabidopsis LEAFY gene promoter. See
also Cardon (1997) Plant J 12:367-77, describing the transcription
factor SPL3, which recognizes a conserved sequence motif in the
promoter region of the A. thaliana floral meristem identity gene
AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29, pp
995-1004, describing the meristem promoter eIF4. Tissue specific
promoters which are active throughout the life cycle of a
particular tissue can be used. In one aspect, the nucleic acids of
the invention are operably linked to a promoter active primarily
only in cotton fiber cells. In one aspect, the nucleic acids of the
invention are operably linked to a promoter active primarily during
the stages of cotton fiber cell elongation, e.g., as described by
Rinehart (1996) supra. The nucleic acids can be operably linked to
the Fbl2A gene promoter to be preferentially expressed in cotton
fiber cells (Ibid). See also, John (1997) Proc. Natl. Acad. Sci.
USA 89:5769-5773; John, et al., U.S. Pat. Nos. 5,608,148 and
5,602,321, describing cotton fiber-specific promoters and methods
for the construction of transgenic cotton plants. Root-specific
promoters may also be used to express the nucleic acids of the
invention. Examples of root-specific promoters include the promoter
from the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev.
Cytol. 123:39-60). Other promoters that can be used to express the
nucleic acids of the invention include, e.g., ovule-specific,
embryo-specific, endosperm-specific, integument-specific, seed
coat-specific promoters, or some combination thereof; a
leaf-specific promoter (see, e.g., Busk (1997) Plant J. 11:1285
1295, describing a leaf-specific promoter in maize); the ORF13
promoter from Agrobacterium rhizogenes (which exhibits high
activity in roots, see, e.g., Hansen (1997) supra); a maize pollen
specific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet.
224:161 168); a tomato promoter active during fruit ripening,
senescence and abscission of leaves and, to a lesser extent, of
flowers can be used (see, e.g., Blume (1997) Plant J. 12:731 746);
a pistil-specific promoter from the potato SK2 gene (see, e.g.,
Ficker (1997) Plant Mol. Biol. 35:425 431); the Blec4 gene from
pea, which is active in epidermal tissue of vegetative and floral
shoot apices of transgenic alfalfa making it a useful tool to
target the expression of foreign genes to the epidermal layer of
actively growing shoots or fibers; the ovule-specific BEL1 gene
(see, e.g., Reiser (1995) Cell 83:735-742, GenBank No. U39944);
and/or, the promoter in Klee, U.S. Pat. No. 5,589,583, describing a
plant promoter region is capable of conferring high levels of
transcription in meristematic tissue and/or rapidly dividing
cells.
[0145] In one aspect, plant promoters which are inducible upon
exposure to plant hormones, such as auxins, are used to express the
nucleic acids of the invention. For example, the invention can use
the auxin-response elements E1 promoter fragment (AuxREs) in the
soybean (Glycine max L.) (Liu (1997) Plant Physiol. 115:397-407);
the auxin-responsive Arabidopsis GST6 promoter (also responsive to
salicylic acid and hydrogen peroxide) (Chen (1996) Plant J. 10:
955-966); the auxin-inducible parC promoter from tobacco (Sakai
(1996) 37:906-913); a plant biotin response element (Streit (1997)
Mol. Plant. Microbe Interact. 10:933-937); and, the promoter
responsive to the stress hormone abscisic acid (Sheen (1996)
Science 274:1900-1902).
[0146] The nucleic acids of the invention can also be operably
linked to plant promoters which are inducible upon exposure to
chemicals reagents which can be applied to the plant, such as
herbicides or antibiotics. For example, the maize In2-2 promoter,
activated by benzenesulfonamide herbicide safeners, can be used (De
Veylder (1997) Plant Cell Physiol. 38:568-577); application of
different herbicide safeners induces distinct gene expression
patterns, including expression in the root, hydathodes, and the
shoot apical meristem. Coding sequence can be under the control of,
e.g., a tetracycline-inducible promoter, e.g., as described with
transgenic tobacco plants containing the Avena sativa L. (oat)
arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);
or, a salicylic acid-responsive element (Stange (1997) Plant J.
11:1315-1324). Using chemically- (e.g., hormone- or pesticide-)
induced promoters, i.e., promoter responsive to a chemical which
can be applied to the transgenic plant in the field, expression of
a polypeptide of the invention can be induced at a particular stage
of development of the plant. Thus, the invention also provides for
transgenic plants containing an inducible gene encoding for
polypeptides of the invention whose host range is limited to target
plant species, such as corn, rice, barley, soybean, tomato, wheat,
potato or other crops, inducible at any stage of development of the
crop.
[0147] One of skill will recognize that a tissue-specific plant
promoter may drive expression of operably linked sequences in
tissues other than the target tissue. Thus, in one aspect, a
tissue-specific promoter is one that drives expression
preferentially in the target tissue or cell type, but may also lead
to some expression in other tissues as well.
[0148] The nucleic acids of the invention can also be operably
linked to plant promoters which are inducible upon exposure to
chemicals reagents. These reagents include, e.g., herbicides,
synthetic auxins, or antibiotics which can be applied, e.g.,
sprayed, onto transgenic plants. Inducible expression of the
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme-producing nucleic acids of the invention
will allow the grower to select plants with the optimal cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme expression and/or activity. The development
of plant parts can thus controlled. In this way the invention
provides the means to facilitate the harvesting of plants and plant
parts. For example, in various embodiments, the maize In2-2
promoter, activated by benzenesulfonamide herbicide safeners, is
used (De Veylder (1997) Plant Cell Physiol. 38:568-577);
application of different herbicide safeners induces distinct gene
expression patterns, including expression in the root, hydathodes,
and the shoot apical meristem. Coding sequences of the invention
are also under the control of a tetracycline-inducible promoter,
e.g., as described with transgenic tobacco plants containing the
Avena sativa L. (oat) arginine decarboxylase gene (Masgrau (1997)
Plant J. 11:465-473); or, a salicylic acid-responsive element
(Stange (1997) Plant J. 11:1315-1324).
[0149] In some aspects, proper polypeptide expression may require
polyadenylation region at the 3'-end of the coding region. The
polyadenylation region can be derived from the natural gene, from a
variety of other plant (or animal or other) genes, or from genes in
the Agrobacterial T-DNA.
[0150] Expression Vectors and Cloning Vehicles
[0151] The invention provides expression vectors and cloning
vehicles comprising nucleic acids of the invention, e.g., sequences
encoding the cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzymes of the invention.
Expression vectors and cloning vehicles of the invention can
comprise viral particles, baculovirus, phage, plasmids, phagemids,
cosmids, fosmids, bacterial artificial chromosomes, viral DNA
(e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and
derivatives of SV40), P1-based artificial chromosomes, yeast
plasmids, yeast artificial chromosomes, and any other vectors
specific for specific hosts of interest (such as bacillus,
Aspergillus and yeast). Vectors of the invention can include
chromosomal, non-chromosomal and synthetic DNA sequences. Large
numbers of suitable vectors are known to those of skill in the art,
and are commercially available. Exemplary vectors are include:
bacterial: pQE.TM. vectors (Qiagen), pBLUESCRIPT.TM. plasmids, pNH
vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3,
pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene),
pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid
or other vector may be used so long as they are replicable and
viable in the host. Low copy number or high copy number vectors may
be employed with the present invention. "Plasmids" can be
commercially available, publicly available on an unrestricted
basis, or can be constructed from available plasmids in accord with
published procedures. Equivalent plasmids to those described herein
are known in the art and will be apparent to the ordinarily skilled
artisan.
[0152] The expression vector can comprise a promoter, a ribosome
binding site for translation initiation and a transcription
terminator. The vector may also include appropriate sequences for
amplifying expression. Mammalian expression vectors can comprise an
origin of replication, any necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
non-transcribed sequences. In some aspects, DNA sequences derived
from the SV40 splice and polyadenylation sites may be used to
provide the required non-transcribed genetic elements.
[0153] In one aspect, the expression vectors contain one or more
selectable marker genes to permit selection of host cells
containing the vector. Such selectable markers include genes
encoding dihydrofolate reductase or genes conferring neomycin
resistance for eukaryotic cell culture, genes conferring
tetracycline or ampicillin resistance in E. coli, and the S.
cerevisiae TRP 1 gene. Promoter regions can be selected from any
desired gene using chloramphenicol transferase (CAT) vectors or
other vectors with selectable markers.
[0154] In one aspect, vectors for expressing the polypeptide or
fragment thereof in eukaryotic cells contain enhancers to increase
expression levels. Enhancers are cis-acting elements of DNA that
can be from about 10 to about 300 bp in length. They can act on a
promoter to increase its transcription. Exemplary enhancers include
the SV40 enhancer on the late side of the replication origin by 100
to 270, the cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the replication origin, and the
adenovirus enhancers.
[0155] A nucleic acid sequence can be inserted into a vector by a
variety of procedures. In general, the sequence is ligated to the
desired position in the vector following digestion of the insert
and the vector with appropriate restriction endonucleases.
Alternatively, blunt ends in both the insert and the vector may be
ligated. A variety of cloning techniques are known in the art,
e.g., as described in Ausubel and Sambrook. Such procedures and
others are deemed to be within the scope of those skilled in the
art.
[0156] The vector can be in the form of a plasmid, a viral
particle, or a phage. Other vectors include chromosomal,
non-chromosomal and synthetic DNA sequences, derivatives of SV40;
bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A
variety of cloning and expression vectors for use with prokaryotic
and eukaryotic hosts are described by, e.g., Sambrook.
[0157] Particular bacterial vectors which can be used include the
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia
Fine Chemicals, Uppsala, Sweden), GEM1 (Promega Biotec, Madison,
Wis., USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, psiX174 pBLUESCRIPT
II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a,
pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia), pKK232-8 and pCM7.
Particular eukaryotic vectors include pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However, any
other vector may be used as long as it is replicable and viable in
the host cell.
[0158] The nucleic acids of the invention can be expressed in
expression cassettes, vectors or viruses and transiently or stably
expressed in plant cells and seeds. One exemplary transient
expression system uses episomal expression systems, e.g.,
cauliflower mosaic virus (CaMV) viral RNA generated in the nucleus
by transcription of an episomal mini-chromosome containing
supercoiled DNA, see, e.g., Covey (1990) Proc. Natl. Acad. Sci. USA
87:1633-1637. Alternatively, coding sequences, i.e., all or
sub-fragments of sequences of the invention can be inserted into a
plant host cell genome becoming an integral part of the host
chromosomal DNA. Sense or antisense transcripts can be expressed in
this manner. A vector comprising the sequences (e.g., promoters or
coding regions) from nucleic acids of the invention can comprise a
marker gene that confers a selectable phenotype on a plant cell or
a seed. For example, the marker may encode biocide resistance,
e.g., antibiotic resistance, such as resistance to kanamycin, G418,
bleomycin, hygromycin, or herbicide resistance, such as resistance
to chlorosulfuron or Basta.
[0159] Expression vectors capable of expressing nucleic acids and
proteins in plants are well known in the art, and can include,
e.g., vectors from Agrobacterium spp., potato virus X (see, e.g.,
Angell (1997) EMBO J. 16:3675-3684), tobacco mosaic virus (see,
e.g., Casper (1996) Gene 173:69-73), tomato bushy stunt virus (see,
e.g., Hillman (1989) Virology 169:42-50), tobacco etch virus (see,
e.g., Dolja (1997) Virology 234:243-252), bean golden mosaic virus
(see, e.g., Morinaga (1993) Microbiol Immunol. 37:471-476),
cauliflower mosaic virus (see, e.g., Cecchini (1997) Mol. Plant
Microbe Interact. 10:1094-1101), maize Ac/Ds transposable element
(see, e.g., Rubin (1997) Mol. Cell. Biol. 17:6294-6302; Kunze
(1996) Curr. Top. Microbiol. Immunol. 204:161-194), and the maize
suppressor-mutator (Spm) transposable element (see, e.g., Schlappi
(1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.
[0160] In one aspect, the expression vector can have two
replication systems to allow it to be maintained in two organisms,
for example in mammalian or insect cells for expression and in a
prokaryotic host for cloning and amplification. Furthermore, for
integrating expression vectors, the expression vector can contain
at least one sequence homologous to the host cell genome. It can
contain two homologous sequences which flank the expression
construct. The integrating vector can be directed to a specific
locus in the host cell by selecting the appropriate homologous
sequence for inclusion in the vector. Constructs for integrating
vectors are well known in the art.
[0161] Expression vectors of the invention may also include a
selectable marker gene to allow for the selection of bacterial
strains that have been transformed, e.g., genes which render the
bacteria resistant to drugs such as ampicillin, chloramphenicol,
erythromycin, kanamycin, neomycin and tetracycline. Selectable
markers can also include biosynthetic genes, such as those in the
histidine, tryptophan and leucine biosynthetic pathways.
[0162] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct RNA synthesis. Particular named bacterial promoters
include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L, and trp.
Eukaryotic promoters include CMV immediate early, HSV thymidine
kinase, early and late SV40, LTRs from retrovirus and mouse
metallothionein-I. Selection of the appropriate vector and promoter
is well within the level of ordinary skill in the art. The
expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
Promoter regions can be selected from any desired gene using
chloramphenicol transferase (CAT) vectors or other vectors with
selectable markers. In addition, the expression vectors in one
aspect contain one or more selectable marker genes to provide a
phenotypic trait for selection of transformed host cells such as
dihydrofolate reductase or neomycin resistance for eukaryotic cell
culture, or such as tetracycline or ampicillin resistance in E.
coli.
[0163] Mammalian expression vectors may also comprise an origin of
replication, any necessary ribosome binding sites, a
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences and 5' flanking
nontranscribed sequences. In some aspects, DNA sequences derived
from the SV40 splice and polyadenylation sites may be used to
provide the required nontranscribed genetic elements.
[0164] Vectors for expressing the polypeptide or fragment thereof
in eukaryotic cells may also contain enhancers to increase
expression levels. Enhancers are cis-acting elements of DNA,
usually from about 10 to about 300 bp in length that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin by 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin and the adenovirus
enhancers.
[0165] In addition, the expression vectors can contain one or more
selectable marker genes to permit selection of host cells
containing the vector. Such selectable markers include genes
encoding dihydrofolate reductase or genes conferring neomycin
resistance for eukaryotic cell culture, genes conferring
tetracycline or ampicillin resistance in E. coli and the S.
cerevisiae TRP1 gene.
[0166] In some aspects, the nucleic acid encoding one of the
polypeptides of the invention, or fragments comprising at least
about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more
consecutive amino acids thereof is assembled in appropriate phase
with a leader sequence capable of directing secretion of the
translated polypeptide or fragment thereof. In one aspect, the
nucleic acid can encode a fusion polypeptide in which one of the
polypeptides of the invention, or fragments comprising at least 5,
10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or more consecutive
amino acids thereof is fused to heterologous peptides or
polypeptides, such as N-terminal identification peptides which
impart desired characteristics, such as increased stability or
simplified purification.
[0167] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is ligated
to the desired position in the vector following digestion of the
insert and the vector with appropriate restriction endonucleases.
Alternatively, blunt ends in both the insert and the vector may be
ligated. A variety of cloning techniques are disclosed in Ausubel
et al. Current Protocols in Molecular Biology, John Wiley 503 Sons,
Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory
Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989. Such
procedures and others are deemed to be within the scope of those
skilled in the art.
[0168] The vector may be, for example, in the form of a plasmid, a
viral particle, or a phage. Other vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, derivatives of SV40;
bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus and pseudorabies. A variety
of cloning and expression vectors for use with prokaryotic and
eukaryotic hosts are described by Sambrook, et al., Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, N.Y.,
(1989).
[0169] Host Cells and Transformed Cells
[0170] The invention also provides a transformed cell comprising a
nucleic acid sequence of the invention, e.g., a sequence encoding a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme of the invention, or a vector of the
invention. The host cell may be any of the host cells familiar to
those skilled in the art, including prokaryotic cells, eukaryotic
cells, such as bacterial cells, fungal cells, yeast cells,
mammalian cells, insect cells, or plant cells. Exemplary bacterial
cells include any species of Streptomyces, Staphylococcus or
Bacillus, or the exemplary species E. coli, Bacillus subtilis,
Bacillus cereus, Salmonella typhimurium. Exemplary insect cells
include any species of Spodoptera or Drosophila, including
Drosophila S2 and Spodoptera Sf9. Exemplary animal cells include
CHO, COS or Bowes melanoma or any mouse or human cell line. The
selection of an appropriate host is within the abilities of those
skilled in the art. Techniques for transforming a wide variety of
higher plant species are well known and described in the technical
and scientific literature. See, e.g., Weising (1988) Ann. Rev.
Genet. 22:421-477; U.S. Pat. No. 5,750,870.
[0171] The vector can be introduced into the host cells using any
of a variety of techniques, including transformation, transfection,
transduction, viral infection, gene guns, or Ti-mediated gene
transfer. Particular methods include calcium phosphate
transfection, DEAE-Dextran mediated transfection, lipofection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0172] In one aspect, the nucleic acids or vectors of the invention
are introduced into the cells for screening, thus, the nucleic
acids enter the cells in a manner suitable for subsequent
expression of the nucleic acid. The method of introduction is
largely dictated by the targeted cell type. Exemplary methods
include CaPO.sub.4 precipitation, liposome fusion, lipofection
(e.g., LIPOFECTIN.TM.), electroporation, viral infection, etc. The
candidate nucleic acids may stably integrate into the genome of the
host cell (for example, with retroviral introduction) or may exist
either transiently or stably in the cytoplasm (i.e. through the use
of traditional plasmids, utilizing standard regulatory sequences,
selection markers, etc.). As many pharmaceutically important
screens require human or model mammalian cell targets, retroviral
vectors capable of transfecting such targets can be used.
[0173] Where appropriate, the engineered host cells can be cultured
in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the invention. Following transformation of a suitable host
strain and growth of the host strain to an appropriate cell
density, the selected promoter may be induced by appropriate means
(e.g., temperature shift or chemical induction) and the cells may
be cultured for an additional period to allow them to produce the
desired polypeptide or fragment thereof.
[0174] Cells can be harvested by centrifugation, disrupted by
physical or chemical means, and the resulting crude extract is
retained for further purification. Microbial cells employed for
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Such methods are well known to those
skilled in the art. The expressed polypeptide or fragment thereof
can be recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
polypeptide. If desired, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0175] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Depending upon the host employed in a recombinant
production procedure, the polypeptides produced by host cells
containing the vector may be glycosylated or may be
non-glycosylated. Polypeptides of the invention may or may not also
include an initial methionine amino acid residue.
[0176] Cell-free translation systems can also be employed to
produce a polypeptide of the invention. Cell-free translation
systems can use mRNAs transcribed from a DNA construct comprising a
promoter operably linked to a nucleic acid encoding the polypeptide
or fragment thereof. In some aspects, the DNA construct may be
linearized prior to conducting an in vitro transcription reaction.
The transcribed mRNA is then incubated with an appropriate
cell-free translation extract, such as a rabbit reticulocyte
extract, to produce the desired polypeptide or fragment
thereof.
[0177] The expression vectors can contain one or more selectable
marker genes to provide a phenotypic trait for selection of
transformed host cells such as dihydrofolate reductase or neomycin
resistance for eukaryotic cell culture, or such as tetracycline or
ampicillin resistance in E. coli.
[0178] Host cells containing the polynucleotides of interest, e.g.,
nucleic acids of the invention, can be cultured in conventional
nutrient media modified as appropriate for activating promoters,
selecting transformants or amplifying genes. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression and will
be apparent to the ordinarily skilled artisan. The clones which are
identified as having the specified enzyme activity may then be
sequenced to identify the polynucleotide sequence encoding an
enzyme having the enhanced activity.
[0179] The invention provides a method for overexpressing a
recombinant cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme in a cell comprising
expressing a vector comprising a nucleic acid of the invention,
e.g., a nucleic acid comprising a nucleic acid sequence with at
least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more sequence identity to an exemplary sequence of the invention
over a region of at least about 100 residues, wherein the sequence
identities are determined by analysis with a sequence comparison
algorithm or by visual inspection, or, a nucleic acid that
hybridizes under stringent conditions to a nucleic acid sequence of
the invention. The overexpression can be effected by any means,
e.g., use of a high activity promoter, a dicistronic vector or by
gene amplification of the vector.
[0180] The nucleic acids of the invention can be expressed, or
overexpressed, in any in vitro or in vivo expression system. Any
cell culture systems can be employed to express, or over-express,
recombinant protein, including bacterial, insect, yeast, fungal or
mammalian cultures. Over-expression can be effected by appropriate
choice of promoters, enhancers, vectors (e.g., use of replicon
vectors, dicistronic vectors (see, e.g., Gurtu (1996) Biochem.
Biophys. Res. Commun. 229:295-8), media, culture systems and the
like. In one aspect, gene amplification using selection markers,
e.g., glutamine synthetase (see, e.g., Sanders (1987) Dev. Biol.
Stand. 66:55-63), in cell systems are used to overexpress the
polypeptides of the invention. The host cell may be any of the host
cells familiar to those skilled in the art, including prokaryotic
cells, eukaryotic cells, mammalian cells, insect cells, or plant
cells. The selection of an appropriate host is within the abilities
of those skilled in the art.
[0181] The vector may be introduced into the host cells using any
of a variety of techniques, including transformation, transfection,
transduction, viral infection, gene guns, or Ti-mediated gene
transfer. Particular methods include calcium phosphate
transfection, DEAE-Dextran mediated transfection, lipofection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0182] Where appropriate, the engineered host cells can be cultured
in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the invention. Following transformation of a suitable host
strain and growth of the host strain to an appropriate cell
density, the selected promoter may be induced by appropriate means
(e.g., temperature shift or chemical induction) and the cells may
be cultured for an additional period to allow them to produce the
desired polypeptide or fragment thereof.
[0183] Cells can be harvested by centrifugation, disrupted by
physical or chemical means and the resulting crude extract is
retained for further purification. Microbial cells employed for
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Such methods are well known to those
skilled in the art. The expressed polypeptide or fragment thereof
can be recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
polypeptide. If desired, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0184] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts
(described by Gluzman, Cell, 23:175, 1981) and other cell lines
capable of expressing proteins from a compatible vector, such as
the C127, 3T3, CHO, HeLa and BHK cell lines.
[0185] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Depending upon the host employed in a recombinant
production procedure, the polypeptides produced by host cells
containing the vector may be glycosylated or may be
non-glycosylated. Polypeptides of the invention may or may not also
include an initial methionine amino acid residue.
[0186] Alternatively, the polypeptides of the invention, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, or 150 or more consecutive amino acids thereof can be
synthetically produced by conventional peptide synthesizers, e.g.,
as discussed below. In other aspects, fragments or portions of the
polypeptides may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides.
[0187] Cell-free translation systems can also be employed to
produce one of the polypeptides of the invention, or fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or
150 or more consecutive amino acids thereof using mRNAs transcribed
from a DNA construct comprising a promoter operably linked to a
nucleic acid encoding the polypeptide or fragment thereof. In some
aspects, the DNA construct may be linearized prior to conducting an
in vitro transcription reaction. The transcribed mRNA is then
incubated with an appropriate cell-free translation extract, such
as a rabbit reticulocyte extract, to produce the desired
polypeptide or fragment thereof.
[0188] Amplification of Nucleic Acids
[0189] In practicing the invention, nucleic acids of the invention
and nucleic acids encoding the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
invention, or modified nucleic acids of the invention, can be
reproduced by amplification, e.g., PCR. Amplification can also be
used to clone or modify the nucleic acids of the invention. Thus,
the invention provides amplification primer sequence pairs for
amplifying nucleic acids of the invention. One of skill in the art
can design amplification primer sequence pairs for any part of or
the full length of these sequences.
[0190] In one aspect, the invention provides a nucleic acid
amplified by an amplification primer pair of the invention, e.g., a
primer pair as set forth by about the first (the 5') 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more residues of a
nucleic acid of the invention, and about the first (the 5') 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25 or more residues of the
complementary strand. The invention provides amplification primer
sequence pairs for amplifying a nucleic acid encoding a polypeptide
having a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme activity, wherein the
primer pair is capable of amplifying a nucleic acid comprising a
sequence of the invention, or fragments or subsequences thereof.
One or each member of the amplification primer sequence pair can
comprise an oligonucleotide comprising at least about 10 to 50 or
more consecutive bases of the sequence, or about 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 or more consecutive bases
of the sequence. The invention provides amplification primer pairs,
wherein the primer pair comprises a first member having a sequence
as set forth by about the first (the 5') 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, or 25 or more residues of a nucleic
acid of the invention, and a second member having a sequence as set
forth by about the first (the 5') 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 or more residues of the complementary
strand of the first member.
[0191] The invention provides cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes
generated by amplification, e.g., polymerase chain reaction (PCR),
using an amplification primer pair of the invention. The invention
provides methods of making a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme by
amplification, e.g., PCR, using an amplification primer pair of the
invention. In one aspect, the amplification primer pair amplifies a
nucleic acid from a library, e.g., a gene library, such as an
environmental library.
[0192] Amplification reactions can also be used to quantify the
amount of nucleic acid in a sample (such as the amount of message
in a cell sample), label the nucleic acid (e.g., to apply it to an
array or a blot), detect the nucleic acid, or quantify the amount
of a specific nucleic acid in a sample. In one aspect of the
invention, message isolated from a cell or a cDNA library are
amplified.
[0193] The skilled artisan can select and design suitable
oligonucleotide amplification primers. Amplification methods are
also well known in the art, and include, e.g., polymerase chain
reaction, PCR (see, e.g., PCR PROTOCOLS, A GUIDE TO METHODS AND
APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR
STRATEGIES (1995), ed. Innis, Academic Press, Inc., N.Y., ligase
chain reaction (LCR) (see, e.g., Wu (1989) Genomics 4:560;
Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);
transcription amplification (see, e.g., Kwoh (1989) Proc. Natl.
Acad. Sci. USA 86:1173); and, self-sustained sequence replication
(see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q
Beta replicase amplification (see, e.g., Smith (1997) J. Clin.
Microbiol. 35:1477-1491), automated Q-beta replicase amplification
assay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and
other RNA polymerase mediated techniques (e.g., NASBA, Cangene,
Mississauga, Ontario); see also Berger (1987) Methods Enzymol.
152:307-316; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and
4,683,202; Sooknanan (1995) Biotechnology 13:563-564.
Determining Sequence Identity in Nucleic Acids and Polypeptides
[0194] The invention provides nucleic acids comprising sequences
having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or more, or complete (100%) sequence identity (homology)
to an exemplary nucleic acid of the invention (see also Tables 1,
2, and 3, Examples 1 and 4, below, and Sequence Listing) over a
region of at least about 50, 75, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,
1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more,
residues. The invention provides polypeptides comprising sequences
having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or more, or complete (100%) sequence identity to an
exemplary polypeptide of the invention (see Tables 1, 2, and 3,
Examples 1 and 4, below, and Sequence Listing). The extent of
sequence identity (homology) may be determined using any computer
program and associated parameters, including those described
herein, such as BLAST 2.2.2. or FASTA version 3.0t78, with the
default parameters.
[0195] Nucleic acid sequences of the invention can comprise at
least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,
or 500 or more consecutive nucleotides of an exemplary sequence of
the invention and sequences substantially identical thereto.
Homologous sequences and fragments of nucleic acid sequences of the
invention can refer to a sequence having at least about 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,
65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence
identity (homology) to these sequences. Homology (sequence
identity) may be determined using any of the computer programs and
parameters described herein, including FASTA version 3.0t78 with
the default parameters. Homologous sequences also include RNA
sequences in which uridines replace the thymines in the nucleic
acid sequences of the invention. The homologous sequences may be
obtained using any of the procedures described herein or may result
from the correction of a sequencing error. It will be appreciated
that the nucleic acid sequences of the invention can be represented
in the traditional single character format (See the inside back
cover of Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman &
Co., New York.) or in any other format which records the identity
of the nucleotides in a sequence.
[0196] In various aspects, sequence comparison programs identified
herein are used in this aspect of the invention, i.e., to determine
if a nucleic acid or polypeptide sequence is within the scope of
the invention. However, protein and/or nucleic acid sequence
identities (homologies) may be evaluated using any sequence
comparison algorithm or program known in the art. Such algorithms
and programs include, but are by no means limited to, TBLASTN,
BLASTP, FASTA, TFASTA and CLUSTALW (see, e.g., Pearson and Lipman,
Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al.,
J. Mol. Biol. 215(3):403-410, 1990; Thompson Nucleic Acids Res.
22(2):4673-4680, 1994; Higgins et al., Methods Enzymol.
266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410,
1990; Altschul et al., Nature Genetics 3:266-272, 1993).
[0197] In one aspect, homology or identity is measured using
sequence analysis software (e.g., Sequence Analysis Software
Package of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
Such software matches similar sequences by assigning degrees of
homology to various deletions, substitutions and other
modifications. In one aspect, the terms "homology" and "identity"
in the context of two or more nucleic acids or polypeptide
sequences, refer to two or more sequences or subsequences that are
the same or have a specified percentage of amino acid residues or
nucleotides that are the same when compared and aligned for maximum
correspondence over a comparison window or designated region as
measured using any number of sequence comparison algorithms or by
manual alignment and visual inspection. In one aspect, for sequence
comparison, one sequence acts as a reference sequence, to which
test sequences are compared. When using a sequence comparison
algorithm, test and reference sequences are entered into a
computer, subsequence coordinates are designated, if necessary and
sequence algorithm program parameters are designated. Default
program parameters can be used, or alternative parameters can be
designated. The sequence comparison algorithm then calculates the
percent sequence identities for the test sequences relative to the
reference sequence, based on the program parameters.
[0198] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequence for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by
the search for similarity method of person & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA
in the Wisconsin Genetics Software Package, Genetics Computer
Group, 575 Science Dr., Madison, Wis.), or by manual alignment and
visual inspection. Other algorithms for determining homology or
identity include, for example, in addition to a BLAST program
(Basic Local Alignment Search Tool at the National Center for
Biological Information), ALIGN, AMAS (Analysis of Multiply Aligned
Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET
(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR,
BIOSCAN (Biological Sequence Comparative Analysis Node), BLIMPS
(BLocks IMProved Searcher), FASTA, Intervals & Points, BMB,
CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS,
Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced
Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC,
FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global
Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive
Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local
Content Program), MACAW (Multiple Alignment Construction &
Analysis Workbench), MAP (Multiple Alignment Program), MBLKP,
MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA
(Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such
alignment programs can also be used to screen genome databases to
identify polynucleotide sequences having substantially identical
sequences. A number of genome databases are available, for example,
a substantial portion of the human genome is available as part of
the Human Genome Sequencing Project (Gibbs, 1995). At least
twenty-one other genomes have already been sequenced, including,
for example, M. genitalium (Fraser et al., 1995), M. jannaschii
(Bult et al., 1996), H. influenzae (Fleischmann et al., 1995), E.
coli (Blattner et al., 1997) and yeast (S. cerevisiae) (Mewes et
al., 1997) and D. melanogaster (Adams et al., 2000). Significant
progress has also been made in sequencing the genomes of model
organism, such as mouse, C. elegans and Arabadopsis sp. Several
databases containing genomic information annotated with some
functional information are maintained by different organizations
and may be accessible via the internet.
[0199] In one aspect, BLAST and BLAST 2.0 algorithms are used,
which are described in Altschul et al., Nuc. Acids Res.
25:3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215:403-410,
1990, respectively. Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T and X determine the sensitivity and speed
of the alignment. The BLASTN program (for nucleotide sequences)
uses as defaults a wordlength (W) of 11, an expectation (E) of 10,
M=5, N=-4 and a comparison of both strands. For amino acid
sequences, the BLASTP program uses as defaults a wordlength of 3
and expectations (E) of 10 and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4 and a
comparison of both strands.
[0200] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin &
Altschul, Proc. Natl. Acad. Sci. USA 90:5873, 1993). One measure of
similarity provided by BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a references sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.2, more in one aspect less than about 0.01 and
most in one aspect less than about 0.001.
[0201] In one aspect, protein and nucleic acid sequence homologies
are evaluated using the Basic Local Alignment Search Tool ("BLAST")
In particular, five specific BLAST programs are used to perform the
following task: [0202] (1) BLASTP and BLAST3 compare an amino acid
query sequence against a protein sequence database; [0203] (2)
BLASTN compares a nucleotide query sequence against a nucleotide
sequence database; [0204] (3) BLASTX compares the six-frame
conceptual translation products of a query nucleotide sequence
(both strands) against a protein sequence database; [0205] (4)
TBLASTN compares a query protein sequence against a nucleotide
sequence database translated in all six reading frames (both
strands); and [0206] (5) TBLASTX compares the six-frame
translations of a nucleotide query sequence against the six-frame
translations of a nucleotide sequence database.
[0207] The BLAST programs identify homologous sequences by
identifying similar segments, which are referred to herein as
"high-scoring segment pairs," between a query amino or nucleic acid
sequence and a test sequence which is in one aspect obtained from a
protein or nucleic acid sequence database. High-scoring segment
pairs are in one aspect identified (i.e., aligned) by means of a
scoring matrix, many of which are known in the art. In one aspect,
the scoring matrix used is the BLOSUM62 matrix (Gonnet (1992)
Science 256:1443-1445; Henikoff and Henikoff (1993) Proteins
17:49-61). Less in one aspect, the PAM or PAM250 matrices may also
be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for
Detecting Distance Relationships: Atlas of Protein Sequence and
Structure, Washington: National Biomedical Research Foundation).
BLAST programs are accessible through the U.S. National Library of
Medicine.
[0208] The parameters used with the above algorithms may be adapted
depending on the sequence length and degree of homology studied. In
some aspects, the parameters may be the default parameters used by
the algorithms in the absence of instructions from the user.
Computer Systems and Computer Program Products
[0209] The invention provides computers, computer systems, computer
readable mediums, computer programs products and the like recorded
or stored thereon the nucleic acid and polypeptide sequences of the
invention. Additionally, in practicing the methods of the
invention, e.g., to determine and identify sequence identities (to
determine whether a nucleic acid is within the scope of the
invention), structural homologies, motifs and the like in silico, a
nucleic acid or polypeptide sequence of the invention can be
stored, recorded, and manipulated on any medium which can be read
and accessed by a computer.
[0210] As used herein, the words "recorded" and "stored" refer to a
process for storing information on a computer medium. A skilled
artisan can readily adopt any known methods for recording
information on a computer readable medium to generate manufactures
comprising one or more of the nucleic acid and/or polypeptide
sequences of the invention. As used herein, the terms "computer,"
"computer program" and "processor" are used in their broadest
general contexts and incorporate all such devices, as described in
detail, below. A "coding sequence of" or a "sequence encodes" a
particular polypeptide or protein, is a nucleic acid sequence which
is transcribed and translated into a polypeptide or protein when
placed under the control of appropriate regulatory sequences.
[0211] The polypeptides of the invention include exemplary
sequences of the invention and sequences substantially identical
thereto, and subsequences (fragments) of any of the preceding
sequences. In one aspect, substantially identical, or homologous,
polypeptide sequences refer to a polypeptide sequence having at
least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more, or complete (100%) sequence identity (homology) to an
exemplary sequence of the invention.
[0212] Homology (sequence identity) may be determined using any of
the computer programs and parameters described herein. A nucleic
acid or polypeptide sequence of the invention can be stored,
recorded and manipulated on any medium which can be read and
accessed by a computer. As used herein, the words "recorded" and
"stored" refer to a process for storing information on a computer
medium. A skilled artisan can readily adopt any of the presently
known methods for recording information on a computer readable
medium to generate manufactures comprising one or more of the
nucleic acid sequences of the invention, one or more of the
polypeptide sequences of the invention. Another aspect of the
invention is a computer readable medium having recorded thereon at
least 2, 5, 10, 15, or 20 or more nucleic acid or polypeptide
sequences of the invention.
[0213] Another aspect of the invention is a computer readable
medium having recorded thereon one or more of the nucleic acid
sequences of the invention. Another aspect of the invention is a
computer readable medium having recorded thereon one or more of the
polypeptide sequences of the invention. Another aspect of the
invention is a computer readable medium having recorded thereon at
least 2, 5, 10, 15, or 20 or more of the nucleic acid or
polypeptide sequences as set forth above.
[0214] Computer readable media include magnetically readable media,
optically readable media, electronically readable media and
magnetic/optical media. For example, the computer readable media
may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital
Versatile Disk (DVD), Random Access Memory (RAM), or Read Only
Memory (ROM) as well as other types of other media known to those
skilled in the art.
[0215] Aspects of the invention include systems (e.g., internet
based systems), e.g., computer systems which store and manipulate
the sequence information described herein. One example of a
computer system 100 is illustrated in block diagram form in FIG. 1.
As used herein, "a computer system" refers to the hardware
components, software components and data storage components used to
analyze a nucleotide sequence of a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention. In one
aspect, the computer system 100 includes a processor for
processing, accessing and manipulating the sequence data. The
processor 105 can be any well-known type of central processing
unit, such as, for example, the Pentium III from Intel Corporation,
or similar processor from Sun, Motorola, Compaq, AMD or
International Business Machines.
[0216] In one aspect, the computer system 100 is a general purpose
system that comprises the processor 105 and one or more internal
data storage components 110 for storing data and one or more data
retrieving devices for retrieving the data stored on the data
storage components. A skilled artisan can readily appreciate that
any one of the currently available computer systems are
suitable.
[0217] In one particular aspect, the computer system 100 includes a
processor 105 connected to a bus which is connected to a main
memory 115 (in one aspect implemented as RAM) and one or more
internal data storage devices 110, such as a hard drive and/or
other computer readable media having data recorded thereon. In some
aspects, the computer system 100 further includes one or more data
retrieving device 118 for reading the data stored on the internal
data storage devices 110.
[0218] The data retrieving device 118 may represent, for example, a
floppy disk drive, a compact disk drive, a magnetic tape drive, or
a modem capable of connection to a remote data storage system
(e.g., via the internet) etc. In some aspects, the internal data
storage device 110 is a removable computer readable medium such as
a floppy disk, a compact disk, a magnetic tape, etc. containing
control logic and/or data recorded thereon. The computer system 100
may advantageously include or be programmed by appropriate software
for reading the control logic and/or the data from the data storage
component once inserted in the data retrieving device.
[0219] The computer system 100 includes a display 120 which is used
to display output to a computer user. It should also be noted that
the computer system 100 can be linked to other computer systems
125a-c in a network or wide area network to provide centralized
access to the computer system 100.
[0220] Software for accessing and processing the nucleotide
sequences of a nucleic acid sequence of the invention, or a
polypeptide sequence of the invention, (such as search tools,
compare tools and modeling tools etc.) may reside in main memory
115 during execution.
[0221] In some aspects, the computer system 100 may further
comprise a sequence comparison algorithm for comparing a nucleic
acid sequence of the invention, or a polypeptide sequence of the
invention, stored on a computer readable medium to a reference
nucleotide or polypeptide sequence(s) stored on a computer readable
medium. A "sequence comparison algorithm" refers to one or more
programs which are implemented (locally or remotely) on the
computer system 100 to compare a nucleotide sequence with other
nucleotide sequences and/or compounds stored within a data storage
means. For example, the sequence comparison algorithm may compare
the nucleotide sequences of a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention, stored on a
computer readable medium to reference sequences stored on a
computer readable medium to identify homologies or structural
motifs.
[0222] FIG. 2 is a flow diagram illustrating one aspect of a
process 200 for comparing a new nucleotide or protein sequence with
a database of sequences in order to determine the homology levels
between the new sequence and the sequences in the database. The
database of sequences can be a private database stored within the
computer system 100, or a public database such as GENBANK that is
available through the Internet.
[0223] The process 200 begins at a start state 201 and then moves
to a state 202 wherein the new sequence to be compared is stored to
a memory in a computer system 100. As discussed above, the memory
could be any type of memory, including RAM or an internal storage
device.
[0224] The process 200 then moves to a state 204 wherein a database
of sequences is opened for analysis and comparison. The process 200
then moves to a state 206 wherein the first sequence stored in the
database is read into a memory on the computer. A comparison is
then performed at a state 210 to determine if the first sequence is
the same as the second sequence. It is important to note that this
step is not limited to performing an exact comparison between the
new sequence and the first sequence in the database. Well-known
methods are known to those of skill in the art for comparing two
nucleotide or protein sequences, even if they are not identical.
For example, gaps can be introduced into one sequence in order to
raise the homology level between the two tested sequences. The
parameters that control whether gaps or other features are
introduced into a sequence during comparison are normally entered
by the user of the computer system.
[0225] Once a comparison of the two sequences has been performed at
the state 210, a determination is made at a decision state 210
whether the two sequences are the same. Of course, the term "same"
is not limited to sequences that are absolutely identical.
Sequences that are within the homology parameters entered by the
user will be marked as "same" in the process 200.
[0226] If a determination is made that the two sequences are the
same, the process 200 moves to a state 214 wherein the name of the
sequence from the database is displayed to the user. This state
notifies the user that the sequence with the displayed name
fulfills the homology constraints that were entered. Once the name
of the stored sequence is displayed to the user, the process 200
moves to a decision state 218 wherein a determination is made
whether more sequences exist in the database. If no more sequences
exist in the database, then the process 200 terminates at an end
state 220. However, if more sequences do exist in the database,
then the process 200 moves to a state 224 wherein a pointer is
moved to the next sequence in the database so that it can be
compared to the new sequence. In this manner, the new sequence is
aligned and compared with every sequence in the database.
[0227] It should be noted that if a determination had been made at
the decision state 212 that the sequences were not homologous, then
the process 200 would move immediately to the decision state 218 in
order to determine if any other sequences were available in the
database for comparison.
[0228] Accordingly, one aspect of the invention is a computer
system comprising a processor, a data storage device having stored
thereon a nucleic acid sequence of the invention, or a polypeptide
sequence of the invention, a data storage device having retrievably
stored thereon reference nucleotide sequences or polypeptide
sequences to be compared to a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention and a
sequence comparer for conducting the comparison. The sequence
comparer may indicate a homology level between the sequences
compared or identify structural motifs in the above described
nucleic acid code a nucleic acid sequence of the invention, or a
polypeptide sequence of the invention, or it may identify
structural motifs in sequences which are compared to these nucleic
acid codes and polypeptide codes. In some aspects, the data storage
device may have stored thereon the sequences of at least 2, 5, 10,
15, 20, 25, 30 or 40 or more of the nucleic acid sequences of the
invention, or the polypeptide sequences of the invention.
[0229] Another aspect of the invention is a method for determining
the level of homology between a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention and a
reference nucleotide sequence. The method including reading the
nucleic acid code or the polypeptide code and the reference
nucleotide or polypeptide sequence through the use of a computer
program which determines homology levels and determining homology
between the nucleic acid code or polypeptide code and the reference
nucleotide or polypeptide sequence with the computer program. The
computer program may be any of a number of computer programs for
determining homology levels, including those specifically
enumerated herein, (e.g., BLAST2N with the default parameters or
with any modified parameters). The method may be implemented using
the computer systems described above. The method may also be
performed by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 or
more of the above described nucleic acid sequences of the
invention, or the polypeptide sequences of the invention through
use of the computer program and determining homology between the
nucleic acid codes or polypeptide codes and reference nucleotide
sequences or polypeptide sequences.
[0230] FIG. 3 is a flow diagram illustrating one aspect of a
process 250 in a computer for determining whether two sequences are
homologous. The process 250 begins at a start state 252 and then
moves to a state 254 wherein a first sequence to be compared is
stored to a memory. The second sequence to be compared is then
stored to a memory at a state 256. The process 250 then moves to a
state 260 wherein the first character in the first sequence is read
and then to a state 262 wherein the first character of the second
sequence is read. It should be understood that if the sequence is a
nucleotide sequence, then the character would normally be either A,
T, C, G or U. If the sequence is a protein sequence, then it is in
one aspect in the single letter amino acid code so that the first
and sequence sequences can be easily compared.
[0231] A determination is then made at a decision state 264 whether
the two characters are the same. If they are the same, then the
process 250 moves to a state 268 wherein the next characters in the
first and second sequences are read. A determination is then made
whether the next characters are the same. If they are, then the
process 250 continues this loop until two characters are not the
same. If a determination is made that the next two characters are
not the same, the process 250 moves to a decision state 274 to
determine whether there are any more characters either sequence to
read.
[0232] If there are not any more characters to read, then the
process 250 moves to a state 276 wherein the level of homology
between the first and second sequences is displayed to the user.
The level of homology is determined by calculating the proportion
of characters between the sequences that were the same out of the
total number of sequences in the first sequence. Thus, if every
character in a first 100 nucleotide sequence aligned with a every
character in a second sequence, the homology level would be
100%.
[0233] Alternatively, the computer program may be a computer
program which compares the nucleotide sequences of a nucleic acid
sequence as set forth in the invention, to one or more reference
nucleotide sequences in order to determine whether the nucleic acid
code of the invention, differs from a reference nucleic acid
sequence at one or more positions. Optionally such a program
records the length and identity of inserted, deleted or substituted
nucleotides with respect to the sequence of either the reference
polynucleotide or a nucleic acid sequence of the invention. In one
aspect, the computer program may be a program which determines
whether a nucleic acid sequence of the invention, contains a single
nucleotide polymorphism (SNP) with respect to a reference
nucleotide sequence.
[0234] Accordingly, another aspect of the invention is a method for
determining whether a nucleic acid sequence of the invention,
differs at one or more nucleotides from a reference nucleotide
sequence comprising the steps of reading the nucleic acid code and
the reference nucleotide sequence through use of a computer program
which identifies differences between nucleic acid sequences and
identifying differences between the nucleic acid code and the
reference nucleotide sequence with the computer program. In some
aspects, the computer program is a program which identifies single
nucleotide polymorphisms. The method may be implemented by the
computer systems described above and the method illustrated in FIG.
3. The method may also be performed by reading at least 2, 5, 10,
15, 20, 25, 30, or 40 or more of the nucleic acid sequences of the
invention and the reference nucleotide sequences through the use of
the computer program and identifying differences between the
nucleic acid codes and the reference nucleotide sequences with the
computer program.
[0235] In other aspects the computer based system may further
comprise an identifier for identifying features within a nucleic
acid sequence of the invention or a polypeptide sequence of the
invention. An "identifier" refers to one or more programs which
identifies certain features within a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention. In one
aspect, the identifier may comprise a program which identifies an
open reading frame in a nucleic acid sequence of the invention.
[0236] FIG. 4 is a flow diagram illustrating one aspect of an
identifier process 300 for detecting the presence of a feature in a
sequence. The process 300 begins at a start state 302 and then
moves to a state 304 wherein a first sequence that is to be checked
for features is stored to a memory 115 in the computer system 100.
The process 300 then moves to a state 306 wherein a database of
sequence features is opened. Such a database would include a list
of each feature's attributes along with the name of the feature.
For example, a feature name could be "Initiation Codon" and the
attribute would be "ATG". Another example would be the feature name
"TAATAA Box" and the feature attribute would be "TAATAA". An
example of such a database is produced by the University of
Wisconsin Genetics Computer Group. Alternatively, the features may
be structural polypeptide motifs such as alpha helices, beta
sheets, or functional polypeptide motifs such as enzymatic active
sites, helix-turn-helix motifs or other motifs known to those
skilled in the art.
[0237] Once the database of features is opened at the state 306,
the process 300 moves to a state 308 wherein the first feature is
read from the database. A comparison of the attribute of the first
feature with the first sequence is then made at a state 310. A
determination is then made at a decision state 316 whether the
attribute of the feature was found in the first sequence. If the
attribute was found, then the process 300 moves to a state 318
wherein the name of the found feature is displayed to the user.
[0238] The process 300 then moves to a decision state 320 wherein a
determination is made whether move features exist in the database.
If no more features do exist, then the process 300 terminates at an
end state 324. However, if more features do exist in the database,
then the process 300 reads the next sequence feature at a state 326
and loops back to the state 310 wherein the attribute of the next
feature is compared against the first sequence. It should be noted,
that if the feature attribute is not found in the first sequence at
the decision state 316, the process 300 moves directly to the
decision state 320 in order to determine if any more features exist
in the database.
[0239] Accordingly, another aspect of the invention is a method of
identifying a feature within a nucleic acid sequence of the
invention, or a polypeptide sequence of the invention, comprising
reading the nucleic acid code(s) or polypeptide code(s) through the
use of a computer program which identifies features therein and
identifying features within the nucleic acid code(s) with the
computer program. In one aspect, computer program comprises a
computer program which identifies open reading frames. The method
may be performed by reading a single sequence or at least 2, 5, 10,
15, 20, 25, 30, or 40 or more of the nucleic acid sequences of the
invention, or the polypeptide sequences of the invention, through
the use of the computer program and identifying features within the
nucleic acid codes or polypeptide codes with the computer
program.
[0240] A nucleic acid sequence of the invention, or a polypeptide
sequence of the invention, may be stored and manipulated in a
variety of data processor programs in a variety of formats. For
example, a nucleic acid sequence of the invention, or a polypeptide
sequence of the invention, may be stored as text in a word
processing file, such as Microsoft WORD.TM. or WORDPERFECT.TM. or
as an ASCII file in a variety of database programs familiar to
those of skill in the art, such as DB2.TM., SYBASE.TM., or
ORACLE.TM.. In addition, many computer programs and databases may
be used as sequence comparison algorithms, identifiers, or sources
of reference nucleotide sequences or polypeptide sequences to be
compared to a nucleic acid sequence of the invention, or a
polypeptide sequence of the invention. The following list is
intended not to limit the invention but to provide guidance to
programs and databases which are useful with the nucleic acid
sequences of the invention, or the polypeptide sequences of the
invention.
[0241] The programs and databases which may be used include, but
are not limited to: MACPATTERN.TM. (EMBL), DISCOVERYBASE.TM.
(Molecular Applications Group), GENEMINE.TM. (Molecular
Applications Group), LOOK.TM. (Molecular Applications Group),
MACLOOK.TM. (Molecular Applications Group), BLAST and BLAST2
(NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403,
1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:
2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245,
1990), CATALYST.TM. (Molecular Simulations Inc.),
Catalyst/SHAPE.TM. (Molecular Simulations Inc.),
Cerius.sup.2.DBAccess.TM. (Molecular Simulations Inc.), HYPOGEN.TM.
(Molecular Simulations Inc.), INSIGHT II.TM. (Molecular Simulations
Inc.), DISCOVER.TM. (Molecular Simulations Inc.), CHARMm.TM.
(Molecular Simulations Inc.), FELIX.TM. (Molecular Simulations
Inc.), DELPHI.TM. (Molecular Simulations Inc.), QuanteMM.TM.,
(Molecular Simulations Inc.), Homology (Molecular Simulations
Inc.), MODELER.TM. (Molecular Simulations Inc.), ISIS.TM.
(Molecular Simulations Inc.), Quanta/Protein Design (Molecular
Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab
Diversity Explorer (Molecular Simulations Inc.), Gene Explorer
(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.),
the MDL Available Chemicals Directory database, the MDL Drug Data
Report data base, the Comprehensive Medicinal Chemistry database,
Derwents's World Drug Index database, the BioByteMasterFile
database, the Genbank database and the Genseqn database. Many other
programs and data bases would be apparent to one of skill in the
art given the present disclosure.
[0242] Motifs which may be detected using the above programs
include sequences encoding leucine zippers, helix-turn-helix
motifs, glycosylation sites, ubiquitination sites, alpha helices
and beta sheets, signal sequences encoding signal peptides which
direct the secretion of the encoded proteins, sequences implicated
in transcription regulation such as homeoboxes, acidic stretches,
enzymatic active sites, substrate binding sites and enzymatic
cleavage sites.
Hybridization of Nucleic Acids
[0243] The invention provides isolated or recombinant nucleic acids
that hybridize under stringent conditions to an exemplary sequence
of the invention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,
SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID
NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ
ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,
SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID
NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ
ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81,
SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID
NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ
ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID
NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117,
SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID
NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135,
SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID
NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,
SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID
NO:163 or SEQ ID NO:165 (see also Tables 1, 2, and 3, Examples 1
and 4, below, and Sequence Listing)). The stringent conditions can
be highly stringent conditions, medium stringent conditions and/or
low stringent conditions, including the high and reduced stringency
conditions described herein. In one aspect, it is the stringency of
the wash conditions that set forth the conditions which determine
whether a nucleic acid is within the scope of the invention, as
discussed below.
[0244] "Hybridization" refers to the process by which a nucleic
acid strand joins with a complementary strand through base pairing.
Hybridization reactions can be sensitive and selective so that a
particular sequence of interest can be identified even in samples
in which it is present at low concentrations. Suitably stringent
conditions can be defined by, for example, the concentrations of
salt or formamide in the prehybridization and hybridization
solutions, or by the hybridization temperature and are well known
in the art. In alternative aspects, stringency can be increased by
reducing the concentration of salt, increasing the concentration of
formamide, or raising the hybridization temperature. In alternative
aspects, nucleic acids of the invention are defined by their
ability to hybridize under various stringency conditions (e.g.,
high, medium, and low), as set forth herein.
[0245] In one aspect, hybridization under high stringency
conditions comprise about 50% formamide at about 37.degree. C. to
42.degree. C. In one aspect, hybridization conditions comprise
reduced stringency conditions in about 35% to 25% formamide at
about 30.degree. C. to 35.degree. C. In one aspect, hybridization
conditions comprise high stringency conditions, e.g., at 42.degree.
C. in 50% formamide, 5.times.SSPE, 0.3% SDS and 200 n/ml sheared
and denatured salmon sperm DNA. In one aspect, hybridization
conditions comprise these reduced stringency conditions, but in 35%
formamide at a reduced temperature of 35.degree. C. The temperature
range corresponding to a particular level of stringency can be
further narrowed by calculating the purine to pyrimidine ratio of
the nucleic acid of interest and adjusting the temperature
accordingly. Variations on the above ranges and conditions are well
known in the art.
[0246] In alternative aspects, nucleic acids of the invention as
defined by their ability to hybridize under stringent conditions
can be between about five residues and the full length of nucleic
acid of the invention; e.g., they can be at least 5, 10, 15, 20,
25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, or more, residues in length. Nucleic acids shorter than
full length are also included. These nucleic acids can be useful
as, e.g., hybridization probes, labeling probes, PCR
oligonucleotide probes, siRNA or miRNA (single or double stranded),
antisense or sequences encoding antibody binding peptides
(epitopes), motifs, active sites and the like.
[0247] In one aspect, nucleic acids of the invention are defined by
their ability to hybridize under high stringency comprises
conditions of about 50% formamide at about 37.degree. C. to
42.degree. C. In one aspect, nucleic acids of the invention are
defined by their ability to hybridize under reduced stringency
comprising conditions in about 35% to 25% formamide at about
30.degree. C. to 35.degree. C.
[0248] Alternatively, nucleic acids of the invention are defined by
their ability to hybridize under high stringency comprising
conditions at 42.degree. C. in 50% formamide, 5.times.SSPE, 0.3%
SDS, and a repetitive sequence blocking nucleic acid, such as cot-1
or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon
sperm DNA). In one aspect, nucleic acids of the invention are
defined by their ability to hybridize under reduced stringency
conditions comprising 35% or 40% formamide at a reduced temperature
of 35.degree. C. or 42.degree. C.
[0249] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(e.g., GC v. AT content) and nucleic acid type (e.g., RNA v. DNA)
of the hybridizing regions of the nucleic acids can be considered
in selecting hybridization conditions. An additional consideration
is whether one of the nucleic acids is immobilized, for example, on
a filter.
[0250] Hybridization may be carried out under conditions of low
stringency, moderate stringency or high stringency. As an example
of nucleic acid hybridization, a polymer membrane containing
immobilized denatured nucleic acids is first prehybridized for 30
minutes at 45.degree. C. in a solution consisting of 0.9 M NaCl, 50
mM NaH.sub.2PO.sub.4, pH 7.0, 5.0 mM Na.sub.2EDTA, 0.5% SDS,
10.times.Denhardt's and 0.5 mg/ml polyriboadenylic acid.
Approximately 2.times.10.sup.7 cpm (specific activity
4-9.times.10.sup.8 cpm/ug) of .sup.32P end-labeled oligonucleotide
probe are then added to the solution. After 12-16 hours of
incubation, the membrane is washed for 30 minutes at room
temperature in 1.times.SET (150 mM NaCl, 20 mM Tris hydrochloride,
pH 7.8, 1 mM Na.sub.2EDTA) containing 0.5% SDS, followed by a 30
minute wash in fresh 1.times.SET at T.sub.m-10.degree. C. for the
oligonucleotide probe. The membrane is then exposed to
auto-radiographic film for detection of hybridization signals. All
of the foregoing hybridizations would be considered to be under
conditions of high stringency.
[0251] Following hybridization, a filter can be washed to remove
any non-specifically bound detectable probe. The stringency used to
wash the filters can also be varied depending on the nature of the
nucleic acids being hybridized, the length of the nucleic acids
being hybridized, the degree of complementarity, the nucleotide
sequence composition (e.g., GC v. AT content) and the nucleic acid
type (e.g., RNA v. DNA). Examples of progressively higher
stringency condition washes are as follows: 2.times.SSC, 0.1% SDS
at room temperature for 15 minutes (low stringency); 0.1.times.SSC,
0.5% SDS at room temperature for 30 minutes to 1 hour (moderate
stringency); 0.1.times.SSC, 0.5% SDS for 15 to 30 minutes at
between the hybridization temperature and 68.degree. C. (high
stringency); and 0.15M NaCl for 15 minutes at 72.degree. C. (very
high stringency). A final low stringency wash can be conducted in
0.1.times.SSC at room temperature. The examples above are merely
illustrative of one set of conditions that can be used to wash
filters. One of skill in the art would know that there are numerous
recipes for different stringency washes. Some other examples are
given below.
[0252] In one aspect, hybridization conditions comprise a wash step
comprising a wash for 30 minutes at room temperature in a solution
comprising 1.times. 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8,
1 mM Na.sub.2EDTA, 0.5% SDS, followed by a 30 minute wash in fresh
solution.
[0253] Nucleic acids which have hybridized to the probe are
identified by autoradiography or other conventional techniques.
[0254] The above procedures may be modified to identify nucleic
acids having decreasing levels of sequence identity (homology) to
the probe sequence. For example, to obtain nucleic acids of
decreasing sequence identity (homology) to the detectable probe,
less stringent conditions may be used. For example, the
hybridization temperature may be decreased in increments of
5.degree. C. from 68.degree. C. to 42.degree. C. in a hybridization
buffer having a Na+ concentration of approximately 1M. Following
hybridization, the filter may be washed with 2.times.SSC, 0.5% SDS
at the temperature of hybridization. These conditions are
considered to be "moderate" conditions above 50.degree. C. and
"low" conditions below 50.degree. C. A specific example of
"moderate" hybridization conditions is when the above hybridization
is conducted at 55.degree. C. A specific example of "low
stringency" hybridization conditions is when the above
hybridization is conducted at 45.degree. C.
[0255] Alternatively, the hybridization may be carried out in
buffers, such as 6.times.SSC, containing formamide at a temperature
of 42.degree. C. In this case, the concentration of formamide in
the hybridization buffer may be reduced in 5% increments from 50%
to 0% to identify clones having decreasing levels of homology to
the probe. Following hybridization, the filter may be washed with
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate" conditions above 25% formamide and
"low" conditions below 25% formamide. A specific example of
"moderate" hybridization conditions is when the above hybridization
is conducted at 30% formamide. A specific example of "low
stringency" hybridization conditions is when the above
hybridization is conducted at 10% formamide.
[0256] However, the selection of a hybridization format may not be
critical--it is the stringency of the wash conditions that set
forth the conditions which determine whether a nucleic acid is
within the scope of the invention. Wash conditions used to identify
nucleic acids within the scope of the invention include, e.g.: a
salt concentration of about 0.02 molar at pH 7 and a temperature of
at least about 50.degree. C. or about 55.degree. C. to about
60.degree. C.; or, a salt concentration of about 0.15 M NaCl at
72.degree. C. for about 15 minutes; or, a salt concentration of
about 0.2.times.SSC at a temperature of at least about 50.degree.
C. or about 55.degree. C. to about 60.degree. C. for about 15 to
about 20 minutes; or, the hybridization complex is washed twice
with a solution with a salt concentration of about 2.times.SSC
containing 0.1% SDS at room temperature for 15 minutes and then
washed twice by 0.1.times.SSC containing 0.1% SDS at 68.degree. C.
for 15 minutes; or, equivalent conditions. See Sambrook, Tijssen
and Ausubel for a description of SSC buffer and equivalent
conditions.
[0257] These methods may be used to isolate or identify nucleic
acids of the invention. For example, the preceding methods may be
used to isolate or identify nucleic acids having a sequence with at
least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more sequence identity (homology) to a nucleic acid sequence
selected from the group consisting of one of the sequences of the
invention, or fragments comprising at least about 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutive
bases thereof and the sequences complementary thereto. Sequence
identity (homology) may be measured using the alignment algorithm.
For example, the homologous polynucleotides may have a coding
sequence which is a naturally occurring allelic variant of one of
the coding sequences described herein. Such allelic variants may
have a substitution, deletion or addition of one or more
nucleotides when compared to the nucleic acids of the invention.
Additionally, the above procedures may be used to isolate nucleic
acids which encode polypeptides having at least about 99%, 95%, at
least 90%, at least 85%, at least 80%, at least 75%, at least 70%,
at least 65%, at least 60%, at least 55%, or at least 50% sequence
identity (homology) to a polypeptide of the invention, or fragments
comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or
150 consecutive amino acids thereof as determined using a sequence
alignment algorithm (e.g., such as the FASTA version 3.0t78
algorithm with the default parameters).
Oligonucleotides Probes and Methods for Using them
[0258] The invention also provides nucleic acid probes that can be
used, e.g., for identifying, amplifying, or isolating nucleic acids
encoding a polypeptide having a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity or fragments thereof or for identifying cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme genes. In one aspect, the probe comprises at least about 10
consecutive bases of a nucleic acid of the invention.
Alternatively, a probe of the invention can be at least about 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150
or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases
of a sequence as set forth in a nucleic acid of the invention. The
probes identify a nucleic acid by binding and/or hybridization. The
probes can be used in arrays of the invention, see discussion
below, including, e.g., capillary arrays. The probes of the
invention can also be used to isolate other nucleic acids or
polypeptides.
[0259] The isolated or recombinant nucleic acids of the invention,
the sequences complementary thereto, or a fragment comprising at
least about 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300,
400, or 500 consecutive bases of one of the sequences of the
invention, or the sequences complementary thereto may also be used
as probes to determine whether a biological sample, such as a soil
sample, contains an organism having a nucleic acid sequence of the
invention or an organism from which the nucleic acid was obtained.
In such procedures, a biological sample potentially harboring the
organism from which the nucleic acid was isolated is obtained and
nucleic acids are obtained from the sample. The nucleic acids are
contacted with the probe under conditions which permit the probe to
specifically hybridize to any complementary sequences from which
are present therein.
[0260] Where necessary, conditions which permit the probe to
specifically hybridize to complementary sequences may be determined
by placing the probe in contact with complementary sequences from
samples known to contain the complementary sequence as well as
control sequences which do not contain the complementary sequence.
Hybridization conditions, such as the salt concentration of the
hybridization buffer, the formamide concentration of the
hybridization buffer, or the hybridization temperature, may be
varied to identify conditions which allow the probe to hybridize
specifically to complementary nucleic acids.
[0261] If the sample contains the organism from which the nucleic
acid was isolated, specific hybridization of the probe is then
detected. Hybridization may be detected by labeling the probe with
a detectable agent such as a radioactive isotope, a fluorescent dye
or an enzyme capable of catalyzing the formation of a detectable
product.
[0262] Many methods for using the labeled probes to detect the
presence of complementary nucleic acids in a sample are familiar to
those skilled in the art. These include Southern Blots, Northern
Blots, colony hybridization procedures and dot blots. Protocols for
each of these procedures are provided in Ausubel et al. Current
Protocols in Molecular Biology, John Wiley 503 Sons, Inc. (1997)
and Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd
Ed., Cold Spring Harbor Laboratory Press (1989.
[0263] Alternatively, more than one probe (at least one of which is
capable of specifically hybridizing to any complementary sequences
which are present in the nucleic acid sample), may be used in an
amplification reaction to determine whether the sample contains an
organism containing a nucleic acid sequence of the invention (e.g.,
an organism from which the nucleic acid was isolated). In one
aspect, the probes comprise oligonucleotides. In one aspect, the
amplification reaction may comprise a PCR reaction. PCR protocols
are described in Ausubel and Sambrook, supra. Alternatively, the
amplification may comprise a ligase chain reaction, 3SR, or strand
displacement reaction. (See Barany, F., "The Ligase Chain Reaction
in a PCR World", PCR Methods and Applications 1:5-16, 1991; E. Fahy
et al., "Self-sustained Sequence Replication (3SR): An Isothermal
Transcription-based Amplification System Alternative to PCR", PCR
Methods and Applications 1:25-33, 1991; and Walker G. T. et al.,
"Strand Displacement Amplification--an Isothermal in vitro DNA
Amplification Technique", Nucleic Acid Research 20:1691-1696,
1992). In such procedures, the nucleic acids in the sample are
contacted with the probes, the amplification reaction is performed
and any resulting amplification product is detected. The
amplification product may be detected by performing gel
electrophoresis on the reaction products and staining the gel with
an intercalator such as ethidium bromide. Alternatively, one or
more of the probes may be labeled with a radioactive isotope and
the presence of a radioactive amplification product may be detected
by autoradiography after gel electrophoresis.
[0264] Probes derived from sequences near the ends of the sequences
of the invention, may also be used in chromosome walking procedures
to identify clones containing genomic sequences located adjacent to
the sequences of the invention. Such methods allow the isolation of
genes which encode additional proteins from the host organism.
[0265] In one aspect, the isolated or recombinant nucleic acids of
the invention, the sequences complementary thereto, or a fragment
comprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150,
200, 300, 400, or 500 or more consecutive bases of one of the
sequences of the invention, or the sequences complementary thereto
are used as probes to identify and isolate related nucleic acids.
In some aspects, the related nucleic acids may be cDNAs or genomic
DNAs from organisms other than the one from which the nucleic acid
was isolated. For example, the other organisms may be related
organisms. In such procedures, a nucleic acid sample is contacted
with the probe under conditions which permit the probe to
specifically hybridize to related sequences. Hybridization of the
probe to nucleic acids from the related organism is then detected
using any of the methods described above.
[0266] By varying the stringency of the hybridization conditions
used to identify nucleic acids, such as cDNAs or genomic DNAs,
which hybridize to the detectable probe, nucleic acids having
different levels of homology to the probe can be identified and
isolated. Stringency may be varied by conducting the hybridization
at varying temperatures below the melting temperatures of the
probes. The melting temperature, T.sub.m, is the temperature (under
defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly complementary probe. Very stringent
conditions are selected to be equal to or about 5.degree. C. lower
than the T.sub.m for a particular probe. The melting temperature of
the probe may be calculated using the following formulas: [0267]
For probes between 14 and 70 nucleotides in length the melting
temperature (T.sub.m) is calculated using the formula:
T.sub.m=81.5+16.6(log [Na+])+0.41(fraction G+C)-(600/N) where N is
the length of the probe. [0268] If the hybridization is carried out
in a solution containing formamide, the melting temperature may be
calculated using the equation: T.sub.m=81.5+16.6(log
[Na+])+0.41(fraction G+C)-(0.63% formamide)-(600/N) where N is the
length of the probe.
[0269] Prehybridization may be carried out in 6.times.SSC,
5.times.Denhardt's reagent, 0.5% SDS, 100 .mu.g denatured
fragmented salmon sperm DNA or 6.times.SSC, 5.times.Denhardt's
reagent, 0.5% SDS, 100 .mu.g denatured fragmented salmon sperm DNA,
50% formamide. The formulas for SSC and Denhardt's solutions are
listed in Sambrook et al., supra.
[0270] In one aspect, hybridization is conducted by adding the
detectable probe to the prehybridization solutions listed above.
Where the probe comprises double stranded DNA, it is denatured
before addition to the hybridization solution. In one aspect, the
filter is contacted with the hybridization solution for a
sufficient period of time to allow the probe to hybridize to cDNAs
or genomic DNAs containing sequences complementary thereto or
homologous thereto. For probes over 200 nucleotides in length, the
hybridization may be carried out at 15-25.degree. C. below the
T.sub.m For shorter probes, such as oligonucleotide probes, the
hybridization may be conducted at 5-10.degree. C. below the
T.sub.m. In one aspect, for hybridizations in 6.times.SSC, the
hybridization is conducted at approximately 68.degree. C. Usually,
for hybridizations in 50% formamide containing solutions, the
hybridization is conducted at approximately 42.degree. C.
Inhibiting Expression of Cellulase Enzymes
[0271] The invention provides nucleic acids complementary to (e.g.,
antisense sequences to) the nucleic acids of the invention, e.g.,
cellulase enzyme-encoding nucleic acids, e.g., nucleic acids
comprising antisense, siRNA, miRNA, ribozymes. Nucleic acids of the
invention comprising antisense sequences can be capable of
inhibiting the transport, splicing or transcription of cellulase
enzyme-encoding genes. The inhibition can be effected through the
targeting of genomic DNA or messenger RNA. The transcription or
function of targeted nucleic acid can be inhibited, for example, by
hybridization and/or cleavage. One exemplary set of inhibitors
provided by the present invention includes oligonucleotides which
are able to either bind cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme gene or
message, in either case preventing or inhibiting the production or
function of a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme. The association can be
through sequence specific hybridization. Another useful class of
inhibitors includes oligonucleotides which cause inactivation or
cleavage of cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme message. The
oligonucleotide can have enzyme activity which causes such
cleavage, such as ribozymes. The oligonucleotide can be chemically
modified or conjugated to an enzyme or composition capable of
cleaving the complementary nucleic acid. A pool of many different
such oligonucleotides can be screened for those with the desired
activity. Thus, the invention provides various compositions for the
inhibition of cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme expression on a nucleic
acid and/or protein level, e.g., antisense, siRNA, miRNA and
ribozymes comprising cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
sequences of the invention and the anti-cellulase, e.g.,
anti-endoglucanase, anti-cellobiohydrolase and/or
anti-beta-glucosidase antibodies of the invention.
[0272] Inhibition of cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
expression can have a variety of industrial applications. For
example, inhibition of cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
expression can slow or prevent spoilage. In one aspect, use of
compositions of the invention that inhibit the expression and/or
activity of cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzymes, e.g., antibodies,
antisense oligonucleotides, ribozymes, siRNA and miRNA are used to
slow or prevent spoilage. Thus, in one aspect, the invention
provides methods and compositions comprising application onto a
plant or plant product (e.g., a cereal, a grain, a fruit, seed,
root, leaf, etc.) antibodies, antisense oligonucleotides,
ribozymes, siRNA and miRNA of the invention to slow or prevent
spoilage. These compositions also can be expressed by the plant
(e.g., a transgenic plant) or another organism (e.g., a bacterium
or other microorganism transformed with a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme gene of the invention).
[0273] The compositions of the invention for the inhibition of
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme expression (e.g., antisense, iRNA,
ribozymes, antibodies) can be used as pharmaceutical compositions,
e.g., as anti-pathogen agents or in other therapies, e.g., as
anti-microbials for, e.g., Salmonella.
[0274] Antisense Oligonucleotides
[0275] The invention provides antisense oligonucleotides capable of
binding cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme message which, in one
aspect, can inhibit cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity by targeting mRNA. Strategies for designing antisense
oligonucleotides are well described in the scientific and patent
literature, and the skilled artisan can design such cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme oligonucleotides using the novel reagents
of the invention. For example, gene walking/RNA mapping protocols
to screen for effective antisense oligonucleotides are well known
in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,
describing an RNA mapping assay, which is based on standard
molecular techniques to provide an easy and reliable method for
potent antisense sequence selection. See also Smith (2000) Eur. J.
Pharm. Sci. 11:191-198.
[0276] Naturally occurring nucleic acids are used as antisense
oligonucleotides. The antisense oligonucleotides can be of any
length; for example, in alternative aspects, the antisense
oligonucleotides are between about 5 to 100, about 10 to 80, about
15 to 60, about 18 to 40. The optimal length can be determined by
routine screening. The antisense oligonucleotides can be present at
any concentration. The optimal concentration can be determined by
routine screening. A wide variety of synthetic, non-naturally
occurring nucleotide and nucleic acid analogues are known which can
address this potential problem. For example, peptide nucleic acids
(PNAs) containing non-ionic backbones, such as N-(2-aminoethyl)
glycine units can be used. Antisense oligonucleotides having
phosphorothioate linkages can also be used, as described in WO
97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol
144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press,
Totowa, N.J., 1996). Antisense oligonucleotides having synthetic
DNA backbone analogues provided by the invention can also include
phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl
phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino),
3'-N-carbamate, and morpholino carbamate nucleic acids, as
described above.
[0277] Combinatorial chemistry methodology can be used to create
vast numbers of oligonucleotides that can be rapidly screened for
specific oligonucleotides that have appropriate binding affinities
and specificities toward any target, such as the sense and
antisense cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme sequences of the invention
(see, e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).
[0278] Inhibitory Ribozymes
[0279] The invention provides ribozymes capable of binding
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme message. These ribozymes can inhibit
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity by, e.g., targeting mRNA.
Strategies for designing ribozymes and selecting the cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme-specific antisense sequence for targeting
are well described in the scientific and patent literature, and the
skilled artisan can design such ribozymes using the novel reagents
of the invention. Ribozymes act by binding to a target RNA through
the target RNA binding portion of a ribozyme which is held in close
proximity to an enzymatic portion of the RNA that cleaves the
target RNA. Thus, the ribozyme recognizes and binds a target RNA
through complementary base-pairing, and once bound to the correct
site, acts enzymatically to cleave and inactivate the target RNA.
Cleavage of a target RNA in such a manner will destroy its ability
to direct synthesis of an encoded protein if the cleavage occurs in
the coding sequence. After a ribozyme has bound and cleaved its RNA
target, it can be released from that RNA to bind and cleave new
targets repeatedly.
[0280] In some circumstances, the enzymatic nature of a ribozyme
can be advantageous over other technologies, such as antisense
technology (where a nucleic acid molecule simply binds to a nucleic
acid target to block its transcription, translation or association
with another molecule) as the effective concentration of ribozyme
necessary to effect a therapeutic treatment can be lower than that
of an antisense oligonucleotide. This potential advantage reflects
the ability of the ribozyme to act enzymatically. Thus, a single
ribozyme molecule is able to cleave many molecules of target RNA.
In one aspect, a ribozyme is a highly specific inhibitor, with the
specificity of inhibition depending not only on the base pairing
mechanism of binding, but also on the mechanism by which the
molecule inhibits the expression of the RNA to which it binds. That
is, the inhibition is caused by cleavage of the RNA target and so
specificity is defined as the ratio of the rate of cleavage of the
targeted RNA over the rate of cleavage of non-targeted RNA. This
cleavage mechanism is dependent upon factors additional to those
involved in base pairing. Thus, the specificity of action of a
ribozyme can be greater than that of antisense oligonucleotide
binding the same RNA site.
[0281] The ribozyme of the invention, e.g., an enzymatic ribozyme
RNA molecule, can be formed in a hammerhead motif, a hairpin motif,
as a hepatitis delta virus motif, a group I intron motif and/or an
RNaseP-like RNA in association with an RNA guide sequence. Examples
of hammerhead motifs are described by, e.g., Rossi (1992) Aids
Research and Human Retroviruses 8:183; hairpin motifs by Hampel
(1989) Biochemistry 28:4929, and Hampel (1990) Nuc. Acids Res.
18:299; the hepatitis delta virus motif by Perrotta (1992)
Biochemistry 31:16; the RNaseP motif by Guerrier-Takada (1983) Cell
35:849; and the group I intron by Cech U.S. Pat. No. 4,987,071. The
recitation of these specific motifs is not intended to be limiting.
Those skilled in the art will recognize that a ribozyme of the
invention, e.g., an enzymatic RNA molecule of this invention, can
have a specific substrate binding site complementary to one or more
of the target gene RNA regions. A ribozyme of the invention can
have a nucleotide sequence within or surrounding that substrate
binding site which imparts an RNA cleaving activity to the
molecule.
[0282] RNA Interference (RNAi)
[0283] In one aspect, the invention provides an RNA inhibitory
molecule, a so-called "RNAi" molecule, comprising a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme sequence of the invention. The RNAi
molecule can comprise a double-stranded RNA (dsRNA) molecule, e.g.,
siRNA and/or miRNA. The RNAi molecule, e.g., siRNA and/or miRNA,
can inhibit expression of a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme gene.
In one aspect, the RNAi molecule, e.g., siRNA and/or miRNA, is
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex
nucleotides in length. While the invention is not limited by any
particular mechanism of action, the RNAi can enter a cell and cause
the degradation of a single-stranded RNA (ssRNA) of similar or
identical sequences, including endogenous mRNAs. When a cell is
exposed to double-stranded RNA (dsRNA), mRNA from the homologous
gene is selectively degraded by a process called RNA interference
(RNAi). A possible basic mechanism behind RNAi is the breaking of a
double-stranded RNA (dsRNA) matching a specific gene sequence into
short pieces called short interfering RNA, which trigger the
degradation of mRNA that matches its sequence. In one aspect, the
RNAi's of the invention are used in gene-silencing therapeutics,
see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one
aspect, the invention provides methods to selectively degrade RNA
using the RNAi's molecules, e.g., siRNA and/or miRNA, of the
invention. The process may be practiced in vitro, ex vivo or in
vivo. In one aspect, the RNAi molecules of the invention can be
used to generate a loss-of-function mutation in a cell, an organ or
an animal. Methods for making and using RNAi molecules, e.g., siRNA
and/or miRNA, for selectively degrade RNA are well known in the
art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109;
6,489,127.
Modification of Nucleic Acids--Making Variant Enzymes of the
Invention
[0284] The invention provides methods of generating variants of the
nucleic acids of the invention, e.g., those encoding a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme. These methods can be repeated or used in
various combinations to generate cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes having
an altered or different activity or an altered or different
stability from that of a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme encoded
by the template nucleic acid. These methods also can be repeated or
used in various combinations, e.g., to generate variations in
gene/message expression, message translation or message stability.
In another aspect, the genetic composition of a cell is altered by,
e.g., modification of a homologous gene ex vivo, followed by its
reinsertion into the cell.
[0285] For example, in one aspect, the invention provides isolated
or recombinant nucleic acids having a sequence comprising at least
one nucleotide base residue modification of SEQ ID NO:163, wherein
the modification comprises one or more of the following changes: a
nucleotide at any one of positions 265 to 267 is modified to CGT,
CGC, CGA, CGG, AGA or AGG; a nucleotide at any one of positions 307
to 309 is modified to GGT, GGC, GGA or GGG; a nucleotide at any one
of positions 328 to 330 is modified to GGT, GGC, GGA or GGG; a
nucleotide at any one of positions 340 to 342 is modified to TTA,
TTG, CTT, CTC, CTA or CTG; a nucleotide at any one of positions 469
to 471 is modified to TCT, TCC, TCA, TCG, AGT or AGC; a nucleotide
at any one of positions 1441 to 1443 is modified to TTT or TTC; a
nucleotide at any one of positions 1648 to 1650 is modified to AAT
or AAC; or, a nucleotide at any one of positions 1768 to 1770 is
modified to CGT, CGC, CGA, CGG, AGA or AGG. In another aspect, the
invention provides isolated or recombinant polypeptides having a
sequence comprising at least one amino acid residue modification of
SEQ ID NO:164, wherein the modification comprises one or more of
the following changes: a methionine at amino acid position 89 is
modified to arginine; a phenylalanine at amino acid position 103 is
modified to glycine; a proline at amino acid position 110 is
modified to glycine; a tyrosine at amino acid position 114 is
modified to leucine; an alanine at amino acid position 157 is
modified to serine; a tryptophan at amino acid position 481 is
modified to phenylalanine; a proline at amino acid position 550 is
modified to asparagine; or a glycine at amino acid position 590 is
modified to arginine.
[0286] In another aspect, the invention provides isolated or
recombinant nucleic acids having a sequence comprising a nucleotide
residue sequence modification of an exemplary sequence of the
invention (e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, etc.) wherein the modification
comprises one or more of the following changes: a nucleotide at the
equivalent of any one of positions 265 to 267 of SEQ ID NO:163 are
changed to CGT, CGC, CGA, CGG, AGA or AGG; a nucleotide at the
equivalent of any one of positions 307 to 309 of SEQ ID NO:163 are
changed to GGT, GGC, GGA or GGG; a nucleotide at the equivalent of
any one of positions 328 to 330 of SEQ ID NO:163 are changed to
GGT, GGC, GGA or GGG; a nucleotide at the equivalent of any one of
positions 340 to 342 of SEQ ID NO:163 are changed to TTA, TTG, CTT,
CTC, CTA or CTG; a nucleotide at the equivalent of any one of
positions 469 to 471 of SEQ ID NO:163 are changed to TCT, TCC, TCA,
TCG, AGT or AGC; a nucleotide at the equivalent of positions 1441
to 1443 of SEQ ID NO:163 are changed to TTT or TTC; a nucleotide at
the equivalent of any one of positions 1648 to 1650 of SEQ ID
NO:163 are changed to AAT or AAC; or a nucleotide at the equivalent
of any one of positions 1768 to 1770 of SEQ ID NO:163 are changed
to CGT, CGC, CGA, CGG, AGA or AGG. In another aspect, the invention
provides isolated or recombinant nucleic acids having a sequence
comprising a nucleotide residue sequence modification of any
nucleic acid of the invention, wherein the modification comprises
one or more of the following changes: a nucleotide at the
equivalent of any one of positions 265 to 267 of SEQ ID NO:163 are
changed to CGT, CGC, CGA, CGG, AGA or AGG; a nucleotide at the
equivalent of any one of positions 307 to 309 of SEQ ID NO:163 are
changed to GGT, GGC, GGA or GGG; a nucleotide at the equivalent of
any one of positions 328 to 330 of SEQ ID NO:163 are changed to
GGT, GGC, GGA or GGG; a nucleotide at the equivalent of any one of
positions 340 to 342 of SEQ ID NO:163 are changed to TTA, TTG, CTT,
CTC, CTA or CTG; a nucleotide at the equivalent of any one of
positions 469 to 471 of SEQ ID NO:163 are changed to TCT, TCC, TCA,
TCG, AGT or AGC; a nucleotide at the equivalent of positions 1441
to 1443 of SEQ ID NO:163 are changed to TTT or TTC; a nucleotide at
the equivalent of any one of positions 1648 to 1650 of SEQ ID
NO:163 are changed to AAT or AAC; or, a nucleotide at the
equivalent of any one of positions 1768 to 1770 of SEQ ID NO:163
are changed to CGT, CGC, CGA, CGG, AGA or AGG.
[0287] In another aspect, the invention provides isolated or
recombinant polypeptides having a sequence comprising an amino acid
residue modification of an exemplary sequence of the invention
(e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, etc.) wherein the modification comprises one or more of the
following changes: an amino acid at the equivalent of the
methionine at amino acid position 89 of SEQ ID NO:164 is changed to
an arginine; an amino acid at the equivalent of the phenylalanine
at amino acid position 103 of SEQ ID NO:164 is changed to a
glycine; an amino acid at the equivalent of the proline at amino
acid position 110 of SEQ ID NO:164 is changed to a glycine; an
amino acid at the equivalent of the tyrosine at amino acid position
114 of SEQ ID NO:164 is changed to a leucine; an amino acid at the
equivalent of the alanine at amino acid position 157 of SEQ ID
NO:164 is changed to a serine; an amino acid at the equivalent of
the tryptophan at amino acid position 481 of SEQ ID NO:164 is
changed to a phenylalanine; an amino acid at the equivalent of the
proline at amino acid position 550 of SEQ ID NO:164 is changed to
an asparagine; or an amino acid at the equivalent of the glycine at
amino acid position 590 of SEQ ID NO:164 is changed to an
arginine.
[0288] In another aspect, the invention provides isolated or
recombinant polypeptides having a sequence comprising an amino acid
residue modification of any polypeptide of the invention, wherein
the modification comprises one or more of the following changes: an
amino acid at the equivalent of the methionine at amino acid
position 89 of SEQ ID NO:164 is changed to an arginine; an amino
acid at the equivalent of the phenylalanine at amino acid position
103 of SEQ ID NO:164 is changed to a glycine; an amino acid at the
equivalent of the proline at amino acid position 110 of SEQ ID
NO:164 is changed to a glycine; an amino acid at the equivalent of
the tyrosine at amino acid position 114 of SEQ ID NO:164 is changed
to a leucine; an amino acid at the equivalent of the alanine at
amino acid position 157 of SEQ ID NO:164 is changed to a serine; an
amino acid at the equivalent of the tryptophan at amino acid
position 481 of SEQ ID NO:164 is changed to a phenylalanine; an
amino acid at the equivalent of the proline at amino acid position
550 of SEQ ID NO:164 is changed to an asparagine; or an amino acid
at the equivalent of the glycine at amino acid position 590 of SEQ
ID NO:164 is changed to an arginine.
[0289] A nucleic acid of the invention can be altered by any means.
For example, random or stochastic methods, or, non-stochastic, or
"directed evolution," methods, see, e.g., U.S. Pat. No. 6,361,974.
Methods for random mutation of genes are well known in the art,
see, e.g., U.S. Pat. No. 5,830,696. For example, mutagens can be
used to randomly mutate a gene. Mutagens include, e.g., ultraviolet
light or gamma irradiation, or a chemical mutagen, e.g., mitomycin,
nitrous acid, photoactivated psoralens, alone or in combination, to
induce DNA breaks amenable to repair by recombination. Other
chemical mutagens include, for example, sodium bisulfite, nitrous
acid, hydroxylamine, hydrazine or formic acid. Other mutagens are
analogues of nucleotide precursors, e.g., nitrosoguanidine,
5-bromouracil, 2-aminopurine, or acridine. These agents can be
added to a PCR reaction in place of the nucleotide precursor
thereby mutating the sequence. Intercalating agents such as
proflavine, acriflavine, quinacrine and the like can also be
used.
[0290] Any technique in molecular biology can be used, e.g., random
PCR mutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA
89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,
e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively,
nucleic acids, e.g., genes, can be reassembled after random, or
"stochastic," fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242;
6,287,862; 6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238;
5,605,793. In alternative aspects, modifications, additions or
deletions are introduced by error-prone PCR, shuffling,
oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR
mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive
ensemble mutagenesis, exponential ensemble mutagenesis,
site-specific mutagenesis, gene reassembly, Gene Site Saturation
Mutagenesis (GSSM), synthetic ligation reassembly (SLR),
recombination, recursive sequence recombination,
phosphothioate-modified DNA mutagenesis, uracil-containing template
mutagenesis, gapped duplex mutagenesis, point mismatch repair
mutagenesis, repair-deficient host strain mutagenesis, chemical
mutagenesis, radiogenic mutagenesis, deletion mutagenesis,
restriction-selection mutagenesis, restriction-purification
mutagenesis, artificial gene synthesis, ensemble mutagenesis,
chimeric nucleic acid multimer creation, Chromosomal Saturation
Mutagenesis (CSM) and/or a combination of these and other
methods.
[0291] The following publications describe a variety of recursive
recombination procedures and/or methods which can be incorporated
into the methods of the invention: Stemmer (1999) "Molecular
breeding of viruses for targeting and other clinical properties"
Tumor Targeting 4:1-4; Ness (1999) Nature Biotechnology 17:893-896;
Chang (1999) "Evolution of a cytokine using DNA family shuffling"
Nature Biotechnology 17:793-797; Minshull (1999) "Protein evolution
by molecular breeding" Current Opinion in Chemical Biology
3:284-290; Christians (1999) "Directed evolution of thymidine
kinase for AZT phosphorylation using DNA family shuffling" Nature
Biotechnology 17:259-264; Crameri (1998) "DNA shuffling of a family
of genes from diverse species accelerates directed evolution"
Nature 391:288-291; Crameri (1997) "Molecular evolution of an
arsenate detoxification pathway by DNA shuffling," Nature
Biotechnology 15:436-438; Zhang (1997) "Directed evolution of an
effective fucosidase from a galactosidase by DNA shuffling and
screening" Proc. Natl. Acad. Sci. USA 94:4504-4509; Patten et al.
(1997) "Applications of DNA Shuffling to Pharmaceuticals and
Vaccines" Current Opinion in Biotechnology 8:724-733; Crameri et
al. (1996) "Construction and evolution of antibody-phage libraries
by DNA shuffling" Nature Medicine 2:100-103; Gates et al. (1996)
"Affinity selective isolation of ligands from peptide libraries
through display on a lac repressor `headpiece dimer`" Journal of
Molecular Biology 255:373-386; Stemmer (1996) "Sexual PCR and
Assembly PCR" In: The Encyclopedia of Molecular Biology. VCH
Publishers, New York. pp. 447-457; Crameri and Stemmer (1995)
"Combinatorial multiple cassette mutagenesis creates all the
permutations of mutant and wildtype cassettes" BioTechniques
18:194-195; Stemmer et al. (1995) "Single-step assembly of a gene
and entire plasmid form large numbers of oligodeoxyribonucleotides"
Gene, 164:49-53; Stemmer (1995) "The Evolution of Molecular
Computation" Science 270: 1510; Stemmer (1995) "Searching Sequence
Space" Bio/Technology 13:549-553; Stemmer (1994) "Rapid evolution
of a protein in vitro by DNA shuffling" Nature 370:389-391; and
Stemmer (1994) "DNA shuffling by random fragmentation and
reassembly: In vitro recombination for molecular evolution." Proc.
Natl. Acad. Sci. USA 91:10747-10751.
[0292] Mutational methods of generating diversity include, for
example, site-directed mutagenesis (Ling et al. (1997) "Approaches
to DNA mutagenesis: an overview" Anal Biochem. 254(2): 157-178;
Dale et al. (1996) "Oligonucleotide-directed random mutagenesis
using the phosphorothioate method" Methods Mol. Biol. 57:369-374;
Smith (1985) "In vitro mutagenesis" Aim. Rev. Genet. 19:423-462;
Botstein & Shortle (1985) "Strategies and applications of in
vitro mutagenesis" Science 229:1193-1201; Carter (1986)
"Site-directed mutagenesis" Biochem. J. 237:1-7; and Kunkel (1987)
"The efficiency of oligonucleotide directed mutagenesis" in Nucleic
Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.
eds., Springer Verlag, Berlin)); mutagenesis using uracil
containing templates (Kunkel (1985) "Rapid and efficient
site-specific mutagenesis without phenotypic selection" Proc. Natl.
Acad. Sci. USA 82:488-492; Kunkel et al. (1987) "Rapid and
efficient site-specific mutagenesis without phenotypic selection"
Methods in Enzymol. 154, 367-382; and Bass et al. (1988) "Mutant
Trp repressors with new DNA-binding specificities" Science
242:240-245); oligonucleotide-directed mutagenesis (Methods in
Enzymol. 100: 468-500 (1983); Methods in Enzymol. 154: 329-350
(1987); Zoller (1982) "Oligonucleotide-directed mutagenesis using
M13-derived vectors: an efficient and general procedure for the
production of point mutations in any DNA fragment" Nucleic Acids
Res. 10:6487-6500; Zoller & Smith (1983)
"Oligonucleotide-directed mutagenesis of DNA fragments cloned into
M13 vectors" Methods in Enzymol. 100:468-500; and Zoller (1987)
Oligonucleotide-directed mutagenesis: a simple method using two
oligonucleotide primers and a single-stranded DNA template" Methods
in Enzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis
(Taylor (1985) "The use of phosphorothioate-modified DNA in
restriction enzyme reactions to prepare nicked DNA" Nucl. Acids
Res. 13: 8749-8764; Taylor (1985) "The rapid generation of
oligonucleotide-directed mutations at high frequency using
phosphorothioate-modified DNA" Nucl. Acids Res. 13: 8765-8787
(1985); Nakamaye (1986) "Inhibition of restriction endonuclease Nci
I cleavage by phosphorothioate groups and its application to
oligonucleotide-directed mutagenesis" Nucl. Acids Res. 14:
9679-9698; Sayers (1988) "Y-T Exonucleases in
phosphorothioate-based oligonucleotide-directed mutagenesis" Nucl.
Acids Res. 16:791-802; and Sayers et al. (1988) "Strand specific
cleavage of phosphorothioate-containing DNA by reaction with
restriction endonucleases in the presence of ethidium bromide"
Nucl. Acids Res. 16: 803-814); mutagenesis using gapped duplex DNA
(Kramer et al. (1984) "The gapped duplex DNA approach to
oligonucleotide-directed mutation construction" Nucl. Acids Res.
12: 9441-9456; Kramer & Fritz (1987) Methods in Enzymol.
"Oligonucleotide-directed construction of mutations via gapped
duplex DNA" 154:350-367; Kramer (1988) "Improved enzymatic in vitro
reactions in the gapped duplex DNA approach to
oligonucleotide-directed construction of mutations" Nucl. Acids
Res. 16: 7207; and Fritz (1988) "Oligonucleotide-directed
construction of mutations: a gapped duplex DNA procedure without
enzymatic reactions in vitro" Nucl. Acids Res. 16: 6987-6999).
[0293] Additional protocols that can be used to practice the
invention include point mismatch repair (Kramer (1984) "Point
Mismatch Repair" Cell 38:879-887), mutagenesis using
repair-deficient host strains (Carter et al. (1985) "Improved
oligonucleotide site-directed mutagenesis using M13 vectors" Nucl.
Acids Res. 13: 4431-4443; and Carter (1987) "Improved
oligonucleotide-directed mutagenesis using M13 vectors" Methods in
Enzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986)
"Use of oligonucleotides to generate large deletions" Nucl. Acids
Res. 14: 5115), restriction-selection and restriction-selection and
restriction-purification (Wells et al. (1986) "Importance of
hydrogen-bond formation in stabilizing the transition state of
subtilisin" Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis
by total gene synthesis (Nambiar et al. (1984) "Total synthesis and
cloning of a gene coding for the ribonuclease S protein" Science
223: 1299-1301; Sakamar and Khorana (1988) "Total synthesis and
expression of a gene for the a-subunit of bovine rod outer segment
guanine nucleotide-binding protein (transducin)" Nucl. Acids Res.
14: 6361-6372; Wells et al. (1985) "Cassette mutagenesis: an
efficient method for generation of multiple mutations at defined
sites" Gene 34:315-323; and Grundstrom et al. (1985)
"Oligonucleotide-directed mutagenesis by microscale `shot-gun` gene
synthesis" Nucl. Acids Res. 13: 3305-3316), double-strand break
repair (Mandecki (1986); Arnold (1993) "Protein engineering for
unusual environments" Current Opinion in Biotechnology 4:450-455.
"Oligonucleotide-directed double-strand break repair in plasmids of
Escherichia coli: a method for site-specific mutagenesis" Proc.
Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many of
the above methods can be found in Methods in Enzymology Volume 154,
which also describes useful controls for trouble-shooting problems
with various mutagenesis methods.
[0294] Protocols that can be used to practice the invention are
described, e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25,
1997), "Methods for In Vitro Recombination;" U.S. Pat. No.
5,811,238 to Stemmer et al. (Sep. 22, 1998) "Methods for Generating
Polynucleotides having Desired Characteristics by Iterative
Selection and Recombination;" U.S. Pat. No. 5,830,721 to Stemmer et
al. (Nov. 3, 1998), "DNA Mutagenesis by Random Fragmentation and
Reassembly;" U.S. Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10,
1998) "End-Complementary Polymerase Reaction;" U.S. Pat. No.
5,837,458 to Minshull, et al. (Nov. 17, 1998), "Methods and
Compositions for Cellular and Metabolic Engineering;" WO 95/22625,
Stemmer and Crameri, "Mutagenesis by Random Fragmentation and
Reassembly;" WO 96/33207 by Stemmer and Lipschutz "End
Complementary Polymerase Chain Reaction;" WO 97/20078 by Stemmer
and Crameri "Methods for Generating Polynucleotides having Desired
Characteristics by Iterative Selection and Recombination;" WO
97/35966 by Minshull and Stemmer, "Methods and Compositions for
Cellular and Metabolic Engineering;" WO 99/41402 by Punnonen et al.
"Targeting of Genetic Vaccine Vectors;" WO 99/41383 by Punnonen et
al. "Antigen Library Immunization;" WO 99/41369 by Punnonen et al.
"Genetic Vaccine Vector Engineering;" WO 99/41368 by Punnonen et
al. "Optimization of Immunomodulatory Properties of Genetic
Vaccines;" EP 752008 by Stemmer and Crameri, "DNA Mutagenesis by
Random Fragmentation and Reassembly;" EP 0932670 by Stemmer
"Evolving Cellular DNA Uptake by Recursive Sequence Recombination;"
WO 99/23107 by Stemmer et al., "Modification of Virus Tropism and
Host Range by Viral Genome Shuffling;" WO 99/21979 by Apt et al.,
"Human Papillomavirus Vectors;" WO 98/31837 by del Cardayre et al.
"Evolution of Whole Cells and Organisms by Recursive Sequence
Recombination;" WO 98/27230 by Patten and Stemmer, "Methods and
Compositions for Polypeptide Engineering;" WO 98/27230 by Stemmer
et al., "Methods for Optimization of Gene Therapy by Recursive
Sequence Shuffling and Selection," WO 00/00632, "Methods for
Generating Highly Diverse Libraries," WO 00/09679, "Methods for
Obtaining in Vitro Recombined Polynucleotide Sequence Banks and
Resulting Sequences," WO 98/42832 by Arnold et al., "Recombination
of Polynucleotide Sequences Using Random or Defined Primers," WO
99/29902 by Arnold et al., "Method for Creating Polynucleotide and
Polypeptide Sequences," WO 98/41653 by Vind, "An in Vitro Method
for Construction of a DNA Library," WO 98/41622 by Borchert et al.,
"Method for Constructing a Library Using DNA Shuffling," and WO
98/42727 by Pati and Zarling, "Sequence Alterations using
Homologous Recombination."
[0295] Protocols that can be used to practice the invention
(providing details regarding various diversity generating methods)
are described, e.g., in U.S. patent application serial no. (USSN)
09/407,800, "SHUFFLING OF CODON ALTERED GENES" by Patten et al.
filed Sep. 28, 1999; "EVOLUTION OF WHOLE CELLS AND ORGANISMS BY
RECURSIVE SEQUENCE RECOMBINATION" by del Cardayre et al., U.S. Pat.
No. 6,379,964; "OLIGONUCLEOTIDE MEDIATED NUCLEIC ACID
RECOMBINATION" by Crameri et al., U.S. Pat. Nos. 6,319,714;
6,368,861; 6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; "USE
OF CODON-VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING"
by Welch et al., U.S. Pat. No. 6,436,675; "METHODS FOR MAKING
CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDES HAVING
DESIRED CHARACTERISTICS" by Selifonov et al., filed Jan. 18, 2000,
(PCT/US00/01202) and, e.g. "METHODS FOR MAKING CHARACTER STRINGS,
POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS"
by Selifonov et al., filed Jul. 18, 2000 (U.S. Ser. No.
09/618,579); "METHODS OF POPULATING DATA STRUCTURES FOR USE IN
EVOLUTIONARY SIMULATIONS" by Selifonov and Stemmer, filed Jan. 18,
2000 (PCT/US00/01138); and "SINGLE-STRANDED NUCLEIC ACID
TEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT
ISOLATION" by Affholter, filed Sep. 6, 2000 (U.S. Ser. No.
09/656,549); and U.S. Pat. Nos. 6,177,263; 6,153,410.
[0296] Non-stochastic, or "directed evolution," methods include,
e.g., saturation mutagenesis, such as Gene Site Saturation
Mutagenesis (GSSM), synthetic ligation reassembly (SLR), or a
combination thereof are used to modify the nucleic acids of the
invention to generate cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes with
new or altered properties (e.g., activity under highly acidic or
alkaline conditions, high or low temperatures, and the like).
Polypeptides encoded by the modified nucleic acids can be screened
for an activity before testing for glucan hydrolysis or other
activity. Any testing modality or protocol can be used, e.g., using
a capillary array platform. See, e.g., U.S. Pat. Nos. 6,361,974;
6,280,926; 5,939,250.
[0297] Gene Site Saturation Mutagenesis, or, GSSM
[0298] The invention also provides methods for making enzyme using
Gene Site Saturation mutagenesis, or, GSSM, as described herein,
and also in U.S. Pat. Nos. 6,171,820 and 6,579,258. In one aspect,
codon primers containing a degenerate N,N,G/T sequence are used to
introduce point mutations into a polynucleotide, e.g., a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme or an antibody of the invention, so as to
generate a set of progeny polypeptides in which a full range of
single amino acid substitutions is represented at each amino acid
position, e.g., an amino acid residue in an enzyme active site or
ligand binding site targeted to be modified. These oligonucleotides
can comprise a contiguous first homologous sequence, a degenerate
N,N,G/T sequence, and, optionally, a second homologous sequence.
The downstream progeny translational products from the use of such
oligonucleotides include all possible amino acid changes at each
amino acid site along the polypeptide, because the degeneracy of
the N,N,G/T sequence includes codons for all 20 amino acids. In one
aspect, one such degenerate oligonucleotide (comprised of, e.g.,
one degenerate N,N,G/T cassette) is used for subjecting each
original codon in a parental polynucleotide template to a full
range of codon substitutions. In another aspect, at least two
degenerate cassettes are used--either in the same oligonucleotide
or not, for subjecting at least two original codons in a parental
polynucleotide template to a full range of codon substitutions. For
example, more than one N,N,G/T sequence can be contained in one
oligonucleotide to introduce amino acid mutations at more than one
site. This plurality of N,N,G/T sequences can be directly
contiguous, or separated by one or more additional nucleotide
sequence(s). In another aspect, oligonucleotides serviceable for
introducing additions and deletions can be used either alone or in
combination with the codons containing an N,N,G/T sequence, to
introduce any combination or permutation of amino acid additions,
deletions, and/or substitutions.
[0299] In one aspect, simultaneous mutagenesis of two or more
contiguous amino acid positions is done using an oligonucleotide
that contains contiguous N,N,G/T triplets, i.e. a degenerate
(N,N,G/T)n sequence. In another aspect, degenerate cassettes having
less degeneracy than the N,N,G/T sequence are used. For example, it
may be desirable in some instances to use (e.g. in an
oligonucleotide) a degenerate triplet sequence comprised of only
one N, where said N can be in the first second or third position of
the triplet. Any other bases including any combinations and
permutations thereof can be used in the remaining two positions of
the triplet. Alternatively, it may be desirable in some instances
to use (e.g. in an oligo) a degenerate N,N,N triplet sequence.
[0300] In one aspect, use of degenerate triplets (e.g., N,N,G/T
triplets) allows for systematic and easy generation of a full range
of possible natural amino acids (for a total of 20 amino acids)
into each and every amino acid position in a polypeptide (in
alternative aspects, the methods also include generation of less
than all possible substitutions per amino acid residue, or codon,
position). For example, for a 100 amino acid polypeptide, 2000
distinct species (i.e. 20 possible amino acids per position X 100
amino acid positions) can be generated. Through the use of an
oligonucleotide or set of oligonucleotides containing a degenerate
N,N,G/T triplet, 32 individual sequences can code for all 20
possible natural amino acids. Thus, in a reaction vessel in which a
parental polynucleotide sequence is subjected to saturation
mutagenesis using at least one such oligonucleotide, there are
generated 32 distinct progeny polynucleotides encoding 20 distinct
polypeptides. In contrast, the use of a non-degenerate
oligonucleotide in site-directed mutagenesis leads to only one
progeny polypeptide product per reaction vessel. Nondegenerate
oligonucleotides can optionally be used in combination with
degenerate primers disclosed; for example, nondegenerate
oligonucleotides can be used to generate specific point mutations
in a working polynucleotide. This provides one means to generate
specific silent point mutations, point mutations leading to
corresponding amino acid changes, and point mutations that cause
the generation of stop codons and the corresponding expression of
polypeptide fragments.
[0301] In one aspect, each saturation mutagenesis reaction vessel
contains polynucleotides encoding at least 20 progeny polypeptide
(e.g., cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzymes) molecules such that all 20 natural
amino acids are represented at the one specific amino acid position
corresponding to the codon position mutagenized in the parental
polynucleotide (other aspects use less than all 20 natural
combinations). The 32-fold degenerate progeny polypeptides
generated from each saturation mutagenesis reaction vessel can be
subjected to clonal amplification (e.g. cloned into a suitable
host, e.g., E. coli host, using, e.g., an expression vector) and
subjected to expression screening. When an individual progeny
polypeptide is identified by screening to display a favorable
change in property (when compared to the parental polypeptide, such
as increased glucan hydrolysis activity under alkaline or acidic
conditions), it can be sequenced to identify the correspondingly
favorable amino acid substitution contained therein.
[0302] In one aspect, upon mutagenizing each and every amino acid
position in a parental polypeptide using saturation mutagenesis as
disclosed herein, favorable amino acid changes may be identified at
more than one amino acid position. One or more new progeny
molecules can be generated that contain a combination of all or
part of these favorable amino acid substitutions. For example, if 2
specific favorable amino acid changes are identified in each of 3
amino acid positions in a polypeptide, the permutations include 3
possibilities at each position (no change from the original amino
acid, and each of two favorable changes) and 3 positions. Thus,
there are 3.times.3.times.3 or 27 total possibilities, including 7
that were previously examined--6 single point mutations (i.e. 2 at
each of three positions) and no change at any position.
[0303] In yet another aspect, site-saturation mutagenesis can be
used together with shuffling, chimerization, recombination and
other mutagenizing processes, along with screening. This invention
provides for the use of any mutagenizing process(es), including
saturation mutagenesis, in an iterative manner. In one
exemplification, the iterative use of any mutagenizing process(es)
is used in combination with screening.
[0304] The invention also provides for the use of proprietary codon
primers (containing a degenerate N,N,N sequence) to introduce point
mutations into a polynucleotide, so as to generate a set of progeny
polypeptides in which a full range of single amino acid
substitutions is represented at each amino acid position (Gene Site
Saturation Mutagenesis (GSSM)). The oligos used are comprised
contiguously of a first homologous sequence, a degenerate N,N,N
sequence and in one aspect but not necessarily a second homologous
sequence. The downstream progeny translational products from the
use of such oligos include all possible amino acid changes at each
amino acid site along the polypeptide, because the degeneracy of
the N,N,N sequence includes codons for all 20 amino acids.
[0305] In one aspect, one such degenerate oligo (comprised of one
degenerate N,N,N cassette) is used for subjecting each original
codon in a parental polynucleotide template to a full range of
codon substitutions. In another aspect, at least two degenerate
N,N,N cassettes are used--either in the same oligo or not, for
subjecting at least two original codons in a parental
polynucleotide template to a full range of codon substitutions.
Thus, more than one N,N,N sequence can be contained in one oligo to
introduce amino acid mutations at more than one site. This
plurality of N,N,N sequences can be directly contiguous, or
separated by one or more additional nucleotide sequence(s). In
another aspect, oligos serviceable for introducing additions and
deletions can be used either alone or in combination with the
codons containing an N,N,N sequence, to introduce any combination
or permutation of amino acid additions, deletions and/or
substitutions.
[0306] In one aspect, it is possible to simultaneously mutagenize
two or more contiguous amino acid positions using an oligo that
contains contiguous N,N,N triplets, i.e. a degenerate (N,N,N).sub.n
sequence. In another aspect, the present invention provides for the
use of degenerate cassettes having less degeneracy than the N,N,N
sequence. For example, it may be desirable in some instances to use
(e.g. in an oligo) a degenerate triplet sequence comprised of only
one N, where the N can be in the first second or third position of
the triplet. Any other bases including any combinations and
permutations thereof can be used in the remaining two positions of
the triplet. Alternatively, it may be desirable in some instances
to use (e.g., in an oligo) a degenerate N,N,N triplet sequence,
N,N,G/T, or an N,N, G/C triplet sequence.
[0307] In one aspect, use of a degenerate triplet (such as N,N,G/T
or an N,N, G/C triplet sequence) is advantageous for several
reasons. In one aspect, this invention provides a means to
systematically and fairly easily generate the substitution of the
full range of possible amino acids (for a total of 20 amino acids)
into each and every amino acid position in a polypeptide. Thus, for
a 100 amino acid polypeptide, the invention provides a way to
systematically and fairly easily generate 2000 distinct species
(i.e., 20 possible amino acids per position times 100 amino acid
positions). It is appreciated that there is provided, through the
use of an oligo containing a degenerate N,N,G/T or an N,N, G/C
triplet sequence, 32 individual sequences that code for 20 possible
amino acids. Thus, in a reaction vessel in which a parental
polynucleotide sequence is subjected to saturation mutagenesis
using one such oligo, there are generated 32 distinct progeny
polynucleotides encoding 20 distinct polypeptides. In contrast, the
use of a non-degenerate oligo in site-directed mutagenesis leads to
only one progeny polypeptide product per reaction vessel.
[0308] This invention also provides for the use of nondegenerate
oligos, which can optionally be used in combination with degenerate
primers disclosed. It is appreciated that in some situations, it is
advantageous to use nondegenerate oligos to generate specific point
mutations in a working polynucleotide. This provides a means to
generate specific silent point mutations, point mutations leading
to corresponding amino acid changes and point mutations that cause
the generation of stop codons and the corresponding expression of
polypeptide fragments.
[0309] Thus, in one aspect of this invention, each saturation
mutagenesis reaction vessel contains polynucleotides encoding at
least 20 progeny polypeptide molecules such that all 20 amino acids
are represented at the one specific amino acid position
corresponding to the codon position mutagenized in the parental
polynucleotide. The 32-fold degenerate progeny polypeptides
generated from each saturation mutagenesis reaction vessel can be
subjected to clonal amplification (e.g., cloned into a suitable E.
coli host using an expression vector) and subjected to expression
screening. When an individual progeny polypeptide is identified by
screening to display a favorable change in property (when compared
to the parental polypeptide), it can be sequenced to identify the
correspondingly favorable amino acid substitution contained
therein.
[0310] In one aspect, upon mutagenizing each and every amino acid
position in a parental polypeptide using saturation mutagenesis as
disclosed herein, a favorable amino acid changes is identified at
more than one amino acid position. One or more new progeny
molecules can be generated that contain a combination of all or
part of these favorable amino acid substitutions. For example, if 2
specific favorable amino acid changes are identified in each of 3
amino acid positions in a polypeptide, the permutations include 3
possibilities at each position (no change from the original amino
acid and each of two favorable changes) and 3 positions. Thus,
there are 3.times.3.times.3 or 27 total possibilities, including 7
that were previously examined--6 single point mutations (i.e., 2 at
each of three positions) and no change at any position.
[0311] The invention provides for the use of saturation mutagenesis
in combination with additional mutagenization processes, such as
process where two or more related polynucleotides are introduced
into a suitable host cell such that a hybrid polynucleotide is
generated by recombination and reductive reassortment.
[0312] In addition to performing mutagenesis along the entire
sequence of a gene, the instant invention provides that mutagenesis
can be use to replace each of any number of bases in a
polynucleotide sequence, wherein the number of bases to be
mutagenized is in one aspect every integer from 15 to 100,000.
Thus, instead of mutagenizing every position along a molecule, one
can subject every or a discrete number of bases (in one aspect a
subset totaling from 15 to 100,000) to mutagenesis. In one aspect,
a separate nucleotide is used for mutagenizing each position or
group of positions along a polynucleotide sequence. A group of 3
positions to be mutagenized may be a codon. The mutations can be
introduced using a mutagenic primer, containing a heterologous
cassette, also referred to as a mutagenic cassette. Exemplary
cassettes can have from 1 to 500 bases. Each nucleotide position in
such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G,
C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any base
that is not A, C, G, or T (E can be referred to as a designer
oligo).
[0313] In one aspect, saturation mutagenesis is comprised of
mutagenizing a complete set of mutagenic cassettes (wherein each
cassette is in one aspect about 1-500 bases in length) in defined
polynucleotide sequence to be mutagenized (wherein the sequence to
be mutagenized is in one aspect from about 15 to 100,000 bases in
length). Thus, a group of mutations (ranging from 1 to 100
mutations) is introduced into each cassette to be mutagenized. A
grouping of mutations to be introduced into one cassette can be
different or the same from a second grouping of mutations to be
introduced into a second cassette during the application of one
round of saturation mutagenesis. Such groupings are exemplified by
deletions, additions, groupings of particular codons and groupings
of particular nucleotide cassettes.
[0314] In one aspect, defined sequences to be mutagenized include a
whole gene, pathway, cDNA, an entire open reading frame (ORF) and
entire promoter, enhancer, repressor/transactivator, origin of
replication, intron, operator, or any polynucleotide functional
group. Generally, a "defined sequences" for this purpose may be any
polynucleotide that a 15 base-polynucleotide sequence and
polynucleotide sequences of lengths between 15 bases and 15,000
bases (this invention specifically names every integer in between).
Considerations in choosing groupings of codons include types of
amino acids encoded by a degenerate mutagenic cassette.
[0315] In one aspect, a grouping of mutations that can be
introduced into a mutagenic cassette, this invention specifically
provides for degenerate codon substitutions (using degenerate
oligos) that code for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 and 20 amino acids at each position and a
library of polypeptides encoded thereby.
[0316] Synthetic Ligation Reassembly (SLR)
[0317] The invention provides a non-stochastic gene modification
system termed "synthetic ligation reassembly," or simply "SLR," a
"directed evolution process," to generate polypeptides, e.g.,
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes or antibodies of the invention, with new
or altered properties.
[0318] SLR is a method of ligating oligonucleotide fragments
together non-stochastically. This method differs from stochastic
oligonucleotide shuffling in that the nucleic acid building blocks
are not shuffled, concatenated or chimerized randomly, but rather
are assembled non-stochastically. See, e.g., U.S. Pat. Nos.
6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776.
In one aspect, SLR comprises the following steps: (a) providing a
template polynucleotide, wherein the template polynucleotide
comprises sequence encoding a homologous gene; (b) providing a
plurality of building block polynucleotides, wherein the building
block polynucleotides are designed to cross-over reassemble with
the template polynucleotide at a predetermined sequence, and a
building block polynucleotide comprises a sequence that is a
variant of the homologous gene and a sequence homologous to the
template polynucleotide flanking the variant sequence; (c)
combining a building block polynucleotide with a template
polynucleotide such that the building block polynucleotide
cross-over reassembles with the template polynucleotide to generate
polynucleotides comprising homologous gene sequence variations.
[0319] SLR does not depend on the presence of high levels of
homology between polynucleotides to be rearranged. Thus, this
method can be used to non-stochastically generate libraries (or
sets) of progeny molecules comprised of over 10.sup.100 different
chimeras. SLR can be used to generate libraries comprised of over
10.sup.1000 different progeny chimeras. Thus, aspects of the
present invention include non-stochastic methods of producing a set
of finalized chimeric nucleic acid molecule shaving an overall
assembly order that is chosen by design. This method includes the
steps of generating by design a plurality of specific nucleic acid
building blocks having serviceable mutually compatible ligatable
ends, and assembling these nucleic acid building blocks, such that
a designed overall assembly order is achieved.
[0320] The mutually compatible ligatable ends of the nucleic acid
building blocks to be assembled are considered to be "serviceable"
for this type of ordered assembly if they enable the building
blocks to be coupled in predetermined orders. Thus, the overall
assembly order in which the nucleic acid building blocks can be
coupled is specified by the design of the ligatable ends. If more
than one assembly step is to be used, then the overall assembly
order in which the nucleic acid building blocks can be coupled is
also specified by the sequential order of the assembly step(s). In
one aspect, the annealed building pieces are treated with an
enzyme, such as a ligase (e.g. T4 DNA ligase), to achieve covalent
bonding of the building pieces.
[0321] In one aspect, the design of the oligonucleotide building
blocks is obtained by analyzing a set of progenitor nucleic acid
sequence templates that serve as a basis for producing a progeny
set of finalized chimeric polynucleotides. These parental
oligonucleotide templates thus serve as a source of sequence
information that aids in the design of the nucleic acid building
blocks that are to be mutagenized, e.g., chimerized or shuffled. In
one aspect of this method, the sequences of a plurality of parental
nucleic acid templates are aligned in order to select one or more
demarcation points. The demarcation points can be located at an
area of homology, and are comprised of one or more nucleotides.
These demarcation points are in one aspect shared by at least two
of the progenitor templates. The demarcation points can thereby be
used to delineate the boundaries of oligonucleotide building blocks
to be generated in order to rearrange the parental polynucleotides.
The demarcation points identified and selected in the progenitor
molecules serve as potential chimerization points in the assembly
of the final chimeric progeny molecules. A demarcation point can be
an area of homology (comprised of at least one homologous
nucleotide base) shared by at least two parental polynucleotide
sequences. Alternatively, a demarcation point can be an area of
homology that is shared by at least half of the parental
polynucleotide sequences, or, it can be an area of homology that is
shared by at least two thirds of the parental polynucleotide
sequences. Even more in one aspect a serviceable demarcation points
is an area of homology that is shared by at least three fourths of
the parental polynucleotide sequences, or, it can be shared by at
almost all of the parental polynucleotide sequences. In one aspect,
a demarcation point is an area of homology that is shared by all of
the parental polynucleotide sequences.
[0322] In one aspect, a ligation reassembly process is performed
exhaustively in order to generate an exhaustive library of progeny
chimeric polynucleotides. In other words, all possible ordered
combinations of the nucleic acid building blocks are represented in
the set of finalized chimeric nucleic acid molecules. At the same
time, in another aspect, the assembly order (i.e. the order of
assembly of each building block in the 5' to 3 sequence of each
finalized chimeric nucleic acid) in each combination is by design
(or non-stochastic) as described above. Because of the
non-stochastic nature of this invention, the possibility of
unwanted side products is greatly reduced.
[0323] In another aspect, the ligation reassembly method is
performed systematically. For example, the method is performed in
order to generate a systematically compartmentalized library of
progeny molecules, with compartments that can be screened
systematically, e.g. one by one. In other words this invention
provides that, through the selective and judicious use of specific
nucleic acid building blocks, coupled with the selective and
judicious use of sequentially stepped assembly reactions, a design
can be achieved where specific sets of progeny products are made in
each of several reaction vessels. This allows a systematic
examination and screening procedure to be performed. Thus, these
methods allow a potentially very large number of progeny molecules
to be examined systematically in smaller groups. Because of its
ability to perform chimerizations in a manner that is highly
flexible yet exhaustive and systematic as well, particularly when
there is a low level of homology among the progenitor molecules,
these methods provide for the generation of a library (or set)
comprised of a large number of progeny molecules. Because of the
non-stochastic nature of the instant ligation reassembly invention,
the progeny molecules generated in one aspect comprise a library of
finalized chimeric nucleic acid molecules having an overall
assembly order that is chosen by design. The saturation mutagenesis
and optimized directed evolution methods also can be used to
generate different progeny molecular species. It is appreciated
that the invention provides freedom of choice and control regarding
the selection of demarcation points, the size and number of the
nucleic acid building blocks, and the size and design of the
couplings. It is appreciated, furthermore, that the requirement for
intermolecular homology is highly relaxed for the operability of
this invention. In fact, demarcation points can even be chosen in
areas of little or no intermolecular homology. For example, because
of codon wobble, i.e. the degeneracy of codons, nucleotide
substitutions can be introduced into nucleic acid building blocks
without altering the amino acid originally encoded in the
corresponding progenitor template. Alternatively, a codon can be
altered such that the coding for an originally amino acid is
altered. This invention provides that such substitutions can be
introduced into the nucleic acid building block in order to
increase the incidence of intermolecular homologous demarcation
points and thus to allow an increased number of couplings to be
achieved among the building blocks, which in turn allows a greater
number of progeny chimeric molecules to be generated.
[0324] Synthetic Gene Reassembly
[0325] In one aspect, the present invention provides a
non-stochastic method termed synthetic gene reassembly, that is
somewhat related to stochastic shuffling, save that the nucleic
acid building blocks are not shuffled or concatenated or chimerized
randomly, but rather are assembled non-stochastically. See, e.g.,
U.S. Pat. No. 6,537,776.
[0326] The synthetic gene reassembly method does not depend on the
presence of a high level of homology between polynucleotides to be
shuffled. The invention can be used to non-stochastically generate
libraries (or sets) of progeny molecules comprised of over
10.sup.100 different chimeras. Conceivably, synthetic gene
reassembly can even be used to generate libraries comprised of over
10.sup.1000 different progeny chimeras.
[0327] Thus, in one aspect, the invention provides a non-stochastic
method of producing a set of finalized chimeric nucleic acid
molecules having an overall assembly order that is chosen by
design, which method is comprised of the steps of generating by
design a plurality of specific nucleic acid building blocks having
serviceable mutually compatible ligatable ends and assembling these
nucleic acid building blocks, such that a designed overall assembly
order is achieved.
[0328] The mutually compatible ligatable ends of the nucleic acid
building blocks to be assembled are considered to be "serviceable"
for this type of ordered assembly if they enable the building
blocks to be coupled in predetermined orders. Thus, in one aspect,
the overall assembly order in which the nucleic acid building
blocks can be coupled is specified by the design of the ligatable
ends and, if more than one assembly step is to be used, then the
overall assembly order in which the nucleic acid building blocks
can be coupled is also specified by the sequential order of the
assembly step(s). In a one aspect of the invention, the annealed
building pieces are treated with an enzyme, such as a ligase (e.g.,
T4 DNA ligase) to achieve covalent bonding of the building
pieces.
[0329] In a another aspect, the design of nucleic acid building
blocks is obtained upon analysis of the sequences of a set of
progenitor nucleic acid templates that serve as a basis for
producing a progeny set of finalized chimeric nucleic acid
molecules. These progenitor nucleic acid templates thus serve as a
source of sequence information that aids in the design of the
nucleic acid building blocks that are to be mutagenized, i.e.
chimerized or shuffled.
[0330] In one exemplification, the invention provides for the
chimerization of a family of related genes and their encoded family
of related products. In a particular exemplification, the encoded
products are enzymes. The cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
present invention can be mutagenized in accordance with the methods
described herein.
[0331] Thus according to one aspect of the invention, the sequences
of a plurality of progenitor nucleic acid templates (e.g.,
polynucleotides of the invention) are aligned in order to select
one or more demarcation points, which demarcation points can be
located at an area of homology. The demarcation points can be used
to delineate the boundaries of nucleic acid building blocks to be
generated. Thus, the demarcation points identified and selected in
the progenitor molecules serve as potential chimerization points in
the assembly of the progeny molecules.
[0332] In one aspect, a serviceable demarcation point is an area of
homology (comprised of at least one homologous nucleotide base)
shared by at least two progenitor templates, but the demarcation
point can be an area of homology that is shared by at least half of
the progenitor templates, at least two thirds of the progenitor
templates, at least three fourths of the progenitor templates and
in one aspect at almost all of the progenitor templates. Even more
in one aspect still a serviceable demarcation point is an area of
homology that is shared by all of the progenitor templates.
[0333] In a one aspect, the gene reassembly process is performed
exhaustively in order to generate an exhaustive library. In other
words, all possible ordered combinations of the nucleic acid
building blocks are represented in the set of finalized chimeric
nucleic acid molecules. At the same time, the assembly order (i.e.
the order of assembly of each building block in the 5' to 3
sequence of each finalized chimeric nucleic acid) in each
combination is by design (or non-stochastic). Because of the
non-stochastic nature of the method, the possibility of unwanted
side products is greatly reduced.
[0334] In another aspect, the method provides that the gene
reassembly process is performed systematically, for example to
generate a systematically compartmentalized library, with
compartments that can be screened systematically, e.g., one by one.
In other words the invention provides that, through the selective
and judicious use of specific nucleic acid building blocks, coupled
with the selective and judicious use of sequentially stepped
assembly reactions, an experimental design can be achieved where
specific sets of progeny products are made in each of several
reaction vessels. This allows a systematic examination and
screening procedure to be performed. Thus, it allows a potentially
very large number of progeny molecules to be examined
systematically in smaller groups.
[0335] Because of its ability to perform chimerizations in a manner
that is highly flexible yet exhaustive and systematic as well,
particularly when there is a low level of homology among the
progenitor molecules, the instant invention provides for the
generation of a library (or set) comprised of a large number of
progeny molecules. Because of the non-stochastic nature of the
instant gene reassembly invention, the progeny molecules generated
in one aspect comprise a library of finalized chimeric nucleic acid
molecules having an overall assembly order that is chosen by
design. In a particularly aspect, such a generated library is
comprised of greater than 10.sup.3 to greater than 10.sup.1000
different progeny molecular species.
[0336] In one aspect, a set of finalized chimeric nucleic acid
molecules, produced as described is comprised of a polynucleotide
encoding a polypeptide. According to one aspect, this
polynucleotide is a gene, which may be a man-made gene. According
to another aspect, this polynucleotide is a gene pathway, which may
be a man-made gene pathway. The invention provides that one or more
man-made genes generated by the invention may be incorporated into
a man-made gene pathway, such as pathway operable in a eukaryotic
organism (including a plant).
[0337] In another exemplification, the synthetic nature of the step
in which the building blocks are generated allows the design and
introduction of nucleotides (e.g., one or more nucleotides, which
may be, for example, codons or introns or regulatory sequences)
that can later be optionally removed in an in vitro process (e.g.,
by mutagenesis) or in an in vivo process (e.g., by utilizing the
gene splicing ability of a host organism). It is appreciated that
in many instances the introduction of these nucleotides may also be
desirable for many other reasons in addition to the potential
benefit of creating a serviceable demarcation point.
[0338] Thus, according to another aspect, the invention provides
that a nucleic acid building block can be used to introduce an
intron. Thus, the invention provides that functional introns may be
introduced into a man-made gene of the invention. The invention
also provides that functional introns may be introduced into a
man-made gene pathway of the invention. Accordingly, the invention
provides for the generation of a chimeric polynucleotide that is a
man-made gene containing one (or more) artificially introduced
intron(s).
[0339] The invention also provides for the generation of a chimeric
polynucleotide that is a man-made gene pathway containing one (or
more) artificially introduced intron(s). In one aspect, the
artificially introduced intron(s) are functional in one or more
host cells for gene splicing much in the way that
naturally-occurring introns serve functionally in gene splicing.
The invention provides a process of producing man-made
intron-containing polynucleotides to be introduced into host
organisms for recombination and/or splicing.
[0340] A man-made gene produced using the invention can also serve
as a substrate for recombination with another nucleic acid.
Likewise, a man-made gene pathway produced using the invention can
also serve as a substrate for recombination with another nucleic
acid. In one aspect, the recombination is facilitated by, or occurs
at, areas of homology between the man-made, intron-containing gene
and a nucleic acid, which serves as a recombination partner. In one
aspect, the recombination partner may also be a nucleic acid
generated by the invention, including a man-made gene or a man-made
gene pathway. Recombination may be facilitated by or may occur at
areas of homology that exist at the one (or more) artificially
introduced intron(s) in the man-made gene.
[0341] In one aspect, the synthetic gene reassembly method of the
invention utilizes a plurality of nucleic acid building blocks,
each of which in one aspect has two ligatable ends. The two
ligatable ends on each nucleic acid building block may be two blunt
ends (i.e. each having an overhang of zero nucleotides), or in one
aspect one blunt end and one overhang, or more in one aspect still
two overhangs. In one aspect, a useful overhang for this purpose
may be a 3' overhang or a 5' overhang. Thus, a nucleic acid
building block may have a 3' overhang or alternatively a 5'
overhang or alternatively two 3' overhangs or alternatively two 5'
overhangs. The overall order in which the nucleic acid building
blocks are assembled to form a finalized chimeric nucleic acid
molecule is determined by purposeful experimental design and is not
random.
[0342] In one aspect, a nucleic acid building block is generated by
chemical synthesis of two single-stranded nucleic acids (also
referred to as single-stranded oligos) and contacting them so as to
allow them to anneal to form a double-stranded nucleic acid
building block. A double-stranded nucleic acid building block can
be of variable size. The sizes of these building blocks can be
small or large. Exemplary sizes for building block range from 1
base pair (not including any overhangs) to 100,000 base pairs (not
including any overhangs). Other exemplary size ranges are also
provided, which have lower limits of from 1 bp to 10,000 bp
(including every integer value in between) and upper limits of from
2 bp to 100,000 bp (including every integer value in between).
[0343] Many methods exist by which a double-stranded nucleic acid
building block can be generated that is serviceable for the
invention; and these are known in the art and can be readily
performed by the skilled artisan. According to one aspect, a
double-stranded nucleic acid building block is generated by first
generating two single stranded nucleic acids and allowing them to
anneal to form a double-stranded nucleic acid building block. The
two strands of a double-stranded nucleic acid building block may be
complementary at every nucleotide apart from any that form an
overhang; thus containing no mismatches, apart from any
overhang(s). According to another aspect, the two strands of a
double-stranded nucleic acid building block are complementary at
fewer than every nucleotide apart from any that form an overhang.
Thus, according to this aspect, a double-stranded nucleic acid
building block can be used to introduce codon degeneracy. In one
aspect the codon degeneracy is introduced using the site-saturation
mutagenesis described herein, using one or more N,N,G/T cassettes
or alternatively using one or more N,N,N cassettes.
[0344] The in vivo recombination method of the invention can be
performed blindly on a pool of unknown hybrids or alleles of a
specific polynucleotide or sequence. However, it is not necessary
to know the actual DNA or RNA sequence of the specific
polynucleotide. The approach of using recombination within a mixed
population of genes can be useful for the generation of any useful
proteins, for example, a cellulase of the invention or a variant
thereof. This approach may be used to generate proteins having
altered specificity or activity. The approach may also be useful
for the generation of hybrid nucleic acid sequences, for example,
promoter regions, introns, exons, enhancer sequences, 31
untranslated regions or 51 untranslated regions of genes. Thus this
approach may be used to generate genes having increased rates of
expression. This approach may also be useful in the study of
repetitive DNA sequences. Finally, this approach may be useful to
make ribozymes or aptamers of the invention.
[0345] In one aspect the invention described herein is directed to
the use of repeated cycles of reductive reassortment, recombination
and selection which allow for the directed molecular evolution of
highly complex linear sequences, such as DNA, RNA or proteins
thorough recombination.
[0346] Optimized Directed Evolution System
[0347] The invention provides a non-stochastic gene modification
system termed "optimized directed evolution system" to generate
polypeptides, e.g., cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes or
antibodies of the invention, with new or altered properties. In one
aspect, optimized directed evolution is directed to the use of
repeated cycles of reductive reassortment, recombination and
selection that allow for the directed molecular evolution of
nucleic acids through recombination.
[0348] Optimized directed evolution allows generation of a large
population of evolved chimeric sequences, wherein the generated
population is significantly enriched for sequences that have a
predetermined number of crossover events. A crossover event is a
point in a chimeric sequence where a shift in sequence occurs from
one parental variant to another parental variant. Such a point is
normally at the juncture of where oligonucleotides from two parents
are ligated together to form a single sequence. This method allows
calculation of the correct concentrations of oligonucleotide
sequences so that the final chimeric population of sequences is
enriched for the chosen number of crossover events. This provides
more control over choosing chimeric variants having a predetermined
number of crossover events.
[0349] In addition, this method provides a convenient means for
exploring a tremendous amount of the possible protein variant space
in comparison to other systems. Previously, if one generated, for
example, 10.sup.13 chimeric molecules during a reaction, it would
be extremely difficult to test such a high number of chimeric
variants for a particular activity. Moreover, a significant portion
of the progeny population would have a very high number of
crossover events which resulted in proteins that were less likely
to have increased levels of a particular activity. By using these
methods, the population of chimerics molecules can be enriched for
those variants that have a particular number of crossover events.
Thus, although one can still generate 10.sup.13 chimeric molecules
during a reaction, each of the molecules chosen for further
analysis most likely has, for example, only three crossover events.
Because the resulting progeny population can be skewed to have a
predetermined number of crossover events, the boundaries on the
functional variety between the chimeric molecules is reduced. This
provides a more manageable number of variables when calculating
which oligonucleotide from the original parental polynucleotides
might be responsible for affecting a particular trait.
[0350] One method for creating a chimeric progeny polynucleotide
sequence is to create oligonucleotides corresponding to fragments
or portions of each parental sequence. Each oligonucleotide in one
aspect includes a unique region of overlap so that mixing the
oligonucleotides together results in a new variant that has each
oligonucleotide fragment assembled in the correct order.
Alternatively protocols for practicing these methods of the
invention can be found in U.S. Pat. Nos. 6,773,900; 6,740,506;
6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.
[0351] The number of oligonucleotides generated for each parental
variant bears a relationship to the total number of resulting
crossovers in the chimeric molecule that is ultimately created. For
example, three parental nucleotide sequence variants might be
provided to undergo a ligation reaction in order to find a chimeric
variant having, for example, greater activity at high temperature.
As one example, a set of 50 oligonucleotide sequences can be
generated corresponding to each portions of each parental variant.
Accordingly, during the ligation reassembly process there could be
up to 50 crossover events within each of the chimeric sequences.
The probability that each of the generated chimeric polynucleotides
will contain oligonucleotides from each parental variant in
alternating order is very low. If each oligonucleotide fragment is
present in the ligation reaction in the same molar quantity it is
likely that in some positions oligonucleotides from the same
parental polynucleotide will ligate next to one another and thus
not result in a crossover event. If the concentration of each
oligonucleotide from each parent is kept constant during any
ligation step in this example, there is a 1/3 chance (assuming 3
parents) that an oligonucleotide from the same parental variant
will ligate within the chimeric sequence and produce no
crossover.
[0352] Accordingly, a probability density function (PDF) can be
determined to predict the population of crossover events that are
likely to occur during each step in a ligation reaction given a set
number of parental variants, a number of oligonucleotides
corresponding to each variant, and the concentrations of each
variant during each step in the ligation reaction. The statistics
and mathematics behind determining the PDF is described below. By
utilizing these methods, one can calculate such a probability
density function, and thus enrich the chimeric progeny population
for a predetermined number of crossover events resulting from a
particular ligation reaction. Moreover, a target number of
crossover events can be predetermined, and the system then
programmed to calculate the starting quantities of each parental
oligonucleotide during each step in the ligation reaction to result
in a probability density function that centers on the predetermined
number of crossover events. These methods are directed to the use
of repeated cycles of reductive reassortment, recombination and
selection that allow for the directed molecular evolution of a
nucleic acid encoding a polypeptide through recombination. This
system allows generation of a large population of evolved chimeric
sequences, wherein the generated population is significantly
enriched for sequences that have a predetermined number of
crossover events. A crossover event is a point in a chimeric
sequence where a shift in sequence occurs from one parental variant
to another parental variant. Such a point is normally at the
juncture of where oligonucleotides from two parents are ligated
together to form a single sequence. The method allows calculation
of the correct concentrations of oligonucleotide sequences so that
the final chimeric population of sequences is enriched for the
chosen number of crossover events. This provides more control over
choosing chimeric variants having a predetermined number of
crossover events.
[0353] In addition, these methods provide a convenient means for
exploring a tremendous amount of the possible protein variant space
in comparison to other systems. By using the methods described
herein, the population of chimerics molecules can be enriched for
those variants that have a particular number of crossover events.
Thus, although one can still generate 10.sup.13 chimeric molecules
during a reaction, each of the molecules chosen for further
analysis most likely has, for example, only three crossover events.
Because the resulting progeny population can be skewed to have a
predetermined number of crossover events, the boundaries on the
functional variety between the chimeric molecules is reduced. This
provides a more manageable number of variables when calculating
which oligonucleotide from the original parental polynucleotides
might be responsible for affecting a particular trait.
[0354] In one aspect, the method creates a chimeric progeny
polynucleotide sequence by creating oligonucleotides corresponding
to fragments or portions of each parental sequence. Each
oligonucleotide in one aspect includes a unique region of overlap
so that mixing the oligonucleotides together results in a new
variant that has each oligonucleotide fragment assembled in the
correct order. See also U.S. Pat. Nos. 6,773,900; 6,740,506;
6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.
[0355] Determining Crossover Events
[0356] Aspects of the invention include a system and software that
receive a desired crossover probability density function (PDF), the
number of parent genes to be reassembled, and the number of
fragments in the reassembly as inputs. The output of this program
is a "fragment PDF" that can be used to determine a recipe for
producing reassembled genes, and the estimated crossover PDF of
those genes. The processing described herein is in one aspect
performed in MATLAB.TM. (The Mathworks, Natick, Mass.) a
programming language and development environment for technical
computing.
[0357] Iterative Processes
[0358] Any process of the invention can be iteratively repeated,
e.g., a nucleic acid encoding an altered or new cellulase
phenotype, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme of the invention, can be identified,
re-isolated, again modified, re-tested for activity. This process
can be iteratively repeated until a desired phenotype is
engineered. For example, an entire biochemical anabolic or
catabolic pathway can be engineered into a cell, including, e.g.,
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity.
[0359] Similarly, if it is determined that a particular
oligonucleotide has no affect at all on the desired trait (e.g., a
new cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme phenotype), it can be removed as a
variable by synthesizing larger parental oligonucleotides that
include the sequence to be removed. Since incorporating the
sequence within a larger sequence prevents any crossover events,
there will no longer be any variation of this sequence in the
progeny polynucleotides. This iterative practice of determining
which oligonucleotides are most related to the desired trait, and
which are unrelated, allows more efficient exploration all of the
possible protein variants that might be provide a particular trait
or activity.
[0360] In Vivo Shuffling
[0361] In various aspects, in vivo shuffling of molecules is used
in methods of the invention to provide variants of polypeptides of
the invention, e.g., antibodies of the invention or cellulases of
the invention, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzymes, and the like. In vivo shuffling
can be performed utilizing the natural property of cells to
recombine multimers. While recombination in vivo has provided the
major natural route to molecular diversity, genetic recombination
remains a relatively complex process that involves 1) the
recognition of homologies; 2) strand cleavage, strand invasion, and
metabolic steps leading to the production of recombinant chiasma;
and finally 3) the resolution of chiasma into discrete recombined
molecules. The formation of the chiasma requires the recognition of
homologous sequences.
[0362] In another aspect, the invention includes a method for
producing a hybrid polynucleotide from at least a first
polynucleotide and a second polynucleotide. The invention can be
used to produce a hybrid polynucleotide by introducing at least a
first polynucleotide and a second polynucleotide (e.g., one, or
both, being an exemplary cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme-encoding sequence of the invention) which share at least one
region of partial sequence homology into a suitable host cell. The
regions of partial sequence homology promote processes which result
in sequence reorganization producing a hybrid polynucleotide. The
term "hybrid polynucleotide", as used herein, is any nucleotide
sequence which results from the method of the present invention and
contains sequence from at least two original polynucleotide
sequences. Such hybrid polynucleotides can result from
intermolecular recombination events which promote sequence
integration between DNA molecules. In addition, such hybrid
polynucleotides can result from intramolecular reductive
reassortment processes which utilize repeated sequences to alter a
nucleotide sequence within a DNA molecule.
[0363] In one aspect, vivo reassortment focuses on
"inter-molecular" processes collectively referred to as
"recombination"; which in bacteria, is generally viewed as a
"RecA-dependent" phenomenon. The invention can rely on
recombination processes of a host cell to recombine and re-assort
sequences, or the cells' ability to mediate reductive processes to
decrease the complexity of quasi-repeated sequences in the cell by
deletion. This process of "reductive reassortment" occurs by an
"intra-molecular", RecA-independent process.
[0364] In another aspect of the invention, novel polynucleotides
can be generated by the process of reductive reassortment. The
method involves the generation of constructs containing consecutive
sequences (original encoding sequences), their insertion into an
appropriate vector and their subsequent introduction into an
appropriate host cell. The reassortment of the individual molecular
identities occurs by combinatorial processes between the
consecutive sequences in the construct possessing regions of
homology, or between quasi-repeated units. The reassortment process
recombines and/or reduces the complexity and extent of the repeated
sequences and results in the production of novel molecular species.
Various treatments may be applied to enhance the rate of
reassortment. These could include treatment with ultra-violet
light, or DNA damaging chemicals and/or the use of host cell lines
displaying enhanced levels of "genetic instability". Thus the
reassortment process may involve homologous recombination or the
natural property of quasi-repeated sequences to direct their own
evolution.
[0365] Repeated or "quasi-repeated" sequences play a role in
genetic instability. In one aspect, "quasi-repeats" are repeats
that are not restricted to their original unit structure.
Quasi-repeated units can be presented as an array of sequences in a
construct; consecutive units of similar sequences. Once ligated,
the junctions between the consecutive sequences become essentially
invisible and the quasi-repetitive nature of the resulting
construct is now continuous at the molecular level. The deletion
process the cell performs to reduce the complexity of the resulting
construct operates between the quasi-repeated sequences. The
quasi-repeated units provide a practically limitless repertoire of
templates upon which slippage events can occur. In one aspect, the
constructs containing the quasi-repeats thus effectively provide
sufficient molecular elasticity that deletion (and potentially
insertion) events can occur virtually anywhere within the
quasi-repetitive units.
[0366] When the quasi-repeated sequences are all ligated in the
same orientation, for instance head to tail or vice versa, the cell
cannot distinguish individual units. Consequently, the reductive
process can occur throughout the sequences. In contrast, when for
example, the units are presented head to head, rather than head to
tail, the inversion delineates the endpoints of the adjacent unit
so that deletion formation will favor the loss of discrete units.
Thus, it is preferable with the present method that the sequences
are in the same orientation. Random orientation of quasi-repeated
sequences will result in the loss of reassortment efficiency, while
consistent orientation of the sequences will offer the highest
efficiency. However, while having fewer of the contiguous sequences
in the same orientation decreases the efficiency, it may still
provide sufficient elasticity for the effective recovery of novel
molecules. Constructs can be made with the quasi-repeated sequences
in the same orientation to allow higher efficiency.
[0367] Sequences can be assembled in a head to tail orientation
using any of a variety of methods, including the following: [0368]
a) Primers that include a poly-A head and poly-T tail which when
made single-stranded would provide orientation can be utilized.
This is accomplished by having the first few bases of the primers
made from RNA and hence easily removed RNaseH. [0369] b) Primers
that include unique restriction cleavage sites can be utilized.
Multiple sites, a battery of unique sequences and repeated
synthesis and ligation steps would be required. [0370] c) The inner
few bases of the primer could be thiolated and an exonuclease used
to produce properly tailed molecules.
[0371] In one aspect, the recovery of the re-assorted sequences
relies on the identification of cloning vectors with a reduced
repetitive index (RI). The re-assorted encoding sequences can then
be recovered by amplification. The products are re-cloned and
expressed. The recovery of cloning vectors with reduced RI can be
affected by: [0372] 1) The use of vectors only stably maintained
when the construct is reduced in complexity. [0373] 2) The physical
recovery of shortened vectors by physical procedures. In this case,
the cloning vector would be recovered using standard plasmid
isolation procedures and size fractionated on either an agarose
gel, or column with a low molecular weight cut off utilizing
standard procedures. [0374] 3) The recovery of vectors containing
interrupted genes which can be selected when insert size decreases.
[0375] 4) The use of direct selection techniques with an expression
vector and the appropriate selection.
[0376] Encoding sequences (for example, genes) from related
organisms may demonstrate a high degree of homology and encode
quite diverse protein products. These types of sequences are
particularly useful in the present invention as quasi-repeats.
However, while the examples illustrated below demonstrate the
reassortment of nearly identical original encoding sequences
(quasi-repeats), this process is not limited to such nearly
identical repeats.
[0377] The following example demonstrates an exemplary method of
the invention. Encoding nucleic acid sequences (quasi-repeats)
derived from three (3) unique species are described. Each sequence
encodes a protein with a distinct set of properties. Each of the
sequences differs by a single or a few base pairs at a unique
position in the sequence. The quasi-repeated sequences are
separately or collectively amplified and ligated into random
assemblies such that all possible permutations and combinations are
available in the population of ligated molecules. The number of
quasi-repeat units can be controlled by the assembly conditions.
The average number of quasi-repeated units in a construct is
defined as the repetitive index (RI).
[0378] Once formed, the constructs may, or may not be size
fractionated on an agarose gel according to published protocols,
inserted into a cloning vector and transfected into an appropriate
host cell. The cells are then propagated and "reductive
reassortment" is effected. The rate of the reductive reassortment
process may be stimulated by the introduction of DNA damage if
desired. Whether the reduction in RI is mediated by deletion
formation between repeated sequences by an "intra-molecular"
mechanism, or mediated by recombination-like events through
"inter-molecular" mechanisms is immaterial. The end result is a
reassortment of the molecules into all possible combinations.
[0379] Optionally, the method comprises the additional step of
screening the library members of the shuffled pool to identify
individual shuffled library members having the ability to bind or
otherwise interact, or catalyze a particular reaction (e.g., such
as catalytic domain of an enzyme) with a predetermined
macromolecule, such as for example a proteinaceous receptor, an
oligosaccharide, virion, or other predetermined compound or
structure.
[0380] The polypeptides that are identified from such libraries can
be used for therapeutic, diagnostic, research and related purposes
(e.g., catalysts, solutes for increasing osmolarity of an aqueous
solution and the like) and/or can be subjected to one or more
additional cycles of shuffling and/or selection.
[0381] In another aspect, it is envisioned that prior to or during
recombination or reassortment, polynucleotides generated by the
method of the invention can be subjected to agents or processes
which promote the introduction of mutations into the original
polynucleotides. The introduction of such mutations would increase
the diversity of resulting hybrid polynucleotides and polypeptides
encoded therefrom. The agents or processes which promote
mutagenesis can include, but are not limited to: (+)--CC-1065, or a
synthetic analog such as (+)--CC-1065-(N3-Adenine (See Sun and
Hurley, (1992); an N-acetylated or deacetylated
4'-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA
synthesis (See, for example, van de Poll et al. (1992)); or a
N-acetylated or deacetylated 4-aminobiphenyl adduct capable of
inhibiting DNA synthesis (See also, van de Poll et al. (1992), pp.
751-758); trivalent chromium, a trivalent chromium salt, a
polycyclic aromatic hydrocarbon (PAH) DNA adduct capable of
inhibiting DNA replication, such as 7-bromomethyl-benz[a]anthracene
("BMA"), tris(2,3-dibromopropyl)phosphate ("Tris-BP"),
1,2-dibromo-3-chloropropane ("DBCP"), 2-bromoacrolein (2BA),
benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide ("BPDE"), a
platinum(II) halogen salt,
N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline ("N-hydroxy-IQ")
and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-j]-pyridine
("N-hydroxy-PhIP"). Exemplary means for slowing or halting PCR
amplification consist of UV light (+)--CC-1065 and
(+)--CC-1065-(N3-Adenine). Particularly encompassed means are DNA
adducts or polynucleotides comprising the DNA adducts from the
polynucleotides or polynucleotides pool, which can be released or
removed by a process including heating the solution comprising the
polynucleotides prior to further processing.
[0382] In another aspect the invention is directed to a method of
producing recombinant proteins having biological activity by
treating a sample comprising double-stranded template
polynucleotides encoding a wild-type protein under conditions
according to the invention which provide for the production of
hybrid or re-assorted polynucleotides.
[0383] Producing Sequence Variants
[0384] The invention also provides additional methods for making
sequence variants of the nucleic acid (e.g., cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme) sequences of the invention. The invention also provides
additional methods for isolating cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes using
the nucleic acids and polypeptides of the invention. In one aspect,
the invention provides for variants of a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme coding sequence (e.g., a gene, cDNA or message) of the
invention, which can be altered by any means, including, e.g.,
random or stochastic methods, or, non-stochastic, or "directed
evolution," methods, as described above.
[0385] The isolated variants may be naturally occurring. Variant
can also be created in vitro. Variants may be created using genetic
engineering techniques such as site directed mutagenesis, random
chemical mutagenesis, Exonuclease III deletion procedures, and
standard cloning techniques. Alternatively, such variants,
fragments, analogs, or derivatives may be created using chemical
synthesis or modification procedures. Other methods of making
variants are also familiar to those skilled in the art. These
include procedures in which nucleic acid sequences obtained from
natural isolates are modified to generate nucleic acids which
encode polypeptides having characteristics which enhance their
value in industrial or laboratory applications. In such procedures,
a large number of variant sequences having one or more nucleotide
differences with respect to the sequence obtained from the natural
isolate are generated and characterized. These nucleotide
differences can result in amino acid changes with respect to the
polypeptides encoded by the nucleic acids from the natural
isolates.
[0386] For example, variants may be created using error prone PCR.
In one aspect of error prone PCR, the PCR is performed under
conditions where the copying fidelity of the DNA polymerase is low,
such that a high rate of point mutations is obtained along the
entire length of the PCR product. Error prone PCR is described,
e.g., in Leung (1989) Technique 1:11-15) and Caldwell (1992) PCR
Methods Applic. 2:28-33. Briefly, in such procedures, nucleic acids
to be mutagenized are mixed with PCR primers, reaction buffer,
MgCl.sub.2, MnCl.sub.2, Taq polymerase and an appropriate
concentration of dNTPs for achieving a high rate of point mutation
along the entire length of the PCR product. For example, the
reaction may be performed using 20 (moles of nucleic acid to be
mutagenized, 30 pmole of each PCR primer, a reaction buffer
comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7
mM MgCl.sub.2, 0.5 mM MnCl.sub.2, 5 units of Taq polymerase, 0.2 mM
dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR may be performed
for 30 cycles of 94.degree. C. for 1 min, 45.degree. C. for 1 min,
and 72.degree. C. for 1 min. However, it will be appreciated that
these parameters may be varied as appropriate. The mutagenized
nucleic acids are cloned into an appropriate vector and the
activities of the polypeptides encoded by the mutagenized nucleic
acids are evaluated.
[0387] In one aspect, variants are created using oligonucleotide
directed mutagenesis to generate site-specific mutations in any
cloned DNA of interest. Oligonucleotide mutagenesis is described,
e.g., in Reidhaar-Olson (1988) Science 241:53-57. Briefly, in such
procedures a plurality of double stranded oligonucleotides bearing
one or more mutations to be introduced into the cloned DNA are
synthesized and inserted into the cloned DNA to be mutagenized. In
one aspect, clones containing the mutagenized DNA are recovered,
expressed, and the activities of the polypeptide encoded therein
assessed.
[0388] Another method for generating variants is assembly PCR.
Assembly PCR involves the assembly of a PCR product from a mixture
of small DNA fragments. A large number of different PCR reactions
occur in parallel in the same vial, with the products of one
reaction priming the products of another reaction. Assembly PCR is
described in, e.g., U.S. Pat. No. 5,965,408.
[0389] In one aspect, sexual PCR mutagenesis is an exemplary method
of generating variants of the invention. In one aspect of sexual
PCR mutagenesis forced homologous recombination occurs between DNA
molecules of different but highly related DNA sequence in vitro, as
a result of random fragmentation of the DNA molecule based on
sequence homology, followed by fixation of the crossover by primer
extension in a PCR reaction. Sexual PCR mutagenesis is described,
e.g., in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751.
Briefly, in such procedures a plurality of nucleic acids to be
recombined are digested with DNase to generate fragments having an
average size of 50-200 nucleotides. Fragments of the desired
average size are purified and resuspended in a PCR mixture. PCR is
conducted under conditions which facilitate recombination between
the nucleic acid fragments. For example, PCR may be performed by
resuspending the purified fragments at a concentration of 10-30
ng/.mu.l in a solution of 0.2 mM of each dNTP, 2.2 mM MgCl.sub.2,
50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5 units
of Taq polymerase per 100:1 of reaction mixture is added and PCR is
performed using the following regime: 94.degree. C. for 60 seconds,
94.degree. C. for 30 seconds, 50-55.degree. C. for 30 seconds,
72.degree. C. for 30 seconds (30-45 times) and 72.degree. C. for 5
minutes. However, it will be appreciated that these parameters may
be varied as appropriate. In some aspects, oligonucleotides may be
included in the PCR reactions. In other aspects, the Klenow
fragment of DNA polymerase I may be used in a first set of PCR
reactions and Taq polymerase may be used in a subsequent set of PCR
reactions. Recombinant sequences are isolated and the activities of
the polypeptides they encode are assessed.
[0390] In one aspect, variants are created by in vivo mutagenesis.
In some aspects, random mutations in a sequence of interest are
generated by propagating the sequence of interest in a bacterial
strain, such as an E. coli strain, which carries mutations in one
or more of the DNA repair pathways. Such "mutator" strains have a
higher random mutation rate than that of a wild-type parent.
Propagating the DNA in one of these strains will eventually
generate random mutations within the DNA. Mutator strains suitable
for use for in vivo mutagenesis are described in PCT Publication
No. WO 91/16427, published Oct. 31, 1991, entitled "Methods for
Phenotype Creation from Multiple Gene Populations".
[0391] Variants may also be generated using cassette mutagenesis.
In cassette mutagenesis a small region of a double stranded DNA
molecule is replaced with a synthetic oligonucleotide "cassette"
that differs from the native sequence. The oligonucleotide often
contains completely and/or partially randomized native
sequence.
[0392] Recursive ensemble mutagenesis may also be used to generate
variants. Recursive ensemble mutagenesis is an algorithm for
protein engineering (protein mutagenesis) developed to produce
diverse populations of phenotypically related mutants whose members
differ in amino acid sequence. This method uses a feedback
mechanism to control successive rounds of combinatorial cassette
mutagenesis. Recursive ensemble mutagenesis is described, e.g., in
Arkin (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815.
[0393] In some aspects, variants are created using exponential
ensemble mutagenesis. Exponential ensemble mutagenesis is a process
for generating combinatorial libraries with a high percentage of
unique and functional mutants, wherein small groups of residues are
randomized in parallel to identify, at each altered position, amino
acids which lead to functional proteins. Exponential ensemble
mutagenesis is described, e.g., in Delegrave (1993) Biotechnology
Res. 11:1548-1552. Random and site-directed mutagenesis are
described, e.g., in Arnold (1993) Current Opinion in Biotechnology
4:450-455.
[0394] In some aspects, the variants are created using shuffling
procedures wherein portions of a plurality of nucleic acids which
encode distinct polypeptides are fused together to create chimeric
nucleic acid sequences which encode chimeric polypeptides as
described in U.S. Pat. No. 5,965,408, filed Jul. 9, 1996, entitled,
"Method of DNA Reassembly by Interrupting Synthesis" and U.S. Pat.
No. 5,939,250, filed May 22, 1996, entitled, "Production of Enzymes
Having Desired Activities by Mutagenesis.
[0395] The variants of the polypeptides of the invention may be
variants in which one or more of the amino acid residues of the
polypeptides of the sequences of the invention are substituted with
a conserved or non-conserved amino acid residue (in one aspect a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code.
[0396] In one aspect, conservative substitutions are those that
substitute a given amino acid in a polypeptide by another amino
acid of like characteristics. In one aspect, conservative
substitutions of the invention comprise the following replacements:
replacements of an aliphatic amino acid such as Alanine, Valine,
Leucine and Isoleucine with another aliphatic amino acid;
replacement of a Serine with a Threonine or vice versa; replacement
of an acidic residue such as Aspartic acid and Glutamic acid with
another acidic residue; replacement of a residue bearing an amide
group, such as Asparagine and Glutamine, with another residue
bearing an amide group; exchange of a basic residue such as Lysine
and Arginine with another basic residue; and replacement of an
aromatic residue such as Phenylalanine, Tyrosine with another
aromatic residue.
[0397] Other variants are those in which one or more of the amino
acid residues of a polypeptide of the invention includes a
substituent group. In one aspect, other variants are those in which
the polypeptide is associated with another compound, such as a
compound to increase the half-life of the polypeptide (for example,
polyethylene glycol). Additional variants are those in which
additional amino acids are fused to the polypeptide, such as a
leader sequence, a secretory sequence, a proprotein sequence or a
sequence which facilitates purification, enrichment, or
stabilization of the polypeptide.
[0398] In some aspects, the fragments, derivatives and analogs
retain the same biological function or activity as the polypeptides
of the invention. In other aspects, the fragment, derivative, or
analog includes a proprotein, such that the fragment, derivative,
or analog can be activated by cleavage of the proprotein portion to
produce an active polypeptide.
[0399] Optimizing Codons to Achieve High Levels of Protein
Expression in Host Cells
[0400] The invention provides methods for modifying cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase, enzyme-encoding nucleic acids to modify (e.g.,
optimize) codon usage. In one aspect, the invention provides
methods for modifying codons in a nucleic acid encoding a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme to increase or decrease its expression in a
host cell. The invention also provides nucleic acids encoding a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme modified to increase its expression in a
host cell, cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme so modified, and methods
of making the modified cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes. The
method comprises identifying a "non-preferred" or a "less
preferred" codon in cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase,
enzyme-encoding nucleic acid and replacing one or more of these
non-preferred or less preferred codons with a "preferred codon"
encoding the same amino acid as the replaced codon and at least one
non-preferred or less preferred codon in the nucleic acid has been
replaced by a preferred codon encoding the same amino acid. A
preferred codon is a codon over-represented in coding sequences in
genes in the host cell and a non-preferred or less preferred codon
is a codon under-represented in coding sequences in genes in the
host cell.
[0401] Host cells for expressing the nucleic acids, expression
cassettes and vectors of the invention include bacteria, yeast,
fungi, plant cells, insect cells and mammalian cells (see
discussion, above). Thus, the invention provides methods for
optimizing codon usage in all of these cells, codon-altered nucleic
acids and polypeptides made by the codon-altered nucleic acids.
Exemplary host cells include gram negative bacteria, such as
Escherichia coli; gram positive bacteria, such as Streptomyces sp.,
Lactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris,
Bacillus subtilis, Bacillus cereus. Exemplary host cells also
include eukaryotic organisms, e.g., various yeast, such as
Saccharomyces sp., including Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces
lactis, Hansenula polymorpha, Aspergillus niger, and mammalian
cells and cell lines and insect cells and cell lines. Thus, the
invention also includes nucleic acids and polypeptides optimized
for expression in these organisms and species.
[0402] For example, the codons of a nucleic acid encoding a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme isolated from a bacterial cell are modified
such that the nucleic acid is optimally expressed in a bacterial
cell different from the bacteria from which the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme was derived, a yeast, a fungi, a plant cell, an insect cell
or a mammalian cell. Methods for optimizing codons are well known
in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.
Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;
Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001)
Infect. Immun. 69:7250-7253, describing optimizing codons in mouse
systems; Outchkourov (2002) Protein Expr. Purif. 24:18-24,
describing optimizing codons in yeast; Feng (2000) Biochemistry
39:15399-15409, describing optimizing codons in E. coli; Humphreys
(2000) Protein Expr. Purif. 20:252-264, describing optimizing codon
usage that affects secretion in E. coli.
Transgenic Non-Human Animals
[0403] The invention provides transgenic non-human animals
comprising a nucleic acid, a polypeptide (e.g., a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme), an expression cassette or vector or a transfected or
transformed cell of the invention. The invention also provides
methods of making and using these transgenic non-human animals.
[0404] The transgenic non-human animals can be, e.g., dogs, goats,
rabbits, sheep, pigs (including all swine, hogs and related
animals), cows, rats and mice, comprising the nucleic acids of the
invention. These animals can be used, e.g., as in vivo models to
study cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme activity, or, as models to screen
for agents that change the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity in vivo. The coding sequences for the polypeptides to be
expressed in the transgenic non-human animals can be designed to be
constitutive, or, under the control of tissue-specific,
developmental-specific or inducible transcriptional regulatory
factors.
[0405] Transgenic non-human animals can be designed and generated
using any method known in the art; see, e.g., U.S. Pat. Nos.
6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166; 6,107,541;
5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698; 5,639,940;
5,573,933; 5,387,742; 5,087,571, describing making and using
transformed cells and eggs and transgenic mice, rats, rabbits,
sheep, pigs and cows. See also, e.g., Pollock (1999) J. Immunol.
Methods 231:147-157, describing the production of recombinant
proteins in the milk of transgenic dairy animals; Baguisi (1999)
Nat. Biotechnol. 17:456-461, demonstrating the production of
transgenic goats. U.S. Pat. No. 6,211,428, describes making and
using transgenic non-human mammals which express in their brains a
nucleic acid construct comprising a DNA sequence. U.S. Pat. No.
5,387,742, describes injecting cloned recombinant or synthetic DNA
sequences into fertilized mouse eggs, implanting the injected eggs
in pseudo-pregnant females, and growing to term transgenic mice.
U.S. Pat. No. 6,187,992, describes making and using a transgenic
mouse.
[0406] "Knockout animals" can also be used to practice the methods
of the invention. For example, in one aspect, the transgenic or
modified animals of the invention comprise a "knockout animal,"
e.g., a "knockout mouse," engineered not to express an endogenous
gene, which is replaced with a gene expressing a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme of the invention, or, a fusion protein comprising a
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme of the invention.
Transgenic Plants and Seeds
[0407] The invention provides transgenic plants and seeds
comprising a nucleic acid, a polypeptide (e.g., a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme), an expression cassette or vector or a transfected or
transformed cell of the invention. The invention also provides
plant products, e.g., oils, seeds, leaves, extracts and the like,
comprising a nucleic acid and/or a polypeptide (e.g., a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme) of the invention. The transgenic plant can
be dicotyledonous (a dicot) or monocotyledonous (a monocot). The
invention also provides methods of making and using these
transgenic plants and seeds. The transgenic plant or plant cell
expressing a polypeptide of the present invention may be
constructed in accordance with any method known in the art. See,
for example, U.S. Pat. No. 6,309,872.
[0408] Nucleic acids and expression constructs of the invention can
be introduced into a plant cell by any means. For example, nucleic
acids or expression constructs can be introduced into the genome of
a desired plant host, or, the nucleic acids or expression
constructs can be episomes. Introduction into the genome of a
desired plant can be such that the host's cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme production is regulated by endogenous transcriptional or
translational control elements. The invention also provides
"knockout plants" where insertion of gene sequence by, e.g.,
homologous recombination, has disrupted the expression of the
endogenous gene. Means to generate "knockout" plants are well-known
in the art, see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA
95:4368-4373; Miao (1995) Plant J 7:359-365. See discussion on
transgenic plants, below.
[0409] The nucleic acids of the invention can be used to confer
desired traits on essentially any plant, e.g., on starch-producing
plants, such as potato, tomato, soybean, beets, corn, wheat, rice,
barley, and the like. Nucleic acids of the invention can be used to
manipulate metabolic pathways of a plant in order to optimize or
alter host's expression of cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme. The
can change cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme activity in a plant.
Alternatively, a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme of the invention can be
used in production of a transgenic plant to produce a compound not
naturally produced by that plant. This can lower production costs
or create a novel product.
[0410] In one aspect, the first step in production of a transgenic
plant involves making an expression construct for expression in a
plant cell. These techniques are well known in the art. They can
include selecting and cloning a promoter, a coding sequence for
facilitating efficient binding of ribosomes to mRNA and selecting
the appropriate gene terminator sequences. One exemplary
constitutive promoter is CaMV35S, from the cauliflower mosaic
virus, which generally results in a high degree of expression in
plants. Other promoters are more specific and respond to cues in
the plant's internal or external environment. An exemplary
light-inducible promoter is the promoter from the cab gene,
encoding the major chlorophyll a/b binding protein.
[0411] In one aspect, the nucleic acid is modified to achieve
greater expression in a plant cell. For example, a sequence of the
invention is likely to have a higher percentage of A-T nucleotide
pairs compared to that seen in a plant, some of which prefer G-C
nucleotide pairs. Therefore, A-T nucleotides in the coding sequence
can be substituted with G-C nucleotides without significantly
changing the amino acid sequence to enhance production of the gene
product in plant cells.
[0412] Selectable marker gene can be added to the gene construct in
order to identify plant cells or tissues that have successfully
integrated the transgene. This may be necessary because achieving
incorporation and expression of genes in plant cells is a rare
event, occurring in just a few percent of the targeted tissues or
cells. Selectable marker genes encode proteins that provide
resistance to agents that are normally toxic to plants, such as
antibiotics or herbicides. Only plant cells that have integrated
the selectable marker gene will survive when grown on a medium
containing the appropriate antibiotic or herbicide. As for other
inserted genes, marker genes also require promoter and termination
sequences for proper function.
[0413] In one aspect, making transgenic plants or seeds comprises
incorporating sequences of the invention and, optionally, marker
genes into a target expression construct (e.g., a plasmid), along
with positioning of the promoter and the terminator sequences. This
can involve transferring the modified gene into the plant through a
suitable method. For example, a construct may be introduced
directly into the genomic DNA of the plant cell using techniques
such as electroporation and microinjection of plant cell
protoplasts, or the constructs can be introduced directly to plant
tissue using ballistic methods, such as DNA particle bombardment.
For example, see, e.g., Christou (1997) Plant Mol. Biol.
35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987)
Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69,
discussing use of particle bombardment to introduce transgenes into
wheat; and Adam (1997) supra, for use of particle bombardment to
introduce YACs into plant cells. For example, Rinehart (1997)
supra, used particle bombardment to generate transgenic cotton
plants. Apparatus for accelerating particles is described U.S. Pat.
No. 5,015,580; and, the commercially available BioRad (Biolistics)
PDS-2000 particle acceleration instrument; see also, John, U.S.
Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describing
particle-mediated transformation of gymnosperms.
[0414] In one aspect, protoplasts can be immobilized and injected
with a nucleic acids, e.g., an expression construct. Although plant
regeneration from protoplasts is not easy with cereals, plant
regeneration is possible in legumes using somatic embryogenesis
from protoplast derived callus. Organized tissues can be
transformed with naked DNA using gene gun technique, where DNA is
coated on tungsten microprojectiles, shot 1/100th the size of
cells, which carry the DNA deep into cells and organelles.
Transformed tissue is then induced to regenerate, usually by
somatic embryogenesis. This technique has been successful in
several cereal species including maize and rice.
[0415] Nucleic acids, e.g., expression constructs, can also be
introduced in to plant cells using recombinant viruses. Plant cells
can be transformed using viral vectors, such as, e.g., tobacco
mosaic virus derived vectors (Rouwendal (1997) Plant Mol. Biol.
33:989-999), see Porta (1996) "Use of viral replicons for the
expression of genes in plants," Mol. Biotechnol. 5:209-221.
[0416] Alternatively, nucleic acids, e.g., an expression construct,
can be combined with suitable T-DNA flanking regions and introduced
into a conventional Agrobacterium tumefaciens host vector. The
virulence functions of the Agrobacterium tumefaciens host will
direct the insertion of the construct and adjacent marker into the
plant cell DNA when the cell is infected by the bacteria.
Agrobacterium tumefaciens-mediated transformation techniques,
including disarming and use of binary vectors, are well described
in the scientific literature. See, e.g., Horsch (1984) Science
233:496-498; Fraley (1983) Proc. Natl. Acad. Sci. USA 80:4803
(1983); Gene Transfer to Plants, Potrykus, ed. (Springer-Verlag,
Berlin 1995). The DNA in an A. tumefaciens cell is contained in the
bacterial chromosome as well as in another structure known as a Ti
(tumor-inducing) plasmid. The Ti plasmid contains a stretch of DNA
termed T-DNA (.about.20 kb long) that is transferred to the plant
cell in the infection process and a series of vir (virulence) genes
that direct the infection process. A. tumefaciens can only infect a
plant through wounds: when a plant root or stem is wounded it gives
off certain chemical signals, in response to which, the vir genes
of A. tumefaciens become activated and direct a series of events
necessary for the transfer of the T-DNA from the Ti plasmid to the
plant's chromosome. The T-DNA then enters the plant cell through
the wound. One speculation is that the T-DNA waits until the plant
DNA is being replicated or transcribed, then inserts itself into
the exposed plant DNA. In order to use A. tumefaciens as a
transgene vector, the tumor-inducing section of T-DNA have to be
removed, while retaining the T-DNA border regions and the vir
genes. The transgene is then inserted between the T-DNA border
regions, where it is transferred to the plant cell and becomes
integrated into the plant's chromosomes.
[0417] The invention provides for the transformation of
monocotyledonous plants using the nucleic acids of the invention,
including important cereals, see Hiei (1997) Plant Mol. Biol.
35:205-218. See also, e.g., Horsch, Science (1984) 233:496; Fraley
(1983) Proc. Natl. Acad. Sci. USA 80:4803; Thykjaer (1997) supra;
Park (1996) Plant Mol. Biol. 32:1135-1148, discussing T-DNA
integration into genomic DNA. See also D'Halluin, U.S. Pat. No.
5,712,135, describing a process for the stable integration of a DNA
comprising a gene that is functional in a cell of a cereal, or
other monocotyledonous plant.
[0418] In one aspect, the third step involves selection and
regeneration of whole plants capable of transmitting the
incorporated target gene to the next generation. Such regeneration
techniques may use manipulation of certain phytohormones in a
tissue culture growth medium. In one aspect, the method uses a
biocide and/or herbicide marker that has been introduced together
with the desired nucleotide sequences. Plant regeneration from
cultured protoplasts is described in Evans et al., Protoplasts
Isolation and Culture, Handbook of Plant Cell Culture, pp. 124-176,
MacMillilan Publishing Company, New York, 1983; and Binding,
Regeneration of Plants, Plant Protoplasts, pp. 21-73, CRC Press,
Boca Raton, 1985. Regeneration can also be obtained from plant
callus, explants, organs, or parts thereof. Such regeneration
techniques are described generally in Klee (1987) Ann. Rev. of
Plant Phys. 38:467-486. To obtain whole plants from transgenic
tissues such as immature embryos, they can be grown under
controlled environmental conditions in a series of media containing
nutrients and hormones, a process known as tissue culture. Once
whole plants are generated and produce seed, evaluation of the
progeny begins.
[0419] In one aspect, after the expression cassette is stably
incorporated in transgenic plants, it can be introduced into other
plants by sexual crossing. Any of a number of standard breeding
techniques can be used, depending upon the species to be crossed.
Since transgenic expression of the nucleic acids of the invention
leads to phenotypic changes, plants comprising the recombinant
nucleic acids of the invention can be sexually crossed with a
second plant to obtain a final product. Thus, the seed of the
invention can be derived from a cross between two transgenic plants
of the invention, or a cross between a plant of the invention and
another plant. The desired effects (e.g., expression of the
polypeptides of the invention to produce a plant in which flowering
behavior is altered) can be enhanced when both parental plants
express the polypeptides (e.g., a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme) of the
invention. The desired effects can be passed to future plant
generations by standard propagation means.
[0420] In one aspect, the nucleic acids and polypeptides of the
invention are expressed in or inserted in any plant or seed.
Transgenic plants of the invention can be dicotyledonous or
monocotyledonous. Examples of monocot transgenic plants of the
invention are grasses, such as meadow grass (blue grass, Poa),
forage grass such as festuca, lolium, temperate grass, such as
Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice,
sorghum, and maize (corn). Examples of dicot transgenic plants of
the invention are tobacco, legumes, such as lupins, potato, sugar
beet, pea, bean and soybean, and cruciferous plants (family
Brassicaceae), such as cauliflower, rape seed, and the closely
related model organism Arabidopsis thaliana. Thus, the transgenic
plants and seeds of the invention include a broad range of plants,
including, but not limited to, species from the genera Anacardium,
Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus,
Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus,
Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis,
Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon,
Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza,
Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus,
Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,
Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and
Zea.
[0421] In alternative embodiments, the nucleic acids of the
invention are expressed in plants which contain fiber cells,
including, e.g., cotton, silk cotton tree (Kapok, Ceiba pentandra),
desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp,
roselle, jute, sisal abaca and flax. In alternative embodiments,
the transgenic plants of the invention can be members of the genus
Gossypium, including members of any Gossypium species, such as G.
arboreum; G. herbaceum, G. barbadense, and G. hirsutum.
[0422] The invention also provides for transgenic plants to be used
for producing large amounts of the polypeptides (e.g., a cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme or antibody) of the invention. For example,
see Palmgren (1997) Trends Genet. 13:348; Chong (1997) Transgenic
Res. 6:289-296 (producing human milk protein beta-casein in
transgenic potato plants using an auxin-inducible, bidirectional
mannopine synthase (mas1',2') promoter with Agrobacterium
tumefaciens-mediated leaf disc transformation methods).
[0423] Using known procedures, one of skill can screen for plants
of the invention by detecting the increase or decrease of transgene
mRNA or protein in transgenic plants. Means for detecting and
quantitation of mRNAs or proteins are well known in the art.
Polypeptides and Peptides
[0424] In one aspect, the invention provides isolated or
recombinant polypeptides having a sequence identity (e.g., at least
about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more, or complete (100%) sequence identity, or homology) to an
exemplary sequence of the invention, e.g., proteins having a
sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ
ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,
SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID
NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ
ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44,
SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID
NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ
ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,
SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID
NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ
ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100,
SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID
NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,
SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID
NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136,
SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID
NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154,
SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID
NO:164 or SEQ ID NO:166 (see also Tables 1, 2, and 3, Examples 1
and 4, below, and Sequence Listing)). The percent sequence identity
can be over the full length of the polypeptide, or, the identity
can be over a region of at least about 50, 60, 70, 80, 90, 100,
150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 or more
residues.
[0425] Polypeptides of the invention can also be shorter than the
full length of exemplary polypeptides. In alternative aspects, the
invention provides polypeptides (peptides, fragments) ranging in
size between about 5 and the full length of a polypeptide, e.g., an
enzyme, such as a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme;
exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200, 250,
300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues,
e.g., contiguous residues of an exemplary cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme of the invention. Peptides of the invention (e.g., a
subsequence of an exemplary polypeptide of the invention) can be
useful as, e.g., labeling probes, antigens (immunogens),
toleragens, motifs, cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme active
sites (e.g., "catalytic domains"), signal sequences and/or prepro
domains.
[0426] In alternative aspects, polypeptides of the invention having
cellulase activity, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase activity are members of a genus
of polypeptides sharing specific structural elements, e.g., amino
acid residues, that correlate with cellulase activity, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
activity. These shared structural elements can be used for the
routine generation of cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase variants.
These shared structural elements of cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
invention can be used as guidance for the routine generation of
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes variants within the scope of the genus of
polypeptides of the invention.
[0427] As used herein, the terms "cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase" encompass any
polypeptide or enzymes capable of catalyzing the complete or
partial breakdown and/or hydrolysis of cellulose (e.g., exemplary
polypeptides of the invention, see also Tables 1, 2, and 3,
Examples 1 and 4, below), or any modification of a cellulose or
lignocellulotic material, e.g., a biomass material comprising
lignocellulose.
[0428] In some aspects, a polypeptide of the invention can have an
alternative enzymatic activity, for example, as set forth in Table
3, below. For example, the polypeptide having a sequence as set
forth in SEQ ID NO:164, encoded, e.g., by SEQ ID NO:163, can have
Alkaline endoglucanase/cellulase activity; the polypeptide having a
sequence as set forth in SEQ ID NO:110, encoded, e.g., by SEQ ID
NO:109, can have xylanase activity; the polypeptide having a
sequence as set forth in SEQ ID NO:12, encoded, e.g., by SEQ ID
NO:11, can have NAD binding oxidoreductase activity; the
polypeptide having a sequence as set forth in SEQ ID NO:118,
encoded, e.g., by SEQ ID NO:117, can have short chain dehydrogenase
activity; the polypeptide having a sequence as set forth in SEQ ID
NO:14, encoded, e.g., by SEQ ID NO:13, can have NADH dependent
dehydrogenase activity; the polypeptide having a sequence as set
forth in SEQ ID NO:138, encoded, e.g., by SEQ ID NO:137, can have
peptidase activity; the polypeptide having a sequence as set forth
in SEQ ID NO:162, encoded, e.g., by SEQ ID NO:161, can have
Alkaline endoglucanase activity, in addition to cellulase activity;
the polypeptide having a sequence as set forth in SEQ ID NO:42,
encoded, e.g., by SEQ ID NO:41, can have cysteinyl tRNA synthetase
activity; the polypeptide having a sequence as set forth in SEQ ID
NO:32, encoded, e.g., by SEQ ID NO:31, can have cellodextrin
phosphorylase activity; the polypeptide having a sequence as set
forth in SEQ ID NO:50, encoded, e.g., by SEQ ID NO:49, can have
fdhd/narq oxidoreductase activity; the polypeptide having a
sequence as set forth in SEQ ID NO:54, encoded, e.g., by SEQ ID
NO:53, can have a radical S-adenosylmethionine (SAM) activity; the
polypeptide having a sequence as set forth in SEQ ID NO:58,
encoded, e.g., by SEQ ID NO:57, can have a subtilisin like protease
activity; etc., as set forth below:
TABLE-US-00001 TABLE 3 Signalp Cleavage EC SEQ ID NO: Enzymatic
Activity Site Signal Sequence Source Number 163, 164 Alkaline
endoglucanase/cellulase 1-30 MSCRTLMSRRVGWGLLLWGGLFL Unknown
RTGSVTG 1, 2 ORF 001-family 1 (.beta.-glucosidase) Unknown 3.2.1.21
101, 102 ORF 003-family 5 (cellulase) 1-29 MRNHLNVPFYFIFFFLIASIFTV
Unknown 3.2.1.4 CSSSTA 103, 104 family 5 (cellulase) 1-20
MLIIGGLLVLLGFSSCGRQA Unknown 3.2.1.4 105, 106 family 5 (cellulase)
Unknown 3.2.1.4 107, 108 family 5 (cellulase) 1-32
MEKQICSNVFSTMLIIGGLLVLL Unknown 3.2.1.4 GFSSCGRQA 109, 110 family
10 (xylanase) 1-28 MKTHSFNLRSRITLLTAALLFIG Unknown 3.2.1.8 ATAGA
11, 12 ORF 003-NAD binding Unknown 1.1.1.18 oxidoreductase 111, 112
family 5 (cellulase) 1-22 MRRLITIILATAVAILSTTSCS Unknown 3.2.1.4
113, 114 ORF 003-family 10 1-27 MKVTRTAVAGIVAAAVLITIGT Unknown
3.2.1.8 STASA 115, 116 ORF 004-short chain dehydrogenase Unknown
1.1.1.100 117, 118 ORF 011-short chain dehydrogenase 1-19
MPKVMLVTGGSRGIGAAVA Unknown 1 . . . 119, 120 ORF 002-oxidoreductase
Unknown 1.4.3.16 121, 122 ORF 004-family 5 (cellulase) Unknown
3.2.1.4 123, 124 ORF 006-family 1 (.beta.-glucosidase) Unknown
3.2.1.21 125, 126 ORF 009-family 1 (.beta.-glucosidase) Unknown
3.2.1.21 127, 128 ORF 004-short chain dehydrogenase Unknown
1.1.1.100 129, 130 ORF 010-short chain dehydrogenase 1-19
MPKVMLVTGGSRGIGAAVA Unknown 1 . . . 13, 14 ORF 005-NADH dependent
Unknown 1.1.1.18 dehydrogenase 131, 132 ORF 007-family 5
(cellulase) Unknown 3.2.1.4 133, 134 ORF 006-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 135, 136 ORF 001-cellulase
(glycosyl Unknown 3.2.1.4 hydrolase family 5) 137, 138 ORF
001-peptidase_M37 Unknown 3.5.1. 139, 140 ORF 001-threonine
dehydrogenase Unknown 1 . . . 141, 142 ORF 005-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 143, 144 ORF 003-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 145, 146 ORF 002-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 147, 148 family 10 (xylanase)
1-26 MLKVLRKPIISGLALALLLPAGA Unknown 3.2.1.8 AGA 149, 150 family 5
(cellulase) Unknown 3.2.1.4 15, 16 ORF-007-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 151, 152 family 5 (cellulase)
Unknown 3.2.1.4 153, 154 family 5 (cellulase) Unknown 3.2.1.4 155,
156 family 5 (cellulase) Unknown 3.2.1.4 157, 158 family 5
(cellulase) Unknown 3.2.1.4 159, 160 family 10 (xylanase) Unknown
3.2.1.8 161, 162 Alkaline endoglucanase/cellulase 1-30
MSCRTLMSRRVGWGLLLWGGLFL Unknown RTGSVTG 165, 166 xylanase 17, 18
ORF 005-.beta.-lactamase 1-23 MRYVLISCLALASLCAQPLPVST Unknown
3.5.2.6 19, 20 ORF 008-family 10 (xylanase) 1-20
MPVLFALFLVASSCAAQSLA Unknown 3.2.1.8 21, 22 ORF 001-family 5
(cellulase) Clostridium 3.2.1.4 thermocellum 23, 24 ORF 003-Family
16 + CBM 1-26 MYKRLLSSVLIIMLLLSAWSPIS Clostridium 3.2.1. VQA
thermocellum 25, 26 ORF 001-family 1 (.beta.-glucosidase)
Clostridium 3.2.1.21 thermocellum 27, 28 ORF 002-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 29, 30 ORF 004-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 3, 4 ORF 008-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 31, 32 ORF 002-cellodextrin
Unknown 2.4.1.20 phosphorylase 33, 34 ORF 006-family 1
(.beta.-glucosidase) Unknown 3.2.1.21 35, 36 ORF 007-family 5
(cellulase) 1-23 MNKILKLFSSLLLFAGICPALQA Unknown 3.2.1.4 37, 38 ORF
011-family 1 (.beta.-glucosidase) Unknown 3.2.1.21 39, 40 ORF
004-putative oxidoreductase Unknown 4.1.1. 41, 42 ORF 004-cysteinyl
tRNA synthetase Unknown 6.1.1.16 43, 44 ORF 011- Unknown 45, 46 ORF
006-family 1 (.beta.-glucosidase) Unknown 3.2.1.21 47, 48 ORF
002-family 1 (.beta.-glucosidase) Unknown 3.2.1.21 49, 50 ORF
006-fdhd/narq oxidoreductase Unknown 5, 6 ORF 012-family 6
(cellulase) 1-29 MTRRSIVRSSSNKWLVLAGAALL Unknown 3.2.1.91 ACTALG
51, 52 ORF 001-family 5 (cellulase) 1-20 MSRGILILVMLSVLSGAALA
Unknown 3.2.1.4 53, 54 ORF 002-Radical SAM family Unknown 1 . . .
55, 56 ORF 004-family 1 (.beta.-glucosidase) Unknown 3.2.1.21 57,
58 ORF 001-subtilisin like protease Unknown 59, 60 family 5
(cellulase) Unknown 3.2.1.4 61, 62 family 5 (cellulase) ORF 1 1-52
MVWTPARSTLAGSSEIPLMTMNI Unknown 3.2.1.4 FPNRKDSRMSLWIKLGILCMMAG
TVMVHG 63, 64 family 5 (cellulase) ORF 4 1-24
MKRREFMLGGAGVAALASTLGVS Unknown 3.2.1.4 A 65, 66 family 10
(xylanase) 1-39 MNTLLPRRRLWSSTAILRTLAAG Unknown 3.2.1.8
ALAAGMVLAPVSAANA 67, 68 family 5 (cellulase)-ORF 2 1-23
MKYIFSYIIMMILIGFIPVYGFG Unknown 3.2.1.4 69, 70 family 26
(mannanase)-ORF4 1-20 MSFKNHILLSLLIVLLFFSA Unknown 3.2.1.78 7, 8
ORF 003-Isocitrate dehydrogenase Unknown 1.1.1.42 71, 72 family 5
(cellulase) 1-21 MKLLKLLIFLLITVIFSDVSA Unknown 3.2.1.4 73, 74
family 10 (xylanase) Unknown 3.2.1.21 75, 76 family 5 (cellulase)
1-21 MLRKLIVSVFGFVMLTSAAAA Unknown 3.2.1.4 77, 78 family 5
(cellulase) 1-28 MKRKRVFIHSLIVFFLMIGSFTS Unknown 3.2.1.4 CGSVA 79,
80 family 5 (cellulase) 1-25 MKYKAIFIYLIVLILFYSINIYA Unknown
3.2.1.4 NA 81, 82 family 5 (cellulase) 1-25 MNLLAQYFSGLFLIFLISIFFVS
Unknown 3.2.1.4 SA 83, 84 ORF 008-dehydrogenase Unknown 3.5.4.25
85, 86 ORF 008-family 1 (.beta.-glucosidase) Unknown 3.2.1.21 87,
88 family 5 (cellulase) 1-23 MRKSVFTLAVFLSALFAFTSCQN Unknown
3.2.1.4 89, 90 family 5 (cellulase) 1-29 MKRSVSIFIACLLMTVLTISGVA
Unknown 3.2.1.4 APEASA 9, 10 ORF 004-family 10 (xylanase) 1-26
MRSVRIVTFALAAALAVPLVTST Unknown 3.2.1.8 ATA 91, 92 ORF 001-family 3
Unknown 3.2.1.52 93, 94 ORF 002-alpha-rhamnosidase Unknown 95, 96
ORF 001-family 3 Unknown 3.2.1.21 97, 98 ORF 003-beta-glucuronidase
Unknown 3.2.1.31 99, 100 ORF 012-family 1 (.beta.-glucosidase)
Unknown 3.2.1.21
[0429] "Amino acid" or "amino acid sequence" as used herein refer
to an oligopeptide, peptide, polypeptide, or protein sequence, or
to a fragment, portion, or subunit of any of these and to naturally
occurring or synthetic molecules. "Amino acid" or "amino acid
sequence" include an oligopeptide, peptide, polypeptide, or protein
sequence, or to a fragment, portion, or subunit of any of these,
and to naturally occurring or synthetic molecules. The term
"polypeptide" as used herein, refers to amino acids joined to each
other by peptide bonds or modified peptide bonds, i.e., peptide
isosteres and may contain modified amino acids other than the 20
gene-encoded amino acids. The polypeptides may be modified by
either natural processes, such as post-translational processing, or
by chemical modification techniques which are well known in the
art. Modifications can occur anywhere in the polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. It will be appreciated that the same type of
modification may be present in the same or varying degrees at
several sites in a given polypeptide. Also a given polypeptide may
have many types of modifications. Modifications include
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of a phosphatidylinositol, cross-linking cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristolyation,
oxidation, pegylation, glucan hydrolase processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation and transfer-RNA mediated addition of amino acids to
protein such as arginylation. (See Creighton, T. E.,
Proteins--Structure and Molecular Properties 2nd Ed., W.H. Freeman
and Company, New York (1993); Posttranslational Covalent
Modification of Proteins, B. C. Johnson, Ed., Academic Press, New
York, pp. 1-12 (1983)). The peptides and polypeptides of the
invention also include all "mimetic" and "peptidomimetic" forms, as
described in further detail, below.
[0430] As used herein, the term "isolated" means that the material
(e.g., a protein or nucleic acid of the invention) is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or polypeptides could be part of a composition
and still be isolated in that such vector or composition is not
part of its natural environment. As used herein, the term
"purified" does not require absolute purity; rather, it is intended
as a relative definition. Individual nucleic acids obtained from a
library have been conventionally purified to electrophoretic
homogeneity. The sequences obtained from these clones could not be
obtained directly either from the library or from total human DNA.
The purified nucleic acids of the invention have been purified from
the remainder of the genomic DNA in the organism by at least
10.sup.4-10.sup.6 fold. In one aspect, the term "purified" includes
nucleic acids which have been purified from the remainder of the
genomic DNA or from other sequences in a library or other
environment by at least one order of magnitude, e.g., in one
aspect, two or three orders, or, four or five orders of
magnitude.
[0431] "Recombinant" polypeptides or proteins refer to polypeptides
or proteins produced by recombinant DNA techniques; i.e., produced
from cells transformed by an exogenous DNA construct encoding the
desired polypeptide or protein. "Synthetic" polypeptides or protein
are those prepared by chemical synthesis. Solid-phase chemical
peptide synthesis methods can also be used to synthesize the
polypeptide or fragments of the invention. Such method have been
known in the art since the early 1960's (Merrifield, R. B., J. Am.
Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,
J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,
Rockford, Ill., pp. 11-12)) and have recently been employed in
commercially available laboratory peptide design and synthesis kits
(Cambridge Research Biochemicals). Such commercially available
laboratory kits have generally utilized the teachings of H. M.
Geysen et al, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and
provide for synthesizing peptides upon the tips of a multitude of
"rods" or "pins" all of which are connected to a single plate.
[0432] The phrase "substantially identical" in the context of two
nucleic acids or polypeptides, refers to two or more sequences that
have, e.g., at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or more nucleotide or amino acid residue (sequence)
identity, when compared and aligned for maximum correspondence, as
measured using one of the known sequence comparison algorithms or
by visual inspection. In alternative aspects, the substantial
identity exists over a region of at least about 100 or more
residues and most commonly the sequences are substantially
identical over at least about 150 to 200 or more residues. In some
aspects, the sequences are substantially identical over the entire
length of the coding regions.
[0433] Additionally a "substantially identical" amino acid sequence
is a sequence that differs from a reference sequence by one or more
conservative or non-conservative amino acid substitutions,
deletions, or insertions. In one aspect, the substitution occurs at
a site that is not the active site of the molecule, or,
alternatively the substitution occurs at a site that is the active
site of the molecule, provided that the polypeptide essentially
retains its functional (enzymatic) properties. A conservative amino
acid substitution, for example, substitutes one amino acid for
another of the same class (e.g., substitution of one hydrophobic
amino acid, such as isoleucine, valine, leucine, or methionine, for
another, or substitution of one polar amino acid for another, such
as substitution of arginine for lysine, glutamic acid for aspartic
acid or glutamine for asparagine). One or more amino acids can be
deleted, for example, from a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase polypeptide,
resulting in modification of the structure of the polypeptide,
without significantly altering its biological activity. For
example, amino- or carboxyl-terminal amino acids that are not
required for cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme biological activity can be
removed. Modified polypeptide sequences of the invention can be
assayed for cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme biological activity by any
number of methods, including contacting the modified polypeptide
sequence with a substrate and determining whether the modified
polypeptide decreases the amount of specific substrate in the assay
or increases the bioproducts of the enzymatic reaction of a
functional cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase polypeptide with the
substrate.
[0434] "Fragments" as used herein are a portion of a naturally
occurring protein which can exist in at least two different
conformations. Fragments can have the same or substantially the
same amino acid sequence as the naturally occurring protein.
Fragments which have different three dimensional structures as the
naturally occurring protein are also included. An example of this,
is a "pro-form" molecule, such as a low activity proprotein that
can be modified by cleavage to produce a mature enzyme with
significantly higher activity.
[0435] In one aspect, the invention provides crystal
(three-dimensional) structures of proteins and peptides, e.g.,
cellulases, of the invention; which can be made and analyzed using
the routine protocols well known in the art, e.g., as described in
MacKenzie (1998) Crystal structure of the family 7 endoglucanase I
(Cel7B) from Humicola insolens at 2.2 A resolution and
identification of the catalytic nucleophile by trapping of the
covalent glycosyl-enzyme intermediate, Biochem. J. 335:409-416;
Sakon (1997) Structure and mechanism of endo/exocellulase E4 from
Thermomonospora fusca, Nat. Struct. Biol 4:810-818; Varrot (1999)
Crystal structure of the catalytic core domain of the family 6
cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 A
resolution, Biochem. J. 337:297-304; illustrating and identifying
specific structural elements as guidance for the routine generation
of cellulase variants of the invention, and as guidance for
identifying enzyme species within the scope of the invention.
[0436] Polypeptides and peptides of the invention can be isolated
from natural sources, be synthetic, or be recombinantly generated
polypeptides. Peptides and proteins can be recombinantly expressed
in vitro or in vivo. The peptides and polypeptides of the invention
can be made and isolated using any method known in the art.
Polypeptide and peptides of the invention can also be synthesized,
whole or in part, using chemical methods well known in the art. See
e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn
(1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K.,
Therapeutic Peptides and Proteins, Formulation, Processing and
Delivery Systems (1995) Technomic Publishing Co., Lancaster, Pa.
For example, peptide synthesis can be performed using various
solid-phase techniques (see e.g., Roberge (1995) Science 269:202;
Merrifield (1997) Methods Enzymol. 289:3-13) and automated
synthesis may be achieved, e.g., using the ABI 431A Peptide
Synthesizer (Perkin Elmer) in accordance with the instructions
provided by the manufacturer.
[0437] The peptides and polypeptides of the invention can also be
glycosylated. The glycosylation can be added post-translationally
either chemically or by cellular biosynthetic mechanisms, wherein
the later incorporates the use of known glycosylation motifs, which
can be native to the sequence or can be added as a peptide or added
in the nucleic acid coding sequence. The glycosylation can be
O-linked or N-linked.
[0438] The peptides and polypeptides of the invention, as defined
above, include all "mimetic" and "peptidomimetic" forms. The terms
"mimetic" and "peptidomimetic" refer to a synthetic chemical
compound which has substantially the same structural and/or
functional characteristics of the polypeptides of the invention.
The mimetic can be either entirely composed of synthetic,
non-natural analogues of amino acids, or, is a chimeric molecule of
partly natural peptide amino acids and partly non-natural analogs
of amino acids. The mimetic can also incorporate any amount of
natural amino acid conservative substitutions as long as such
substitutions also do not substantially alter the mimetic's
structure and/or activity. As with polypeptides of the invention
which are conservative variants or members of a genus of
polypeptides of the invention (e.g., having about 50% or more
sequence identity to an exemplary sequence of the invention),
routine experimentation will determine whether a mimetic is within
the scope of the invention, i.e., that its structure and/or
function is not substantially altered. Thus, in one aspect, a
mimetic composition is within the scope of the invention if it has
a cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzymes activity.
[0439] Polypeptide mimetic compositions of the invention can
contain any combination of non-natural structural components. In
alternative aspect, mimetic compositions of the invention include
one or all of the following three structural groups: a) residue
linkage groups other than the natural amide bond ("peptide bond")
linkages; b) non-natural residues in place of naturally occurring
amino acid residues; or c) residues which induce secondary
structural mimicry, i.e., to induce or stabilize a secondary
structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix
conformation, and the like. For example, a polypeptide of the
invention can be characterized as a mimetic when all or some of its
residues are joined by chemical means other than natural peptide
bonds. Individual peptidomimetic residues can be joined by peptide
bonds, other chemical bonds or coupling means, such as, e.g.,
glutaraldehyde, N-hydroxysuccinimide esters, bifunctional
maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or
N,N'-diisopropylcarbodiimide (DIC). Linking groups that can be an
alternative to the traditional amide bond ("peptide bond") linkages
include, e.g., ketomethylene (e.g., --C(.dbd.O)--CH.sub.2-- for
--C(.dbd.O)--NH--), aminomethylene (CH.sub.2--NH), ethylene, olefin
(CH.dbd.CH), ether (CH.sub.2--O), thioether (CH.sub.2--S),
tetrazole (CN.sub.4--), thiazole, retroamide, thioamide, or ester
(see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino
Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone
Modifications," Marcell Dekker, N.Y.).
[0440] A polypeptide of the invention can also be characterized as
a mimetic by containing all or some non-natural residues in place
of naturally occurring amino acid residues. Non-natural residues
are well described in the scientific and patent literature; a few
exemplary non-natural compositions useful as mimetics of natural
amino acid residues and guidelines are described below. Mimetics of
aromatic amino acids can be generated by replacing by, e.g., D- or
L-naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine;
D- or L-1, -2, 3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine;
D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or
L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;
D-(trifluoromethyl)-phenylglycine;
D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or
L-p-biphenylphenylalanine; D- or L-p-methoxy-biphenylphenylalanine;
D- or L-2-indole(alkyl)alanines; and, D- or L-alkylainines, where
alkyl can be substituted or unsubstituted methyl, ethyl, propyl,
hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl,
or a non-acidic amino acids. Aromatic rings of a non-natural amino
acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,
benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic
rings.
[0441] Mimetics of acidic amino acids can be generated by
substitution by, e.g., non-carboxylate amino acids while
maintaining a negative charge; (phosphono)alanine; sulfated
threonine. Carboxyl side groups (e.g., aspartyl or glutamyl) can
also be selectively modified by reaction with carbodiimides
(R'--N--C--N--R') such as, e.g.,
1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or
glutamyl can also be converted to asparaginyl and glutaminyl
residues by reaction with ammonium ions. Mimetics of basic amino
acids can be generated by substitution with, e.g., (in addition to
lysine and arginine) the amino acids ornithine, citrulline, or
(guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where
alkyl is defined above. Nitrile derivative (e.g., containing the
CN-moiety in place of COOH) can be substituted for asparagine or
glutamine. Asparaginyl and glutaminyl residues can be deaminated to
the corresponding aspartyl or glutamyl residues. Arginine residue
mimetics can be generated by reacting arginyl with, e.g., one or
more conventional reagents, including, e.g., phenylglyoxal,
2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin, in one aspect
under alkaline conditions. Tyrosine residue mimetics can be
generated by reacting tyrosyl with, e.g., aromatic diazonium
compounds or tetranitromethane. N-acetylimidizol and
tetranitromethane can be used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Cysteine residue mimetics can be
generated by reacting cysteinyl residues with, e.g.,
alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide
and corresponding amines; to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteine residue mimetics can also
be generated by reacting cysteinyl residues with, e.g.,
bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic
acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl
disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate;
2-chloromercuri-4 nitrophenol; or,
chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimetics can be
generated (and amino terminal residues can be altered) by reacting
lysinyl with, e.g., succinic or other carboxylic acid anhydrides.
Lysine and other alpha-amino-containing residue mimetics can also
be generated by reaction with imidoesters, such as methyl
picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,
trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione,
and transamidase-catalyzed reactions with glyoxylate. Mimetics of
methionine can be generated by reaction with, e.g., methionine
sulfoxide. Mimetics of proline include, e.g., pipecolic acid,
thiazolidine carboxylic acid, 3- or 4-hydroxy proline,
dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
Histidine residue mimetics can be generated by reacting histidyl
with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
Other mimetics include, e.g., those generated by hydroxylation of
proline and lysine; phosphorylation of the hydroxyl groups of seryl
or threonyl residues; methylation of the alpha-amino groups of
lysine, arginine and histidine; acetylation of the N-terminal
amine; methylation of main chain amide residues or substitution
with N-methyl amino acids; or amidation of C-terminal carboxyl
groups.
[0442] In one aspect, a residue, e.g., an amino acid, of a
polypeptide of the invention can also be replaced by an amino acid
(or peptidomimetic residue) of the opposite chirality. In one
aspect, any amino acid naturally occurring in the L-configuration
(which can also be referred to as the R or S, depending upon the
structure of the chemical entity) can be replaced with the amino
acid of the same chemical structural type or a peptidomimetic, but
of the opposite chirality, referred to as the D-amino acid, but
also can be referred to as the R- or S-form.
[0443] The invention also provides methods for modifying the
polypeptides of the invention by either natural processes, such as
post-translational processing (e.g., phosphorylation, acylation,
etc), or by chemical modification techniques, and the resulting
modified polypeptides. Modifications can occur anywhere in the
polypeptide, including the peptide backbone, the amino acid
side-chains and the amino or carboxyl termini. It will be
appreciated that the same type of modification may be present in
the same or varying degrees at several sites in a given
polypeptide. Also a given polypeptide may have many types of
modifications. In one aspect, modifications include acetylation,
acylation, ADP-ribosylation, amidation, covalent attachment of
flavin, covalent attachment of a heme moiety, covalent attachment
of a nucleotide or nucleotide derivative, covalent attachment of a
lipid or lipid derivative, covalent attachment of a
phosphatidylinositol, cross-linking cyclization, disulfide bond
formation, demethylation, formation of covalent cross-links,
formation of cysteine, formation of pyroglutamate, formylation,
gamma-carboxylation, glycosylation, GPI anchor formation,
hydroxylation, iodination, methylation, myristolyation, oxidation,
pegylation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, and transfer-RNA mediated
addition of amino acids to protein such as arginylation. See, e.g.,
Creighton, T. E., Proteins--Structure and Molecular Properties 2nd
Ed., W.H. Freeman and Company, New York (1993); Posttranslational
Covalent Modification of Proteins, B. C. Johnson, Ed., Academic
Press, New York, pp. 1-12 (1983).
[0444] Solid-phase chemical peptide synthesis methods can also be
used to synthesize the polypeptide or fragments of the invention.
Such method have been known in the art since the early 1960's
(Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154, 1963) (See
also Stewart, J. M. and Young, J. D., Solid Phase Peptide
Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill., pp.
11-12)) and have recently been employed in commercially available
laboratory peptide design and synthesis kits (Cambridge Research
Biochemicals). Such commercially available laboratory kits have
generally utilized the teachings of H. M. Geysen et al, Proc. Natl.
Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing
peptides upon the tips of a multitude of "rods" or "pins" all of
which are connected to a single plate. When such a system is
utilized, a plate of rods or pins is inverted and inserted into a
second plate of corresponding wells or reservoirs, which contain
solutions for attaching or anchoring an appropriate amino acid to
the pin's or rod's tips. By repeating such a process step, i.e.,
inverting and inserting the rod's and pin's tips into appropriate
solutions, amino acids are built into desired peptides. In
addition, a number of available FMOC peptide synthesis systems are
available. For example, assembly of a polypeptide or fragment can
be carried out on a solid support using an Applied Biosystems, Inc.
Model 431A.TM. automated peptide synthesizer. Such equipment
provides ready access to the peptides of the invention, either by
direct synthesis or by synthesis of a series of fragments that can
be coupled using other known techniques.
[0445] The polypeptides of the invention include cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes in an active or inactive form. For example, the
polypeptides of the invention include proproteins before
"maturation" or processing of prepro sequences, e.g., by a
proprotein-processing enzyme, such as a proprotein convertase to
generate an "active" mature protein. The polypeptides of the
invention include cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes
inactive for other reasons, e.g., before "activation" by a
post-translational processing event, e.g., an endo- or
exo-peptidase or proteinase action, a phosphorylation event, an
amidation, a glycosylation or a sulfation, a dimerization event,
and the like. The polypeptides of the invention include all active
forms, including active subsequences, e.g., catalytic domains or
active sites, of the enzyme.
[0446] The invention includes immobilized cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes, anti-cellulase, e.g., anti-endoglucanase,
anti-cellobiohydrolase and/or anti-beta-glucosidase antibodies and
fragments thereof. The invention provides methods for inhibiting
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme activity, e.g., using dominant negative
mutants or anti-cellulase, e.g., anti-endoglucanase,
anti-cellobiohydrolase and/or anti-beta-glucosidase antibodies of
the invention. The invention includes heterocomplexes, e.g., fusion
proteins, heterodimers, etc., comprising the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes of the invention.
[0447] Polypeptides of the invention can have a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity under various conditions, e.g., extremes in pH
and/or temperature, oxidizing agents, and the like. The invention
provides methods leading to alternative cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme preparations with different catalytic efficiencies and
stabilities, e.g., towards temperature, oxidizing agents and
changing wash conditions. In one aspect, cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme variants can be produced using techniques of site-directed
mutagenesis and/or random mutagenesis. In one aspect, directed
evolution can be used to produce a great variety of cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme variants with alternative specificities and
stability.
[0448] The proteins of the invention are also useful as research
reagents to identify cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
modulators, e.g., activators or inhibitors of cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity. Briefly, test samples (compounds, broths,
extracts, and the like) are added to cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme assays to determine their ability to inhibit substrate
cleavage. Inhibitors identified in this way can be used in industry
and research to reduce or prevent undesired proteolysis. As with
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes, inhibitors can be combined to increase
the spectrum of activity.
[0449] The enzymes of the invention are also useful as research
reagents to digest proteins or in protein sequencing. For example,
the cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzymes may be used to break polypeptides
into smaller fragments for sequencing using, e.g. an automated
sequencer.
[0450] The invention also provides methods of discovering new
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes using the nucleic acids, polypeptides and
antibodies of the invention. In one aspect, phagemid libraries are
screened for expression-based discovery of cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes. In another aspect, lambda phage libraries are screened for
expression-based discovery of cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes.
Screening of the phage or phagemid libraries can allow the
detection of toxic clones; improved access to substrate; reduced
need for engineering a host, by-passing the potential for any bias
resulting from mass excision of the library; and, faster growth at
low clone densities. Screening of phage or phagemid libraries can
be in liquid phase or in solid phase. In one aspect, the invention
provides screening in liquid phase. This gives a greater
flexibility in assay conditions; additional substrate flexibility;
higher sensitivity for weak clones; and ease of automation over
solid phase screening.
[0451] The invention provides screening methods using the proteins
and nucleic acids of the invention and robotic automation to enable
the execution of many thousands of biocatalytic reactions and
screening assays in a short period of time, e.g., per day, as well
as ensuring a high level of accuracy and reproducibility (see
discussion of arrays, below). As a result, a library of derivative
compounds can be produced in a matter of weeks. For further
teachings on modification of molecules, including small molecules,
see PCT/US94/09174; U.S. Pat. No. 6,245,547.
[0452] In one aspect, polypeptides or fragments of the invention
are obtained through biochemical enrichment or purification
procedures. The sequence of potentially homologous polypeptides or
fragments may be determined by cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme assays
(see, e.g., Examples 1, 2 and 3, below), gel electrophoresis and/or
microsequencing. The sequence of the prospective polypeptide or
fragment of the invention can be compared to an exemplary
polypeptide of the invention, or a fragment, e.g., comprising at
least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 or
more consecutive amino acids thereof using any of the programs
described above.
[0453] Another aspect of the invention is an assay for identifying
fragments or variants of the invention, which retain the enzymatic
function of the polypeptides of the invention. For example the
fragments or variants of said polypeptides, may be used to catalyze
biochemical reactions, which indicate that the fragment or variant
retains the enzymatic activity of a polypeptide of the invention.
An exemplary assay for determining if fragments of variants retain
the enzymatic activity of the polypeptides of the invention
includes the steps of: contacting the polypeptide fragment or
variant with a substrate molecule under conditions which allow the
polypeptide fragment or variant to function and detecting either a
decrease in the level of substrate or an increase in the level of
the specific reaction product of the reaction between the
polypeptide and substrate.
[0454] The present invention exploits the unique catalytic
properties of enzymes. Whereas the use of biocatalysts (i.e.,
purified or crude enzymes, non-living or living cells) in chemical
transformations normally requires the identification of a
particular biocatalyst that reacts with a specific starting
compound, the present invention uses selected biocatalysts and
reaction conditions that are specific for functional groups that
are present in many starting compounds, such as small molecules.
Each biocatalyst is specific for one functional group, or several
related functional groups and can react with many starting
compounds containing this functional group.
[0455] In one aspect, the biocatalytic reactions produce a
population of derivatives from a single starting compound. These
derivatives can be subjected to another round of biocatalytic
reactions to produce a second population of derivative compounds.
Thousands of variations of the original small molecule or compound
can be produced with each iteration of biocatalytic
derivatization.
[0456] Enzymes react at specific sites of a starting compound
without affecting the rest of the molecule, a process which is very
difficult to achieve using traditional chemical methods. This high
degree of biocatalytic specificity provides the means to identify a
single active compound within the library. The library is
characterized by the series of biocatalytic reactions used to
produce it, a so-called "biosynthetic history". Screening the
library for biological activities and tracing the biosynthetic
history identifies the specific reaction sequence producing the
active compound. The reaction sequence is repeated and the
structure of the synthesized compound determined. This mode of
identification, unlike other synthesis and screening approaches,
does not require immobilization technologies and compounds can be
synthesized and tested free in solution using virtually any type of
screening assay. It is important to note, that the high degree of
specificity of enzyme reactions on functional groups allows for the
"tracking" of specific enzymatic reactions that make up the
biocatalytically produced library.
[0457] In one aspect, procedural steps are performed using robotic
automation enabling the execution of many thousands of biocatalytic
reactions and/or screening assays per day as well as ensuring a
high level of accuracy and reproducibility. Robotic automation can
also be used to screen for cellulase activity to determine if a
polypeptide is within the scope of the invention. As a result, in
one aspect, a library of derivative compounds can be produced in a
matter of weeks which would take years to produce using
"traditional" chemical or enzymatic screening methods.
[0458] In a particular aspect, the invention provides a method for
modifying small molecules, comprising contacting a polypeptide
encoded by a polynucleotide described herein or enzymatically
active fragments thereof with a small molecule to produce a
modified small molecule. A library of modified small molecules is
tested to determine if a modified small molecule is present within
the library, which exhibits a desired activity. A specific
biocatalytic reaction which produces the modified small molecule of
desired activity is identified by systematically eliminating each
of the biocatalytic reactions used to produce a portion of the
library and then testing the small molecules produced in the
portion of the library for the presence or absence of the modified
small molecule with the desired activity. The specific biocatalytic
reactions which produce the modified small molecule of desired
activity is optionally repeated. The biocatalytic reactions are
conducted with a group of biocatalysts that react with distinct
structural moieties found within the structure of a small molecule,
each biocatalyst is specific for one structural moiety or a group
of related structural moieties; and each biocatalyst reacts with
many different small molecules which contain the distinct
structural moiety.
[0459] Cellulase, e.g., Endoglucanase, Cellobiohydrolase and/or
Beta-Glucosidase Enzyme Signal Sequences, Prepro and Catalytic
Domains
[0460] The invention provides cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme signal
sequences (e.g., signal peptides (SPs)), prepro domains and
catalytic domains (CDs). The SPs, prepro domains and/or CDs of the
invention can be isolated or recombinant peptides or can be part of
a fusion protein, e.g., as a heterologous domain in a chimeric
protein. The invention provides nucleic acids encoding these
catalytic domains (CDs), prepro domains and signal sequences (SPs,
e.g., a peptide having a sequence comprising/consisting of amino
terminal residues of a polypeptide of the invention).
[0461] The invention provides isolated or recombinant signal
sequences (e.g., signal peptides) consisting of or comprising a
sequence as set forth in residues 1 to 14, 1 to 15, 1 to 16, 1 to
17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24,
1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to
32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40,
1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46, or 1 to 47,
or more, of a polypeptide of the invention, e.g., exemplary
polypeptides of the invention, see also Table 3, Examples 1 and 4,
below, and Sequence Listing. For example, Table 3, above, sets
forth exemplary signal (leader) sequences of the invention, e.g.,
as in the polypeptide having a sequence as set forth in SEQ ID
NO:164, encoded, e.g., by SEQ ID NO:163, has a signal sequence
comprising (or consisting of) the amino terminal 30 residues, or,
MSCRTLMSRRVGWGLLLWGGLFLRTGSVTG. Additional signal sequences are
similarly set forth in Table 3.
[0462] In one aspect, the invention provides signal sequences
comprising the first 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more amino
terminal residues of a polypeptide of the invention.
[0463] The invention includes polypeptides with or without a signal
sequence and/or a prepro sequence. The invention includes
polypeptides with heterologous signal sequences and/or prepro
sequences. The prepro sequence (including a sequence of the
invention used as a heterologous prepro domain) can be located on
the amino terminal or the carboxy terminal end of the protein. The
invention also includes isolated or recombinant signal sequences,
prepro sequences and catalytic domains (e.g., "active sites")
comprising sequences of the invention. The polypeptide comprising a
signal sequence of the invention can be a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme of the invention or another cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme or
another enzyme or other polypeptide. Methods for identifying
"prepro" domain sequences and signal sequences are well known in
the art, see, e.g., Van de Ven (1993) Crit. Rev. Oncog.
4(2):115-136. For example, to identify a prepro sequence, the
protein is purified from the extracellular space and the N-terminal
protein sequence is determined and compared to the unprocessed
form.
[0464] The cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme signal sequences (SPs)
and/or prepro sequences of the invention can be isolated or
recombinant peptides, or, sequences joined to another cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme or a non-cellulase, e.g.,
non-endoglucanase, non-cellobiohydrolase and/or
non-beta-glucosidase polypeptide, e.g., as a fusion (chimeric)
protein. In one aspect, the invention provides polypeptides
comprising cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme signal sequences of the
invention. In one aspect, polypeptides comprising cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme signal sequences SPs and/or prepro of the invention comprise
sequences heterologous to a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme of the
invention (e.g., a fusion protein comprising an SP and/or prepro of
the invention and sequences from another cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme or a non-cellulase, e.g., non-endoglucanase,
non-cellobiohydrolase and/or non-beta-glucosidase protein). In one
aspect, the invention provides cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
invention with heterologous SPs and/or prepro sequences, e.g.,
sequences with a yeast signal sequence. A cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme of the invention can comprise a heterologous SP and/or
prepro in a vector, e.g., a pPIC series vector (Invitrogen,
Carlsbad, Calif.).
[0465] In one aspect, SPs and/or prepro sequences of the invention
are identified following identification of novel cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
polypeptides. The pathways by which proteins are sorted and
transported to their proper cellular location are often referred to
as protein targeting pathways. One of the most important elements
in all of these targeting systems is a short amino acid sequence at
the amino terminus of a newly synthesized polypeptide called the
signal sequence. This signal sequence directs a protein to its
appropriate location in the cell and is removed during transport or
when the protein reaches its final destination. Most lysosomal,
membrane, or secreted proteins have an amino-terminal signal
sequence that marks them for translocation into the lumen of the
endoplasmic reticulum. The signal sequences can vary in length from
about 10 to 65, or more, amino acid residues. Various methods of
recognition of signal sequences are known to those of skill in the
art. For example, in one aspect, novel cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme signal peptides are identified by a method referred to as
SignalP. SignalP uses a combined neural network which recognizes
both signal peptides and their cleavage sites. (Nielsen (1997)
"Identification of prokaryotic and eukaryotic signal peptides and
prediction of their cleavage sites." Protein Engineering
10:1-6.
[0466] In some aspects cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
invention do not have SPs and/or prepro sequences or "domains." In
one aspect, the invention provides the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes of the invention lacking all or part of an SP and/or a
prepro domain. In one aspect, the invention provides a nucleic acid
sequence encoding a signal sequence (SP) and/or prepro from one
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme operably linked to a nucleic acid sequence
of a different cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme or, optionally, a signal
sequence (SPs) and/or prepro domain from a non-cellulase, e.g.,
non-endoglucanase, non-cellobiohydrolase and/or
non-beta-glucosidase protein may be desired.
[0467] The invention also provides isolated or recombinant
polypeptides comprising signal sequences (SPs), prepro domain
and/or catalytic domains (CDs) of the invention and heterologous
sequences. The heterologous sequences are sequences not naturally
associated (e.g., to a enzyme) with an SP, prepro domain and/or CD.
The sequence to which the SP, prepro domain and/or CD are not
naturally associated can be on the SP's, prepro domain and/or CD's
amino terminal end, carboxy terminal end, and/or on both ends of
the SP and/or CD. In one aspect, the invention provides an isolated
or recombinant polypeptide comprising (or consisting of) a
polypeptide comprising a signal sequence (SP), prepro domain and/or
catalytic domain (CD) of the invention with the proviso that it is
not associated with any sequence to which it is naturally
associated (e.g., a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
sequence). Similarly in one aspect, the invention provides isolated
or recombinant nucleic acids encoding these polypeptides. Thus, in
one aspect, the isolated or recombinant nucleic acid of the
invention comprises coding sequence for a signal sequence (SP),
prepro domain and/or catalytic domain (CD) of the invention and a
heterologous sequence (i.e., a sequence not naturally associated
with the a signal sequence (SP), prepro domain and/or catalytic
domain (CD) of the invention). The heterologous sequence can be on
the 3' terminal end, 5' terminal end, and/or on both ends of the
SP, prepro domain and/or CD coding sequence.
[0468] Hybrid (Chimeric) Cellulase, e.g., Endoglucanase,
Cellobiohydrolase and/or Beta-Glucosidase Enzymes and Peptide
Libraries
[0469] In one aspect, the invention provides hybrid cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes and fusion proteins, including peptide
libraries, comprising sequences of the invention. The peptide
libraries of the invention can be used to isolate peptide
modulators (e.g., activators or inhibitors) of targets, such as
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme substrates, receptors, enzymes. The peptide
libraries of the invention can be used to identify formal binding
partners of targets, such as ligands, e.g., cytokines, hormones and
the like. In one aspect, the invention provides chimeric proteins
comprising a signal sequence (SP), prepro domain and/or catalytic
domain (CD) of the invention or a combination thereof and a
heterologous sequence (see above).
[0470] In one aspect, the fusion proteins of the invention (e.g.,
the peptide moiety) are conformationally stabilized (relative to
linear peptides) to allow a higher binding affinity for targets.
The invention provides fusions of cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
invention and other peptides, including known and random peptides.
They can be fused in such a manner that the structure of the
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes is not significantly perturbed and the
peptide is metabolically or structurally conformationally
stabilized. This allows the creation of a peptide library that is
easily monitored both for its presence within cells and its
quantity.
[0471] Amino acid sequence variants of the invention can be
characterized by a predetermined nature of the variation, a feature
that sets them apart from a naturally occurring form, e.g., an
allelic or interspecies variation of a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme sequence. In one aspect, the variants of the invention
exhibit the same qualitative biological activity as the naturally
occurring analogue. Alternatively, the variants can be selected for
having modified characteristics. In one aspect, while the site or
region for introducing an amino acid sequence variation is
predetermined, the mutation per se need not be predetermined. For
example, in order to optimize the performance of a mutation at a
given site, random mutagenesis may be conducted at the target codon
or region and the expressed cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
variants screened for the optimal combination of desired activity.
Techniques for making substitution mutations at predetermined sites
in DNA having a known sequence are well known, as discussed herein
for example, M13 primer mutagenesis and PCR mutagenesis. Screening
of the mutants can be done using, e.g., assays of glucan
hydrolysis. In alternative aspects, amino acid substitutions can be
single residues; insertions can be on the order of from about 1 to
20 amino acids, although considerably larger insertions can be
done. Deletions can range from about 1 to about 20, 30, 40, 50, 60,
70 residues or more. To obtain a final derivative with the optimal
properties, substitutions, deletions, insertions or any combination
thereof may be used. Generally, these changes are done on a few
amino acids to minimize the alteration of the molecule. However,
larger changes may be tolerated in certain circumstances.
[0472] The invention provides cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes where
the structure of the polypeptide backbone, the secondary or the
tertiary structure, e.g., an alpha-helical or beta-sheet structure,
has been modified. In one aspect, the charge or hydrophobicity has
been modified. In one aspect, the bulk of a side chain has been
modified. Substantial changes in function or immunological identity
are made by selecting substitutions that are less conservative. For
example, substitutions can be made which more significantly affect:
the structure of the polypeptide backbone in the area of the
alteration, for example a alpha-helical or a beta-sheet structure;
a charge or a hydrophobic site of the molecule, which can be at an
active site; or a side chain. The invention provides substitutions
in polypeptide of the invention where (a) a hydrophilic residues,
e.g. seryl or threonyl, is substituted for (or by) a hydrophobic
residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b)
a cysteine or proline is substituted for (or by) any other residue;
(c) a residue having an electropositive side chain, e.g. lysyl,
arginyl, or histidyl, is substituted for (or by) an electronegative
residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky
side chain, e.g. phenylalanine, is substituted for (or by) one not
having a side chain, e.g. glycine. The variants can exhibit the
same qualitative biological activity (i.e., a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity) although variants can be selected to modify the
characteristics of the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes as
needed.
[0473] In one aspect, cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
invention comprise epitopes or purification tags, signal sequences
or other fusion sequences, etc. In one aspect, the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes of the invention can be fused to a random peptide to form a
fusion polypeptide. By "fused" or "operably linked" herein is meant
that the random peptide and the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme are
linked together, in such a manner as to minimize the disruption to
the stability of the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
structure, e.g., it retains cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity. The fusion polypeptide (or fusion polynucleotide encoding
the fusion polypeptide) can comprise further components as well,
including multiple peptides at multiple loops.
[0474] In one aspect, the peptides and nucleic acids encoding them
are randomized, either fully randomized or they are biased in their
randomization, e.g. in nucleotide/residue frequency generally or
per position. "Randomized" means that each nucleic acid and peptide
consists of essentially random nucleotides and amino acids,
respectively. In one aspect, the nucleic acids which give rise to
the peptides can be chemically synthesized, and thus may
incorporate any nucleotide at any position. Thus, when the nucleic
acids are expressed to form peptides, any amino acid residue may be
incorporated at any position. The synthetic process can be designed
to generate randomized nucleic acids, to allow the formation of all
or most of the possible combinations over the length of the nucleic
acid, thus forming a library of randomized nucleic acids. The
library can provide a sufficiently structurally diverse population
of randomized expression products to affect a probabilistically
sufficient range of cellular responses to provide one or more cells
exhibiting a desired response. Thus, the invention provides an
interaction library large enough so that at least one of its
members will have a structure that gives it affinity for some
molecule, protein, or other factor.
[0475] In one aspect, a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme of the
invention is a multidomain enzyme that comprises a signal peptide,
a carbohydrate binding module, a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
catalytic domain, a linker and/or another catalytic domain.
[0476] The invention provides a methods and sequences for
generating chimeric polypeptides which may encode biologically
active hybrid polypeptides (e.g., hybrid cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes). In one aspect, the original polynucleotides (e.g., an
exemplary nucleic acid of the invention) encode biologically active
polypeptides. In one aspect, a method of the invention produces new
hybrid polypeptides by utilizing cellular processes which integrate
the sequence of the original polynucleotides such that the
resulting hybrid polynucleotide encodes a polypeptide demonstrating
activities derived, but different, from the original biologically
active polypeptides (e.g., cellulase or antibody of the invention).
For example, the original polynucleotides may encode a particular
enzyme (e.g., cellulase) from or found in different microorganisms.
An enzyme encoded by a first polynucleotide from one organism or
variant may, for example, function effectively under a particular
environmental condition, e.g. high salinity. An enzyme encoded by a
second polynucleotide from a different organism or variant may
function effectively under a different environmental condition,
such as extremely high temperatures. A hybrid polynucleotide
containing sequences from the first and second original
polynucleotides may encode an enzyme which exhibits characteristics
of both enzymes encoded by the original polynucleotides. Thus, the
enzyme encoded by the hybrid polynucleotide of the invention may
function effectively under environmental conditions shared by each
of the enzymes encoded by the first and second polynucleotides,
e.g., high salinity and extreme temperatures.
[0477] In one aspect, a hybrid polypeptide generated by a method of
the invention may exhibit specialized enzyme activity not displayed
in the original enzymes. For example, following recombination
and/or reductive reassortment of polynucleotides encoding
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes, the resulting hybrid polypeptide encoded
by a hybrid polynucleotide can be screened for specialized
non-cellulase, e.g., non-endoglucanase, non-cellobiohydrolase
and/or non-beta-glucosidase enzyme activities, e.g., hydrolase,
peptidase, phosphorylase, etc., activities, obtained from each of
the original enzymes. In one aspect, the hybrid polypeptide is
screened to ascertain those chemical functionalities which
distinguish the hybrid polypeptide from the original parent
polypeptides, such as the temperature, pH or salt concentration at
which the hybrid polypeptide functions.
[0478] In one aspect, the invention relates to a method for
producing a biologically active hybrid polypeptide and screening
such a polypeptide for enhanced activity by: [0479] 1) introducing
at least a first polynucleotide in operable linkage and a second
polynucleotide in operable linkage, the at least first
polynucleotide and second polynucleotide sharing at least one
region of partial sequence homology, into a suitable host cell;
[0480] 2) growing the host cell under conditions which promote
sequence reorganization resulting in a hybrid polynucleotide in
operable linkage; [0481] 3) expressing a hybrid polypeptide encoded
by the hybrid polynucleotide; [0482] 4) screening the hybrid
polypeptide under conditions which promote identification of
enhanced biological activity; and [0483] 5) isolating the a
polynucleotide encoding the hybrid polypeptide.
Isolating and Discovering Cellulase Enzymes
[0484] The invention provides methods for isolating and discovering
cellulases, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzymes and the nucleic acids that encode
them. Polynucleotides or enzymes may be isolated from individual
organisms ("isolates"), collections of organisms that have been
grown in defined media ("enrichment cultures"), or, uncultivated
organisms ("environmental samples"). The organisms can be isolated
by, e.g., in vivo biopanning (see discussion, below). The use of a
culture-independent approach to derive polynucleotides encoding
novel bioactivities from environmental samples is most preferable
since it allows one to access untapped resources of biodiversity.
Polynucleotides or enzymes also can be isolated from any one of
numerous organisms, e.g. bacteria. In addition to whole cells,
polynucleotides or enzymes also can be isolated from crude enzyme
preparations derived from cultures of these organisms, e.g.,
bacteria.
[0485] "Environmental libraries" are generated from environmental
samples and represent the collective genomes of naturally occurring
organisms archived in cloning vectors that can be propagated in
suitable prokaryotic hosts. Because the cloned DNA is initially
extracted directly from environmental samples, the libraries are
not limited to the small fraction of prokaryotes that can be grown
in pure culture. Additionally, a normalization of the environmental
DNA present in these samples could allow more equal representation
of the DNA from all of the species present in the original sample.
This can dramatically increase the efficiency of finding
interesting genes from minor constituents of the sample which may
be under-represented by several orders of magnitude compared to the
dominant species.
[0486] In one aspect, gene libraries generated from one or more
uncultivated microorganisms are screened for an activity of
interest. Potential pathways encoding bioactive molecules of
interest are first captured in prokaryotic cells in the form of
gene expression libraries. In one aspect, polynucleotides encoding
activities of interest are isolated from such libraries and
introduced into a host cell. The host cell is grown under
conditions which promote recombination and/or reductive
reassortment creating potentially active biomolecules with novel or
enhanced activities.
[0487] In vivo biopanning may be performed utilizing a FACS-based
and non-optical (e.g., magnetic) based machines. In one aspect,
complex gene libraries are constructed with vectors which contain
elements which stabilize transcribed RNA. For example, the
inclusion of sequences which result in secondary structures such as
hairpins which are designed to flank the transcribed regions of the
RNA would serve to enhance their stability, thus increasing their
half life within the cell. The probe molecules used in the
biopanning process consist of oligonucleotides labeled with
reporter molecules that only fluoresce upon binding of the probe to
a target molecule. These probes are introduced into the recombinant
cells from the library using one of several transformation methods.
The probe molecules bind to the transcribed target mRNA resulting
in DNA/RNA heteroduplex molecules. Binding of the probe to a target
will yield a fluorescent signal which is detected and sorted by the
FACS machine during the screening process.
[0488] In one aspect, subcloning is performed to further isolate
sequences of interest. In subcloning, a portion of DNA is
amplified, digested, generally by restriction enzymes, to cut out
the desired sequence, the desired sequence is ligated into a
recipient vector and is amplified. At each step in subcloning, the
portion is examined for the activity of interest, in order to
ensure that DNA that encodes the structural protein has not been
excluded. The insert may be purified at any step of the subcloning,
for example, by gel electrophoresis prior to ligation into a vector
or where cells containing the recipient vector and cells not
containing the recipient vector are placed on selective media
containing, for example, an antibiotic, which will kill the cells
not containing the recipient vector. Specific methods of subcloning
cDNA inserts into vectors are well-known in the art (Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring
Harbor Laboratory Press (1989)). In another aspect, the enzymes of
the invention are subclones. Such subclones may differ from the
parent clone by, for example, length, a mutation, a tag or a
label.
[0489] The microorganisms from which the polynucleotide may be
discovered, isolated or prepared include prokaryotic
microorganisms, such as Eubacteria and Archaebacteria and lower
eukaryotic microorganisms such as fungi, some algae and protozoa.
Polynucleotides may be discovered, isolated or prepared from
environmental samples in which case the nucleic acid may be
recovered without culturing of an organism or recovered from one or
more cultured organisms. In one aspect, such microorganisms may be
extremophiles, such as hyperthermophiles, psychrophiles,
psychrotrophs, halophiles, barophiles and acidophiles.
Polynucleotides encoding enzymes isolated from extremophilic
microorganisms can be used. Enzymes of this invention can function
at temperatures above 100.degree. C., e.g., as those found in
terrestrial hot springs and deep sea thermal vents, or at
temperatures below 0.degree. C., e.g., as those found in arctic
waters, in a saturated salt environment, e.g., as those found in
the Dead Sea, at pH values around 0, e.g., as those found in coal
deposits and geothermal sulfur-rich springs, or at pH values
greater than 11, e.g., as those found in sewage sludge. In one
aspect, enzymes of the invention have high activity throughout a
wide range of temperatures and pHs.
[0490] Polynucleotides selected and isolated as hereinabove
described are introduced into a suitable host cell. A suitable host
cell is any cell which is capable of promoting recombination and/or
reductive reassortment. The selected polynucleotides are in one
aspect already in a vector which includes appropriate control
sequences. The host cell can be a higher eukaryotic cell, such as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
or in one aspect, the host cell can be a prokaryotic cell, such as
a bacterial cell. Introduction of the construct into the host cell
can be effected by calcium phosphate transfection, DEAE-Dextran
mediated transfection, or electroporation.
[0491] Exemplary hosts include bacterial cells, such as E. coli,
Streptomyces, Salmonella typhimurium; fungal cells, such as yeast;
insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells
such as CHO, COS or Bowes melanoma; adenoviruses; and plant cells;
see discussion, above. The selection of an appropriate host is
deemed to be within the scope of those skilled in the art from the
teachings herein.
[0492] Various mammalian cell culture systems can be employed to
express recombinant protein; examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described in "SV40-transformed simian cells support the replication
of early SV40 mutants" (Gluzman, 1981) and other cell lines capable
of expressing a compatible vector, for example, the C127, 3T3, CHO,
HeLa and BHK cell lines. Mammalian expression vectors can comprise
an origin of replication, a suitable promoter and enhancer and also
any necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences and
5' flanking nontranscribed sequences. DNA sequences derived from
the SV40 splice and polyadenylation sites may be used to provide
the required nontranscribed genetic elements.
[0493] In another aspect, nucleic acids, polypeptides and methods
of the invention are used in biochemical pathways, or to generate
novel polynucleotides encoding biochemical pathways from one or
more operons or gene clusters or portions thereof. For example,
bacteria and many eukaryotes have a coordinated mechanism for
regulating genes whose products are involved in related processes.
The genes are clustered, in structures referred to as "gene
clusters," on a single chromosome and are transcribed together
under the control of a single regulatory sequence, including a
single promoter which initiates transcription of the entire
cluster. Thus, a gene cluster is a group of adjacent genes that are
either identical or related, usually as to their function (an
example of a biochemical pathway encoded by gene clusters are
polyketides).
[0494] In one aspect, gene cluster DNA is isolated from different
organisms and ligated into vectors, e.g., vectors containing
expression regulatory sequences which can control and regulate the
production of a detectable protein or protein-related array
activity from the ligated gene clusters. Use of vectors which have
an exceptionally large capacity for exogenous DNA introduction can
be appropriate for use with such gene clusters and are described by
way of example herein to include the f-factor (or fertility factor)
of E. coli. This f-factor of E. coli is a plasmid which affects
high-frequency transfer of itself during conjugation and is ideal
to achieve and stably propagate large DNA fragments, such as gene
clusters from mixed microbial samples. One aspect is to use cloning
vectors, referred to as "fosmids" or bacterial artificial
chromosome (BAC) vectors. These are derived from E. coli f-factor
which is able to stably integrate large segments of genomic DNA.
When integrated with DNA from a mixed uncultured environmental
sample, this makes it possible to achieve large genomic fragments
in the form of a stable "environmental DNA library." Another type
of vector for use in the present invention is a cosmid vector.
Cosmid vectors were originally designed to clone and propagate
large segments of genomic DNA. Cloning into cosmid vectors is
described in detail in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press
(1989). Once ligated into an appropriate vector, two or more
vectors containing different polyketide synthase gene clusters can
be introduced into a suitable host cell. Regions of partial
sequence homology shared by the gene clusters will promote
processes which result in sequence reorganization resulting in a
hybrid gene cluster. The novel hybrid gene cluster can then be
screened for enhanced activities not found in the original gene
clusters.
[0495] Methods for screening for various enzyme activities are
known to those of skill in the art and are discussed throughout the
present specification, see, e.g., Examples 1, 2 and 3, below. Such
methods may be employed when isolating the polypeptides and
polynucleotides of the invention.
[0496] In one aspect, the invention provides methods for
discovering and isolating cellulases, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase, or compounds
to modify the activity of these enzymes, using a whole cell
approach (see discussion, below). Putative clones encoding
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase from genomic DNA library can be screened.
Screening Methodologies and "On-Line" Monitoring Devices
[0497] In practicing the methods of the invention, a variety of
apparatus and methodologies can be used to in conjunction with the
polypeptides and nucleic acids of the invention, e.g., to screen
polypeptides for cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme activity, to screen
compounds as potential modulators, e.g., activators or inhibitors,
of a cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzyme activity, for antibodies that bind
to a polypeptide of the invention, for nucleic acids that hybridize
to a nucleic acid of the invention, to screen for cells expressing
a polypeptide of the invention and the like. In addition to the
array formats described in detail below for screening samples,
alternative formats can also be used to practice the methods of the
invention. Such formats include, for example, mass spectrometers,
chromatographs, e.g., high-throughput HPLC and other forms of
liquid chromatography, and smaller formats, such as 1536-well
plates, 384-well plates and so on. High throughput screening
apparatus can be adapted and used to practice the methods of the
invention, see, e.g., U.S. Patent Application Nos. 20020001809;
20050272044.
[0498] Capillary Arrays
[0499] Nucleic acids or polypeptides of the invention can be
immobilized to or applied to an array. Arrays can be used to screen
for or monitor libraries of compositions (e.g., small molecules,
antibodies, nucleic acids, etc.) for their ability to bind to or
modulate the activity of a nucleic acid or a polypeptide of the
invention. Capillary arrays, such as the GIGAMATRIX.TM., Diversa
Corporation, San Diego, Calif.; and arrays described in, e.g., U.S.
Patent Application No. 20020080350 A1; WO 0231203 A; WO 0244336 A,
provide an alternative apparatus for holding and screening samples.
In one aspect, the capillary array includes a plurality of
capillaries formed into an array of adjacent capillaries, wherein
each capillary comprises at least one wall defining a lumen for
retaining a sample. The lumen may be cylindrical, square, hexagonal
or any other geometric shape so long as the walls form a lumen for
retention of a liquid or sample. The capillaries of the capillary
array can be held together in close proximity to form a planar
structure. The capillaries can be bound together, by being fused
(e.g., where the capillaries are made of glass), glued, bonded, or
clamped side-by-side. Additionally, the capillary array can include
interstitial material disposed between adjacent capillaries in the
array, thereby forming a solid planar device containing a plurality
of through-holes.
[0500] A capillary array can be formed of any number of individual
capillaries, for example, a range from 100 to 4,000,000
capillaries. Further, a capillary array having about 100,000 or
more individual capillaries can be formed into the standard size
and shape of a Microtiter.RTM. plate for fitment into standard
laboratory equipment. The lumens are filled manually or
automatically using either capillary action or microinjection using
a thin needle. Samples of interest may subsequently be removed from
individual capillaries for further analysis or characterization.
For example, a thin, needle-like probe is positioned in fluid
communication with a selected capillary to either add or withdraw
material from the lumen.
[0501] In a single-pot screening assay, the assay components are
mixed yielding a solution of interest, prior to insertion into the
capillary array. The lumen is filled by capillary action when at
least a portion of the array is immersed into a solution of
interest. Chemical or biological reactions and/or activity in each
capillary are monitored for detectable events. A detectable event
is often referred to as a "hit", which can usually be distinguished
from "non-hit"producing capillaries by optical detection. Thus,
capillary arrays allow for massively parallel detection of
"hits".
[0502] In a multi-pot screening assay, a polypeptide or nucleic
acid, e.g., a ligand, can be introduced into a first component,
which is introduced into at least a portion of a capillary of a
capillary array. An air bubble can then be introduced into the
capillary behind the first component. A second component can then
be introduced into the capillary, wherein the second component is
separated from the first component by the air bubble. The first and
second components can then be mixed by applying hydrostatic
pressure to both sides of the capillary array to collapse the
bubble. The capillary array is then monitored for a detectable
event resulting from reaction or non-reaction of the two
components.
[0503] In a binding screening assay, a sample of interest can be
introduced as a first liquid labeled with a detectable particle
into a capillary of a capillary array, wherein the lumen of the
capillary is coated with a binding material for binding the
detectable particle to the lumen. The first liquid may then be
removed from the capillary tube, wherein the bound detectable
particle is maintained within the capillary, and a second liquid
may be introduced into the capillary tube. The capillary is then
monitored for a detectable event resulting from reaction or
non-reaction of the particle with the second liquid.
[0504] Arrays, or "Biochips"
[0505] Nucleic acids or polypeptides of the invention can be
immobilized to or applied to an array. Arrays can be used to screen
for or monitor libraries of compositions (e.g., small molecules,
antibodies, nucleic acids, etc.) for their ability to bind to or
modulate the activity of a nucleic acid or a polypeptide of the
invention. For example, in one aspect of the invention, a monitored
parameter is transcript expression of a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme gene. One or more, or, all the transcripts of a cell can be
measured by hybridization of a sample comprising transcripts of the
cell, or, nucleic acids representative of or complementary to
transcripts of a cell, by hybridization to immobilized nucleic
acids on an array, or "biochip." By using an "array" of nucleic
acids on a microchip, some or all of the transcripts of a cell can
be simultaneously quantified. Alternatively, arrays comprising
genomic nucleic acid can also be used to determine the genotype of
a newly engineered strain made by the methods of the invention.
Polypeptide arrays" can also be used to simultaneously quantify a
plurality of proteins. The present invention can be practiced with
any known "array," also referred to as a "microarray" or "nucleic
acid array" or "polypeptide array" or "antibody array" or
"biochip," or variation thereof. Arrays are generically a plurality
of "spots" or "target elements," each target element comprising a
defined amount of one or more biological molecules, e.g.,
oligonucleotides, immobilized onto a defined area of a substrate
surface for specific binding to a sample molecule, e.g., mRNA
transcripts.
[0506] The terms "array" or "microarray" or "biochip" or "chip" as
used herein is a plurality of target elements, each target element
comprising a defined amount of one or more polypeptides (including
antibodies) or nucleic acids immobilized onto a defined area of a
substrate surface, as discussed in further detail, below.
[0507] In practicing the methods of the invention, any known array
and/or method of making and using arrays can be incorporated in
whole or in part, or variations thereof, as described, for example,
in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606;
6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452;
5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752;
5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752;
5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313;
WO 96/17958; see also, e.g., Johnston (1998) Curr. Biol.
8:R171-R174; Schummer (1997) Biotechniques 23:1087-1092; Kern
(1997) Biotechniques 23:120-124; Solinas-Toldo (1997) Genes,
Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature Genetics
Supp. 21:25-32. See also published U.S. patent applications Nos.
20010018642; 20010019827; 20010016322; 20010014449; 20010014448;
20010012537; 20010008765.
Antibodies and Antibody-Based Screening Methods
[0508] The invention provides isolated or recombinant antibodies
that specifically bind to a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme of the
invention. These antibodies can be used to isolate, identify or
quantify the cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzymes of the invention or
related polypeptides. These antibodies can be used to isolate other
polypeptides within the scope the invention or other related
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes. The antibodies can be designed to bind to
an active site of a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme. Thus,
the invention provides methods of inhibiting cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes using the antibodies of the invention (see discussion above
regarding applications for anti-cellulase, e.g.,
anti-endoglucanase, anti-cellobiohydrolase and/or
anti-beta-glucosidase enzyme compositions of the invention).
[0509] The term "antibody" includes a peptide or polypeptide
derived from, modeled after or substantially encoded by an
immunoglobulin gene or immunoglobulin genes, or fragments thereof,
capable of specifically binding an antigen or epitope, see, e.g.
Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven
Press, N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273;
Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term
antibody includes antigen-binding portions, i.e., "antigen binding
sites," (e.g., fragments, subsequences, complementarity determining
regions (CDRs)) that retain capacity to bind antigen, including (i)
a Fab fragment, a monovalent fragment consisting of the VL, VH, CL
and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab fragments linked by a disulfide bridge at the
hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains; (iv) a Fv fragment consisting of the VL and VH domains of
a single arm of an antibody, (v) a dAb fragment (Ward et al.,
(1989) Nature 341:544-546), which consists of a VH domain; and (vi)
an isolated complementarity determining region (CDR). Single chain
antibodies are also included by reference in the term
"antibody."
[0510] The invention provides fragments of the enzymes of the
invention (e.g., peptides) including immunogenic fragments (e.g.,
subsequences) of a polypeptide of the invention. The invention
provides compositions comprising a polypeptide or peptide of the
invention and adjuvants or carriers and the like.
[0511] The antibodies can be used in immunoprecipitation, staining,
immunoaffinity columns, and the like. If desired, nucleic acid
sequences encoding for specific antigens can be generated by
immunization followed by isolation of polypeptide or nucleic acid,
amplification or cloning and immobilization of polypeptide onto an
array of the invention. Alternatively, the methods of the invention
can be used to modify the structure of an antibody produced by a
cell to be modified, e.g., an antibody's affinity can be increased
or decreased. Furthermore, the ability to make or modify antibodies
can be a phenotype engineered into a cell by the methods of the
invention.
[0512] Methods of immunization, producing and isolating antibodies
(polyclonal and monoclonal) are known to those of skill in the art
and described in the scientific and patent literature, see, e.g.,
Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y.
(1991); Stites (eds.) BASIC AND CLINICAL IMMUNOLOGY (7th ed.) Lange
Medical Publications, Los Altos, Calif. ("Stites"); Goding,
MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.) Academic
Press, New York, N.Y. (1986); Kohler (1975) Nature 256:495; Harlow
(1988) ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor
Publications, New York. Antibodies also can be generated in vitro,
e.g., using recombinant antibody binding site expressing phage
display libraries, in addition to the traditional in vivo methods
using animals. See, e.g., Hoogenboom (1997) Trends Biotechnol.
15:62-70; Katz (1997) Annu. Rev. Biophys. Biomol. Struct.
26:27-45.
[0513] The polypeptides of the invention or fragments comprising at
least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof, may also be used to generate
antibodies which bind specifically to the polypeptides or
fragments. The resulting antibodies may be used in immunoaffinity
chromatography procedures to isolate or purify the polypeptide or
to determine whether the polypeptide is present in a biological
sample. In such procedures, a protein preparation, such as an
extract, or a biological sample is contacted with an antibody
capable of specifically binding to one of the polypeptides of the
invention, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.
[0514] In immunoaffinity procedures, the antibody is attached to a
solid support, such as a bead or other column matrix. The protein
preparation is placed in contact with the antibody under conditions
in which the antibody specifically binds to one of the polypeptides
of the invention, or fragment thereof. After a wash to remove
non-specifically bound proteins, the specifically bound
polypeptides are eluted.
[0515] The ability of proteins in a biological sample to bind to
the antibody may be determined using any of a variety of procedures
familiar to those skilled in the art. For example, binding may be
determined by labeling the antibody with a detectable label such as
a fluorescent agent, an enzymatic label, or a radioisotope.
Alternatively, binding of the antibody to the sample may be
detected using a secondary antibody having such a detectable label
thereon. Particular assays include ELISA assays, sandwich assays,
radioimmunoassays and Western Blots.
[0516] Polyclonal antibodies generated against the polypeptides of
the invention, or fragments comprising at least 5, 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof can
be obtained by direct injection of the polypeptides into an animal
or by administering the polypeptides to an animal, for example, a
nonhuman. The antibody so obtained can bind the polypeptide itself.
In this manner, even a sequence encoding only a fragment of the
polypeptide can be used to generate antibodies which may bind to
the whole native polypeptide. Such antibodies can then be used to
isolate the polypeptide from cells expressing that polypeptide.
[0517] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, Nature, 256:495-497, 1975), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
4:72, 1983) and the EBV-hybridoma technique (Cole, et al., 1985, in
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96).
[0518] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to the polypeptides of the invention, or
fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50,
75, 100, or 150 consecutive amino acids thereof. Alternatively,
transgenic mice may be used to express humanized antibodies to
these polypeptides or fragments thereof.
[0519] Antibodies generated against the polypeptides of the
invention, or fragments comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof may be
used in screening for similar polypeptides from other organisms and
samples. In such techniques, polypeptides from the organism are
contacted with the antibody and those polypeptides which
specifically bind the antibody are detected. Any of the procedures
described above may be used to detect antibody binding. One such
screening assay is described in "Methods for Measuring Cellulase
Activities", Methods in Enzymology, Vol 160, pp. 87-116.
Kits
[0520] The invention provides kits comprising the compositions,
e.g., nucleic acids, expression cassettes, vectors, cells,
transgenic seeds or plants or plant parts, polypeptides (e.g., a
cellulase enzyme) and/or antibodies of the invention. The kits also
can contain instructional material teaching the methodologies and
industrial, medical and dietary uses of the invention, as described
herein.
Whole Cell Engineering and Measuring Metabolic Parameters
[0521] The methods of the invention provide whole cell evolution,
or whole cell engineering, of a cell to develop a new cell strain
having a new phenotype, e.g., a new or modified cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme activity, by modifying the genetic composition of the cell.
See U.S. patent application no. 20040033975.
[0522] The genetic composition can be modified by addition to the
cell of a nucleic acid of the invention, e.g., a coding sequence
for an enzyme of the invention. See, e.g., WO0229032;
WO0196551.
[0523] To detect the new phenotype, at least one metabolic
parameter of a modified cell is monitored in the cell in a "real
time" or "on-line" time frame. In one aspect, a plurality of cells,
such as a cell culture, is monitored in "real time" or "on-line."
In one aspect, a plurality of metabolic parameters is monitored in
"real time" or "on-line." Metabolic parameters can be monitored
using the cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzymes of the invention.
[0524] Metabolic flux analysis (MFA) is based on a known
biochemistry framework. A linearly independent metabolic matrix is
constructed based on the law of mass conservation and on the
pseudo-steady state hypothesis (PSSH) on the intracellular
metabolites. In practicing the methods of the invention, metabolic
networks are established, including the: [0525] identity of all
pathway substrates, products and intermediary metabolites [0526]
identity of all the chemical reactions interconverting the pathway
metabolites, the stoichiometry of the pathway reactions, [0527]
identity of all the enzymes catalyzing the reactions, the enzyme
reaction kinetics, [0528] the regulatory interactions between
pathway components, e.g. allosteric interactions, enzyme-enzyme
interactions etc, [0529] intracellular compartmentalization of
enzymes or any other supramolecular organization of the enzymes,
and, [0530] the presence of any concentration gradients of
metabolites, enzymes or effector molecules or diffusion barriers to
their movement.
[0531] Once the metabolic network for a given strain is built,
mathematic presentation by matrix notion can be introduced to
estimate the intracellular metabolic fluxes if the on-line
metabolome data is available. Metabolic phenotype relies on the
changes of the whole metabolic network within a cell. Metabolic
phenotype relies on the change of pathway utilization with respect
to environmental conditions, genetic regulation, developmental
state and the genotype, etc. In one aspect of the methods of the
invention, after the on-line MFA calculation, the dynamic behavior
of the cells, their phenotype and other properties are analyzed by
investigating the pathway utilization. For example, if the glucose
supply is increased and the oxygen decreased during the yeast
fermentation, the utilization of respiratory pathways will be
reduced and/or stopped, and the utilization of the fermentative
pathways will dominate. Control of physiological state of cell
cultures will become possible after the pathway analysis. The
methods of the invention can help determine how to manipulate the
fermentation by determining how to change the substrate supply,
temperature, use of inducers, etc. to control the physiological
state of cells to move along desirable direction. In practicing the
methods of the invention, the MFA results can also be compared with
transcriptome and proteome data to design experiments and protocols
for metabolic engineering or gene shuffling, etc.
[0532] In practicing the methods of the invention, any modified or
new phenotype can be conferred and detected, including new or
improved characteristics in the cell. Any aspect of metabolism or
growth can be monitored.
[0533] Monitoring Expression of an mRNA Transcript
[0534] In one aspect of the invention, the engineered phenotype
comprises increasing or decreasing the expression of an mRNA
transcript (e.g., a cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
message) or generating new (e.g., cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme)
transcripts in a cell. This increased or decreased expression can
be traced by testing for the presence of a cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme of the invention or by cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity assays. mRNA transcripts, or messages, also can be
detected and quantified by any method known in the art, including,
e.g., Northern blots, quantitative amplification reactions,
hybridization to arrays, and the like. Quantitative amplification
reactions include, e.g., quantitative PCR, including, e.g.,
quantitative reverse transcription polymerase chain reaction, or
RT-PCR; quantitative real time RT-PCR, or "real-time kinetic
RT-PCR" (see, e.g., Kreuzer (2001) Br. J. Haematol. 114:313-318;
Xia (2001) Transplantation 72:907-914).
[0535] In one aspect of the invention, the engineered phenotype is
generated by knocking out expression of a homologous gene. The
gene's coding sequence or one or more transcriptional control
elements can be knocked out, e.g., promoters or enhancers. Thus,
the expression of a transcript can be completely ablated or only
decreased.
[0536] In one aspect of the invention, the engineered phenotype
comprises increasing the expression of a homologous gene. This can
be effected by knocking out of a negative control element,
including a transcriptional regulatory element acting in cis- or
trans-, or, mutagenizing a positive control element. One or more,
or, all the transcripts of a cell can be measured by hybridization
of a sample comprising transcripts of the cell, or, nucleic acids
representative of or complementary to transcripts of a cell, by
hybridization to immobilized nucleic acids on an array.
[0537] Monitoring Expression of a Polypeptides, Peptides and Amino
Acids
[0538] In one aspect of the invention, the engineered phenotype
comprises increasing or decreasing the expression of a polypeptide
(e.g., a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme) or generating new
polypeptides in a cell. This increased or decreased expression can
be traced by determining the amount of cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme present or by cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
activity assays. Polypeptides, peptides and amino acids also can be
detected and quantified by any method known in the art, including,
e.g., nuclear magnetic resonance (NMR), spectrophotometry,
radiography (protein radiolabeling), electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), hyperdiffusion chromatography,
various immunological methods, e.g. immunoprecipitation,
immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs),
enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent
assays, gel electrophoresis (e.g., SDS-PAGE), staining with
antibodies, fluorescent activated cell sorter (FACS), pyrolysis
mass spectrometry, Fourier-Transform Infrared Spectrometry, Raman
spectrometry, GC-MS, and LC-Electrospray and
cap-LC-tandem-electrospray mass spectrometries, and the like. Novel
bioactivities can also be screened using methods, or variations
thereof, described in U.S. Pat. No. 6,057,103. Furthermore, as
discussed below in detail, one or more, or, all the polypeptides of
a cell can be measured using a protein array.
Industrial, Energy, Pharmaceutical and Other Applications
[0539] Polypeptides of the invention (e.g., having cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase) can catalyze the breakdown of cellulose. The
enzymes of the invention can be highly selective catalysts. The
invention provides industrial processes using enzymes of the
invention, e.g., in the pharmaceutical or nutrient (diet)
supplement industry, the energy industry (e.g., to make "clean"
biofuels), in the food and feed industries, e.g., in methods for
making food and feed products and food and feed additives. In one
aspect, the invention provides processes using enzymes of the
invention in the medical industry, e.g., to make pharmaceuticals or
dietary aids or supplements, or food supplements and additives. In
addition, the invention provides methods for using the enzymes of
the invention in bioethanol, including "clean" fuel,
production.
[0540] The enzymes of the invention can catalyze reactions with
exquisite stereo-, regio- and chemo-selectivities. The cellulase,
e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes of the invention can be engineered to
function in various solvents, operate at extreme pHs (for example,
high pHs and low pHs) extreme temperatures (for example, high
temperatures and low temperatures), extreme salinity levels (for
example, high salinity and low salinity) and catalyze reactions
with compounds that are structurally unrelated to their natural,
physiological substrates.
[0541] Biomass Conversion and Production of Clean Bio Fuels
[0542] The invention provides enzymes and methods for the
conversion of biomass (e.g., lignocellulosic materials) to fuels
(e.g., bioethanol) and chemicals. Thus, the compositions and
methods of the invention provide effective and sustainable
alternatives to use of petroleum-based products. The invention
provides organisms expressing enzymes of the invention for
participation in chemical cycles involving natural biomass
conversion. In one aspect, enzymes and methods for the conversion
are used in enzyme ensembles for the efficient depolymerization of
cellulosic and hemicellulosic polymers to metabolizable carbon
moieties. As discussed above, the invention provides methods for
discovering and implementing the most effective of enzymes to
enable these important new "biomass conversion" and alternative
energy industrial processes.
[0543] In one aspect, the polypeptides of the invention, e.g.,
proteins having cellulase activity, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity, are
used in processes for converting lignocellulosic biomass to
ethanol. The invention also provides processes for making ethanol
("bioethanol") from compositions comprising lignocellulosic
biomass. The lignocellulose biomass material can be obtained from
agricultural crops, as a byproduct of food or feed production, or
as lignocellulosic waste products, such as plant residues and waste
paper. Examples of suitable plant residues for treatment with
polypeptides of the invention include stems, leaves, hulls, husks,
cobs and the like, as well as wood, wood chips, wood pulp, and
sawdust. Examples of paper waste suitable for treatment with
polypeptides of the invention include discard photocopy paper,
computer printer paper, notebook paper, notepad paper, typewriter
paper, and the like, as well as newspapers, magazines, cardboard,
and paper-based packaging materials.
[0544] In one aspect, the enzymes and methods of the invention can
be used in conjunction with more "traditional" means of making
ethanol from biomass, e.g., as methods comprising hydrolyzing
lignocellulosic materials by subjecting dried lignocellulosic
material in a reactor to a catalyst comprised of a dilute solution
of a strong acid and a metal salt; this can lower the activation
energy, or the temperature, of cellulose hydrolysis to obtain
higher sugar yields; see, e.g., U.S. Pat. Nos. 6,660,506;
6,423,145.
[0545] Another exemplary method that incorporated use of enzymes of
the invention comprises hydrolyzing lignocellulosic material
containing hemicellulose, cellulose and lignin by subjecting the
material to a first stage hydrolysis step in an aqueous medium at a
temperature and a pressure chosen to effect primarily
depolymerization of hemicellulose without major depolymerization of
cellulose to glucose. This step results in a slurry in which the
liquid aqueous phase contains dissolved monosaccharides resulting
from depolymerization of hemicellulose and a solid phase containing
cellulose and lignin. A second stage hydrolysis step can comprise
conditions such that at least a major portion of the cellulose is
depolymerized, such step resulting in a liquid aqueous phase
containing dissolved/soluble depolymerization products of
cellulose. See, e.g., U.S. Pat. No. 5,536,325. Enzymes of the
invention can be added at any stage of this exemplary process.
[0546] Another exemplary method that incorporated use of enzymes of
the invention comprises processing a lignocellulose-containing
biomass material by one or more stages of dilute acid hydrolysis
with about 0.4% to 2% strong acid; and treating an unreacted solid
lignocellulosic component of the acid hydrolyzed biomass material
by alkaline delignification to produce precursors for biodegradable
thermoplastics and derivatives. See, e.g., U.S. Pat. No. 6,409,841.
Enzymes of the invention can be added at any stage of this
exemplary process.
[0547] Another exemplary method that incorporated use of enzymes of
the invention comprises prehydrolyzing lignocellulosic material in
a prehydrolysis reactor; adding an acidic liquid to the solid
lignocellulosic material to make a mixture; heating the mixture to
reaction temperature; maintaining reaction temperature for time
sufficient to fractionate the lignocellulosic material into a
solubilized portion containing at least about 20% of the lignin
from the lignocellulosic material and a solid fraction containing
cellulose; removing a solubilized portion from the solid fraction
while at or near reaction temperature wherein the cellulose in the
solid fraction is rendered more amenable to enzymatic digestion;
and recovering a solubilized portion. See, e.g., U.S. Pat. No.
5,705,369. Enzymes of the invention can be added at any stage of
this exemplary process.
[0548] The invention provides methods for making motor fuel
compositions (e.g., for spark ignition motors) based on liquid
hydrocarbons blended with a fuel grade alcohol made by using an
enzyme or a method of the invention. In one aspect, the fuels made
by use of an enzyme of the invention comprise, e.g., coal gas
liquid- or natural gas liquid-ethanol blends. In one aspect, a
co-solvent is biomass-derived 2-methyltetrahydrofuran (MTHF). See,
e.g., U.S. Pat. No. 6,712,866.
[0549] Methods of the invention for the enzymatic degradation of
lignocellulose, e.g., for production of ethanol from
lignocellulosic material, can also comprise use of ultrasonic
treatment of the biomass material; see, e.g., U.S. Pat. No.
6,333,181.
[0550] Another exemplary process for making a biofuel comprising
ethanol using enzymes of the invention comprises pretreating a
starting material comprising a lignocellulosic feedstock comprising
at least hemicellulose and cellulose. In one aspect, the starting
material comprises potatoes, soybean (rapeseed), barley, rye, corn,
oats, wheat, beets or sugar cane or a component or waste or food or
feed production byproduct. The starting material ("feedstock") is
reacted at conditions which disrupt the plant's fiber structure to
effect at least a partial hydrolysis of the hemicellulose and
cellulose. Disruptive conditions can comprise, e.g., subjecting the
starting material to an average temperature of 180.degree. C. to
270.degree. C. at pH 0.5 to 2.5 for a period of about 5 seconds to
60 minutes; or, temperature of 220.degree. C. to 270.degree. C., at
pH 0.5 to 2.5 for a period of 5 seconds to 120 seconds, or
equivalent. This generates a feedstock with increased accessibility
to being digested by an enzyme, e.g., a cellulase enzyme of the
invention. U.S. Pat. No. 6,090,595.
[0551] Exemplary conditions for cellulase hydrolysis of
lignocellulosic material include reactions at temperatures between
about 30.degree. C. and 48.degree. C., and/or a pH between about
4.0 and 6.0. Other exemplary conditions include a temperature
between about 30.degree. C. and 60.degree. C. and a pH between
about 4.0 and 8.0.
[0552] Animal Feeds and Food or Feed Additives
[0553] In addition to providing dietary aids or supplements, or
food supplements and additives for human use, the invention also
provides compositions and methods for treating animal feeds and
foods and food or feed additives using a polypeptide of the
invention, e.g., a protein having cellulase activity, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes of the invention, and/or the antibodies of the invention.
The invention provides animal feeds, foods, and additives
comprising cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzymes of the invention and/or
antibodies of the invention. The animal can be any farm animal or
any animal.
[0554] The animal feed additive of the invention may be a
granulated enzyme product that may readily be mixed with feed
components. Alternatively, feed additives of the invention can form
a component of a pre-mix. The granulated enzyme product of the
invention may be coated or uncoated. The particle size of the
enzyme granulates can be compatible with that of feed and pre-mix
components. This provides a safe and convenient mean of
incorporating enzymes into feeds. Alternatively, the animal feed
additive of the invention may be a stabilized liquid composition.
This may be an aqueous or oil-based slurry. See, e.g., U.S. Pat.
No. 6,245,546.
[0555] Cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzymes of the present invention, in the
modification of animal feed or a food, can process the food or feed
either in vitro (by modifying components of the feed or food) or in
vivo. Polypeptides of the invention can be added to animal feed or
food compositions.
[0556] In one aspect, an enzyme of the invention is added in
combination with another enzyme, e.g., beta-galactosidases,
catalases, laccases, other cellulases, endoglycosidases,
endo-beta-1,4-laccases, amyloglucosidases, other glucosidases,
glucose isomerases, glycosyltransferases, lipases, phospholipases,
lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases,
cutinases, peroxidases, amylases, glucoamylases, pectinases,
reductases, oxidases, decarboxylases, phenoloxidases, ligninases,
pullulanases, arabinanases, hemicellulases, mannanases,
xylolaccases, xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, peptidases,
proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, other cellobiohydrolases and/or transglutaminases.
These enzyme digestion products are more digestible by the animal.
Thus, cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzymes of the invention can contribute to
the available energy of the feed or food, or to the digestibility
of the food or feed by breaking down cellulose.
[0557] In another aspect, cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme of the
invention can be supplied by expressing the enzymes directly in
transgenic feed crops (as, e.g., transgenic plants, seeds and the
like), such as grains, cereals, corn, soy bean, rape seed, lupin
and the like. As discussed above, the invention provides transgenic
plants, plant parts and plant cells comprising a nucleic acid
sequence encoding a polypeptide of the invention. In one aspect,
the nucleic acid is expressed such that the cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzyme of the invention is produced in recoverable quantities. The
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzyme can be recovered from any plant or plant
part. Alternatively, the plant or plant part containing the
recombinant polypeptide can be used as such for improving the
quality of a food or feed, e.g., improving nutritional value,
palatability, etc.
[0558] In one aspect, the enzyme delivery matrix of the invention
is in the form of discrete plural particles, pellets or granules.
By "granules" is meant particles that are compressed or compacted,
such as by a pelletizing, extrusion, or similar compacting to
remove water from the matrix. Such compression or compacting of the
particles also promotes intraparticle cohesion of the particles.
For example, the granules can be prepared by pelletizing the
grain-based substrate in a pellet mill. The pellets prepared
thereby are ground or crumbled to a granule size suitable for use
as an adjuvant in animal feed. Since the matrix is itself approved
for use in animal feed, it can be used as a diluent for delivery of
enzymes in animal feed.
[0559] In one aspect, the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme
contained in the invention enzyme delivery matrix and methods is a
thermostable cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme, as described herein, so
as to resist inactivation of the cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzyme during
manufacture where elevated temperatures and/or steam may be
employed to prepare the palletized enzyme delivery matrix. During
digestion of feed containing the invention enzyme delivery matrix,
aqueous digestive fluids will cause release of the active enzyme.
Other types of thermostable enzymes and nutritional supplements
that are thermostable can also be incorporated in the delivery
matrix for release under any type of aqueous conditions.
[0560] In one aspect, a coating is applied to the enzyme matrix
particles for many different purposes, such as to add a flavor or
nutrition supplement to animal feed, to delay release of animal
feed supplements and enzymes in gastric conditions, and the like.
In one aspect, the coating is applied to achieve a functional goal,
for example, whenever it is desirable to slow release of the enzyme
from the matrix particles or to control the conditions under which
the enzyme will be released. The composition of the coating
material can be such that it is selectively broken down by an agent
to which it is susceptible (such as heat, acid or base, enzymes or
other chemicals). Alternatively, two or more coatings susceptible
to different such breakdown agents may be consecutively applied to
the matrix particles.
[0561] The invention is also directed towards a process for
preparing an enzyme-releasing matrix. In accordance with the
invention, the process comprises providing discrete plural
particles of a grain-based substrate in a particle size suitable
for use as an enzyme-releasing matrix, wherein the particles
comprise a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme encoded by an amino acid
sequence of the invention. In one aspect, the process includes
compacting or compressing the particles of enzyme-releasing matrix
into granules, which most in one aspect is accomplished by
pelletizing. The mold inhibitor and cohesiveness agent, when used,
can be added at any suitable time, and in one aspect are mixed with
the grain-based substrate in the desired proportions prior to
pelletizing of the grain-based substrate. Moisture content in the
pellet mill feed in one aspect is in the ranges set forth above
with respect to the moisture content in the finished product, and
in one aspect is about 14-15%. In one aspect, moisture is added to
the feedstock in the form of an aqueous preparation of the enzyme
to bring the feedstock to this moisture content. The temperature in
the pellet mill in one aspect is brought to about 82.degree. C.
with steam. The pellet mill may be operated under any conditions
that impart sufficient work to the feedstock to provide pellets.
The pelleting process itself is a cost-effective process for
removing water from the enzyme-containing composition.
[0562] The compositions and methods of the invention can be
practiced in conjunction with administration of prebiotics, which
are high molecular weight sugars, e.g., fructo-oligosaccharides
(FOS); galacto-oligosaccharides (GOS), GRAS (Generally Recognized
As Safe) material. These prebiotics can be metabolized by some
probiotic lactic acid bacteria (LAB). They are non-digestible by
the majority of intestinal microbes.
[0563] Treating Foods and Food Processing
[0564] The invention provides foods and feeds comprising enzymes of
the invention, and methods for using enzymes of the invention in
processing foods and feeds. Cellulases, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
invention have numerous applications in food processing industry.
The invention provides methods for hydrolyzing cellulose-comprising
compositions, including, e.g., a plant cell, a bacterial cell, a
yeast cell, an insect cell, or an animal cell, or any plant or
plant part, or any food or feed, a waste product and the like.
[0565] For example, the invention provides feeds or foods
comprising a cellulase, e.g., endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase enzyme the invention, e.g., in a
feed, a liquid, e.g., a beverage (such as a fruit juice or a beer),
a bread or a dough or a bread product, or a drink (e.g., a beer) or
a beverage precursor (e.g., a wort).
[0566] The food treatment processes of the invention can also
include the use of any combination of other enzymes such as
tryptophanases or tyrosine decarboxylases, laccases, catalases,
laccases, other cellulases, endoglycosidases,
endo-beta-1,4-laccases, amyloglucosidases, other glucosidases,
glucose isomerases, glycosyltransferases, lipases, phospholipases,
lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases,
cutinases, peroxidases, amylases, glucoamylases, pectinases,
reductases, oxidases, decarboxylases, phenoloxidases, ligninases,
pullulanases, arabinanases, hemicellulases, mannanases,
xylolaccases, xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, peptidases,
proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, other cellobiohydrolases and/or
transglutaminases.
[0567] In one aspect, the invention provides enzymes and processes
for hydrolyzing liquid (liquefied) and granular starch. Such starch
can be derived from any source, e.g., beet, cane sugar, potato,
corn, wheat, milo, sorghum, rye or bulgher. The invention applies
to any plant starch source, e.g., a grain starch source, which is
useful in liquefaction (for example, to make bioethanol), including
any other grain or vegetable source known to produce starch
suitable for liquefaction. The methods of the invention comprise
liquefying starch (e.g., making bioethanol) from any natural
material, such as rice, germinated rice, corn, barley, milo, wheat,
legumes, potato, beet, cane sugar and sweet potato. The liquefying
process can substantially hydrolyze the starch to produce a syrup.
The temperature range of the liquefaction can be any liquefaction
temperature which is known to be effective in liquefying starch.
For example, the temperature of the starch can be between about
80.degree. C. to about 115.degree. C., between about 100.degree. C.
to about 110.degree. C., and from about 105.degree. C. to about
108.degree. C. The bioethanols made using the enzymes and processes
of the invention can be used as fuels or in fuels (e.g., auto
fuels), e.g., as discussed below, in addition to their use in (or
for making) foods and feeds, including alcoholic beverages.
[0568] Waste Treatment
[0569] The invention provides enzymes for use in waste treatment.
Cellulases, e.g., endoglucanase, cellobiohydrolase, mannanase
and/or beta-glucosidase enzymes of the invention can be used in a
variety of waste treatment or related industrial applications,
e.g., in waste treatment related to biomass conversion to generate
fuels. For example, in one aspect, the invention provides a solid
and/or liquid waste digestion process using cellulase, e.g.,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
enzymes of the invention. The methods can comprise reducing the
mass and volume of substantially untreated solid waste. Solid waste
can be treated with an enzymatic digestive process in the presence
of an enzymatic solution (including cellulase, e.g., endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase enzymes of the
invention) at a controlled temperature. This results in a reaction
without appreciable bacterial fermentation from added
microorganisms. The solid waste is converted into a liquefied waste
and any residual solid waste. The resulting liquefied waste can be
separated from said any residual solidified waste. See e.g., U.S.
Pat. No. 5,709,796.
[0570] In one aspect, the compositions and methods of the invention
are used for odor removal, odor prevention or odor reduction, e.g.,
in animal waste lagoons, e.g., on swine farms, in other animal
waste management systems, or in any industrial or food processing
application.
[0571] The enzymes and methods for the conversion of biomass (e.g.,
lignocellulosic materials) to fuels (e.g., bioethanol) can
incorporate the treatment/recycling of municipal solid waste
material, including waste obtained directly from a municipality or
municipal solid waste that was previously land-filled and
subsequently recovered, or sewage sludge, e.g., in the form of
sewage sludge cake which contains substantial amounts of cellulosic
material. Since sewage sludge cakes will normally not contain
substantial amounts of recyclable materials (aluminum, glass,
plastics, etc.), they can be directly treated with concentrated
sulfuric acid (to reduce the heavy metal content of the cellulosic
component of the waste) and processed in the ethanol production
system. See, e.g., U.S. Pat. Nos. 6,267,309; 5,975,439.
[0572] Another exemplary method using enzymes of the invention for
recovering organic and inorganic matter from waste material
comprises sterilizing a solid organic matter and softening it by
subjecting it to heat and pressure. This exemplary process may be
carried out by first agitating waste material and then subjecting
it to heat and pressure, which sterilizes it and softens the
organic matter contained therein. In one aspect, after heating
under pressure, the pressure may be suddenly released from a
perforated chamber to forces the softened organic matter outwardly
through perforations of the container, thus separating the organic
matter from the solid inorganic matter. The softened sterilized,
organic matter is then fermented in fermentation chamber, e.g.,
using enzymes of the invention, e.g., to form a mash. The mash may
be subjected to further processing by centrifuge, distillation
column and/or anaerobic digester to recover fuels such as ethanol
and methane, and animal feed supplements. See, e.g., U.S. Pat. No.
6,251,643.
[0573] Enzymes of the invention can also be used in processes,
e.g., pretreatments, to reduce the odor of an industrial waste, or
a waste generated from an animal production facility, and the like.
For example, enzymes of the invention can be used to treat an
animal waste in a waste holding facility to enhance efficient
degradation of large amounts of organic matter with reduced odor.
The process can also include inoculation with sulfide-utilizing
bacteria and organic digesting bacteria and lytic enzymes (in
addition to an enzyme of the invention). See, e.g., U.S. Pat. No.
5,958,758.
[0574] Enzymes of the invention can also be used in mobile systems,
e.g., batch type reactors, for bioremediation of aqueous, hazardous
wastes, e.g., as described in U.S. Pat. No. 5,833,857. Batch type
reactors can be large vessels having circulatory capability wherein
bacteria (e.g., expressing an enzyme of the invention) are
maintained in an efficient state by nutrients being feed into the
reactor. Such systems can be used where effluent can be delivered
to the reactor or the reactor is built into a waste water treatment
system. Enzymes of the invention can also be used in treatment
systems for use at small or temporary remote locations, e.g.,
portable, high volume, highly efficient, versatile waste water
treatment systems.
[0575] The waste treatment processes of the invention can include
the use of any combination of other enzymes such as other
cellulase, e.g., endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase enzymes, catalases, laccases, other cellulases,
endoglycosidases, endo-beta-1,4-laccases, amyloglucosidases, other
glucosidases, glucose isomerases, glycosyltransferases, lipases,
phospholipases, lipooxygenases, beta-laccases,
endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,
glucoamylases, pectinases, reductases, oxidases, decarboxylases,
phenoloxidases, ligninases, pullulanases, phytases, arabinanases,
hemicellulases, mannanases, xylolaccases, xylanases, pectin acetyl
esterases, rhamnogalacturonan acetyl esterases, proteases,
peptidases, proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, other cellobiohydrolases and/or
transglutaminases.
[0576] Detergent Compositions
[0577] The invention provides detergent compositions comprising one
or more polypeptides of the invention (e.g., enzymes having
cellulase, endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity) and methods of making and using these
compositions. The invention incorporates all methods of making and
using detergent compositions, see, e.g., U.S. Pat. Nos. 6,413,928;
6,399,561; 6,365,561; 6,380,147. The detergent compositions can be
a one and two part aqueous composition, a non-aqueous liquid
composition, a cast solid, a granular form, a particulate form, a
compressed tablet, a gel and/or a paste and a slurry form. The
invention also provides methods capable of a rapid removal of gross
food soils, films of food residue and other minor food compositions
using these detergent compositions. Enzymes of the invention can
facilitate the removal of starchy stains by means of catalytic
hydrolysis of the starch polysaccharide. Enzymes of the invention
can be used in dishwashing detergents in textile laundering
detergents.
[0578] The actual active enzyme content depends upon the method of
manufacture of a detergent composition and is not critical,
assuming the detergent solution has the desired enzymatic activity.
In one aspect, the amount of glucosidase present in the final
solution ranges from about 0.001 mg to 0.5 mg per gram of the
detergent composition. The particular enzyme chosen for use in the
process and products of this invention depends upon the conditions
of final utility, including the physical product form, use pH, use
temperature, and soil types to be degraded or altered. The enzyme
can be chosen to provide optimum activity and stability for any
given set of utility conditions. In one aspect, the polypeptides of
the present invention are active in the pH ranges of from about 4
to about 12 and in the temperature range of from about 20.degree.
C. to about 95.degree. C. The detergents of the invention can
comprise cationic, semi-polar nonionic or zwitterionic surfactants;
or, mixtures thereof.
[0579] Enzymes of the present invention (e.g., enzymes having
cellulase, endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity) can be formulated into powdered and
liquid detergents having pH between 4.0 and 12.0 at levels of about
0.01 to about 5% (preferably 0.1% to 0.5%) by weight. These
detergent compositions can also include other enzymes such as known
proteases, cellulases, lipases or endoglycosidases, as well as
builders and stabilizers. The addition of enzymes of the invention
to conventional cleaning compositions does not create any special
use limitation. In other words, any temperature and pH suitable for
the detergent is also suitable for the present compositions as long
as the pH is within the above range, and the temperature is below
the described enzyme's denaturing temperature. In addition, the
polypeptides of the invention can be used in a cleaning composition
without detergents, again either alone or in combination with
builders and stabilizers.
[0580] The present invention provides cleaning compositions
including detergent compositions for cleaning hard surfaces,
detergent compositions for cleaning fabrics, dishwashing
compositions, oral cleaning compositions, denture cleaning
compositions, and contact lens cleaning solutions.
[0581] In one aspect, the invention provides a method for washing
an object comprising contacting the object with a polypeptide of
the invention under conditions sufficient for washing. A
polypeptide of the invention may be included as a detergent
additive. The detergent composition of the invention may, for
example, be formulated as a hand or machine laundry detergent
composition comprising a polypeptide of the invention. A laundry
additive suitable for pre-treatment of stained fabrics can comprise
a polypeptide of the invention. A fabric softener composition can
comprise a polypeptide of the invention. Alternatively, a
polypeptide of the invention can be formulated as a detergent
composition for use in general household hard surface cleaning
operations. In alternative aspects, detergent additives and
detergent compositions of the invention may comprise one or more
other enzymes such as a protease, a lipase, a cutinase, another
glucosidase, a carbohydrase, another cellulase, a pectinase, a
mannanase, an arabinase, a galactanase, a xylanase, an oxidase,
e.g., a lactase, and/or a peroxidase. The properties of the
enzyme(s) of the invention are chosen to be compatible with the
selected detergent (i.e. pH-optimum, compatibility with other
enzymatic and non-enzymatic ingredients, etc.) and the enzyme(s) is
present in effective amounts. In one aspect, enzymes of the
invention are used to remove malodorous materials from fabrics.
Various detergent compositions and methods for making them that can
be used in practicing the invention are described in, e.g., U.S.
Pat. Nos. 6,333,301; 6,329,333; 6,326,341; 6,297,038; 6,309,871;
6,204,232; 6,197,070; 5,856,164.
[0582] The detergents and related processes of the invention can
also include the use of any combination of other enzymes such as
tryptophanases or tyrosine decarboxylases, laccases, catalases,
laccases, other cellulases, endoglycosidases,
endo-beta-1,4-laccases, amyloglucosidases, other glucosidases,
glucose isomerases, glycosyltransferases, lipases, phospholipases,
lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases,
cutinases, peroxidases, amylases, glucoamylases, pectinases,
reductases, oxidases, decarboxylases, phenoloxidases, ligninases,
pullulanases, arabinanases, hemicellulases, mannanases,
xylolaccases, xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, peptidases,
proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, other cellobiohydrolases and/or
transglutaminases.
[0583] Treating Fabrics and Textiles
[0584] The invention provides methods of treating fabrics and
textiles using one or more polypeptides of the invention, e.g.,
enzymes having cellulase, endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase activity. The polypeptides of the
invention can be used in any fabric-treating method, which are well
known in the art, see, e.g., U.S. Pat. No. 6,077,316. For example,
in one aspect, the feel and appearance of a fabric is improved by a
method comprising contacting the fabric with an enzyme of the
invention in a solution. In one aspect, the fabric is treated with
the solution under pressure.
[0585] In one aspect, the enzymes of the invention are applied
during or after the weaving of textiles, or during the desizing
stage, or one or more additional fabric processing steps. During
the weaving of textiles, the threads are exposed to considerable
mechanical strain. Prior to weaving on mechanical looms, warp yarns
are often coated with sizing starch or starch derivatives in order
to increase their tensile strength and to prevent breaking. The
enzymes of the invention can be applied to remove these sizing
starch or starch derivatives. After the textiles have been woven, a
fabric can proceed to a desizing stage. This can be followed by one
or more additional fabric processing steps. Desizing is the act of
removing size from textiles. After weaving, the size coating must
be removed before further processing the fabric in order to ensure
a homogeneous and wash-proof result. The invention provides a
method of desizing comprising enzymatic hydrolysis of the size by
the action of an enzyme of the invention.
[0586] The enzymes of the invention (e.g., enzymes having
cellulase, endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity) can be used to desize fabrics, including
cotton-containing fabrics, as detergent additives, e.g., in aqueous
compositions. The invention provides methods for producing a
stonewashed look on indigo-dyed denim fabric and garments. For the
manufacture of clothes, the fabric can be cut and sewn into clothes
or garments, which is afterwards finished. In particular, for the
manufacture of denim jeans, different enzymatic finishing methods
have been developed. The finishing of denim garment normally is
initiated with an enzymatic desizing step, during which garments
are subjected to the action of amylolytic enzymes in order to
provide softness to the fabric and make the cotton more accessible
to the subsequent enzymatic finishing steps. The invention provides
methods of finishing denim garments (e.g., a "bio-stoning
process"), enzymatic desizing and providing softness to fabrics
using the Enzymes of the invention. The invention provides methods
for quickly softening denim garments in a desizing and/or finishing
process.
[0587] The invention also provides disinfectants comprising enzymes
of the invention (e.g., enzymes having cellulase, endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity).
[0588] The fabric or textile treatment processes of the invention
can also include the use of any combination of other enzymes such
as tryptophanases or tyrosine decarboxylases, laccases, catalases,
laccases, other cellulases, endoglycosidases,
endo-beta-1,4-laccases, amyloglucosidases, other glucosidases,
glucose isomerases, glycosyltransferases, lipases, phospholipases,
lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases,
cutinases, peroxidases, amylases, glucoamylases, pectinases,
reductases, oxidases, decarboxylases, phenoloxidases, ligninases,
pullulanases, arabinanases, hemicellulases, mannanases,
xylolaccases, xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, peptidases,
proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, other cellobiohydrolases and/or
transglutaminases.
[0589] Paper or Pulp Treatment
[0590] The enzymes of the invention (e.g., enzymes having
cellulase, endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity) can be in paper or pulp treatment or
paper deinking. For example, in one aspect, the invention provides
a paper treatment process using enzymes of the invention. In one
aspect, the enzymes of the invention can be used to modify starch
in the paper thereby converting it into a liquefied form. In
another aspect, paper components of recycled photocopied paper
during chemical and enzymatic deinking processes. In one aspect,
Enzymes of the invention can be used in combination with other
enzymes, including other cellulases (including other
endoglucanases, cellobiohydrolases and/or beta-glucosidases). The
wood, paper, paper product or pulp can be treated by the following
three processes: 1) disintegration in the presence of an enzyme of
the invention, 2) disintegration with a deinking chemical and an
enzyme of the invention, and/or 3) disintegration after soaking
with an enzyme of the invention. The recycled paper treated with an
enzyme of the invention can have a higher brightness due to removal
of toner particles as compared to the paper treated with just
cellulase. While the invention is not limited by any particular
mechanism, the effect of an enzyme of the invention may be due to
its behavior as surface-active agents in pulp suspension.
[0591] The invention provides methods of treating paper and paper
pulp using one or more polypeptides of the invention. The
polypeptides of the invention can be used in any paper- or
pulp-treating method, which are well known in the art, see, e.g.,
U.S. Pat. Nos. 6,241,849; 6,066,233; 5,582,681. For example, in one
aspect, the invention provides a method for deinking and
decolorizing a printed paper containing a dye, comprising pulping a
printed paper to obtain a pulp slurry, and dislodging an ink from
the pulp slurry in the presence of an enzyme of the invention
(other enzymes can also be added). In another aspect, the invention
provides a method for enhancing the freeness of pulp, e.g., pulp
made from secondary fiber, by adding an enzymatic mixture
comprising an enzyme of the invention (can also include other
enzymes, e.g., pectinase enzymes) to the pulp and treating under
conditions to cause a reaction to produce an enzymatically treated
pulp. The freeness of the enzymatically treated pulp is increased
from the initial freeness of the secondary fiber pulp without a
loss in brightness.
[0592] The paper, wood or pulp treatment or recycling processes of
the invention can also include the use of any combination of other
enzymes such as tryptophanases or tyrosine decarboxylases,
laccases, catalases, laccases, other cellulases, endoglycosidases,
endo-beta-1,4-laccases, amyloglucosidases, other glucosidases,
glucose isomerases, glycosyltransferases, lipases, phospholipases,
lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases,
cutinases, peroxidases, amylases, glucoamylases, pectinases,
reductases, oxidases, decarboxylases, phenoloxidases, ligninases,
pullulanases, arabinanases, hemicellulases, mannanases,
xylolaccases, xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, peptidases,
proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, other cellobiohydrolases and/or
transglutaminases.
[0593] Repulping: Treatment of Lignocellulosic Materials
[0594] The invention also provides a method for the treatment of
lignocellulosic fibers, wherein the fibers are treated with a
polypeptide of the invention (e.g., enzymes having cellulase,
endoglucanase, cellobiohydrolase, mannanase and/or beta-glucosidase
activity), in an amount which is efficient for improving the fiber
properties. The enzymes of the invention may also be used in the
production or recycling of lignocellulosic materials such as pulp,
paper and cardboard, from starch reinforced waste paper and
cardboard, especially where repulping or recycling occurs at pH
above 7 and where the enzymes of the invention can facilitate the
disintegration of the waste material through degradation of the
reinforcing starch. The enzymes of the invention can be useful in a
process for producing a papermaking pulp from starch-coated printed
paper. The process may be performed as described in, e.g., WO
95/14807. An exemplary process comprises disintegrating the paper
to produce a pulp, treating with a starch-degrading enzyme before,
during or after the disintegrating, and separating ink particles
from the pulp after disintegrating and enzyme treatment. See also
U.S. Pat. No. 6,309,871 and other US patents cited herein. Thus,
the invention includes a method for enzymatic deinking of recycled
paper pulp, wherein the polypeptide is applied in an amount which
is efficient for effective de-inking of the fiber surface.
[0595] Brewing and Fermenting
[0596] The invention provides methods of brewing (e.g., fermenting)
beer comprising an enzyme of the invention, e.g., enzymes having
cellulase, endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity. In one exemplary process,
starch-containing raw materials are disintegrated and processed to
form a malt. An enzyme of the invention is used at any point in the
fermentation process. For example, enzymes of the invention can be
used in the processing of barley malt. The major raw material of
beer brewing is barley malt. This can be a three stage process.
First, the barley grain can be steeped to increase water content,
e.g., to around about 40%. Second, the grain can be germinated by
incubation at 15-25.degree. C. for 3 to 6 days when enzyme
synthesis is stimulated under the control of gibberellins. During
this time enzyme levels rise significantly. In one aspect, enzymes
of the invention are added at this (or any other) stage of the
process. The action of the enzyme results in an increase in
fermentable reducing sugars. This can be expressed as the diastatic
power, DP, which can rise from around 80 to 190 in 5 days at
12.degree. C.
[0597] Enzymes of the invention can be used in any beer producing
process, as described, e.g., in U.S. Pat. Nos. 5,762,991;
5,536,650; 5,405,624; 5,021,246; 4,788,066.
[0598] Increasing the Flow of Production Fluids from a Subterranean
Formation
[0599] The invention also includes a method using an enzyme of the
invention (e.g., enzymes having cellulase, endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity),
wherein the method increases the flow of production fluids from a
subterranean formation by removing viscous, starch-containing,
damaging fluids formed during production operations; these fluids
can be found within the subterranean formation which surrounds a
completed well bore. Thus, this method of the invention results in
production fluids being able to flow from the well bore. This
method of the invention also addresses the problem of damaging
fluids reducing the flow of production fluids from a formation
below expected flow rates. In one aspect, the invention provides
for formulating an enzyme treatment (using an enzyme of the
invention) by blending together an aqueous fluid and a polypeptide
of the invention; pumping the enzyme treatment to a desired
location within the well bore; allowing the enzyme treatment to
degrade the viscous, starch-containing, damaging fluid, whereby the
fluid can be removed from the subterranean formation to the well
surface; and wherein the enzyme treatment is effective to attack
the alpha glucosidic linkages in the starch-containing fluid.
[0600] The subterranean formation enzyme treatment processes of the
invention can also include the use of any combination of other
enzymes such as tryptophanases or tyrosine decarboxylases,
laccases, catalases, laccases, other cellulases, endoglycosidases,
endo-beta-1,4-laccases, amyloglucosidases, other glucosidases,
glucose isomerases, glycosyltransferases, lipases, phospholipases,
lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases,
cutinases, peroxidases, amylases, glucoamylases, pectinases,
reductases, oxidases, decarboxylases, phenoloxidases, ligninases,
pullulanases, arabinanases, hemicellulases, mannanases,
xylolaccases, xylanases, pectin acetyl esterases,
rhamnogalacturonan acetyl esterases, proteases, peptidases,
proteinases, polygalacturonases, rhamnogalacturonases,
galactanases, pectin lyases, transglutaminases, pectin
methylesterases, other cellobiohydrolases and/or
transglutaminases.
[0601] Pharmaceutical Compositions and Dietary Supplements
[0602] The invention also provides pharmaceutical compositions and
dietary supplements (e.g., dietary aids) comprising a cellulase of
the invention (e.g., enzymes having endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity). The
cellulase activity comprises endoglucanase, cellobiohydrolase,
mannanase and/or beta-glucosidase activity. In one aspect, the
pharmaceutical compositions and dietary supplements (e.g., dietary
aids) are formulated for oral ingestion, e.g., to improve the
digestibility of foods and feeds having a high cellulose or
lignocellulosic component.
[0603] Periodontal treatment compounds can comprise an enzyme of
the invention, e.g., as described in U.S. Pat. No. 6,776,979.
Compositions and methods for the treatment or prophylaxis of acidic
gut syndrome can comprise an enzyme of the invention, e.g., as
described in U.S. Pat. No. 6,468,964.
[0604] In another aspect, wound dressings, implants and the like
comprise antimicrobial (e.g., antibiotic-acting) enzymes, including
an enzyme of the invention (including, e.g., exemplary sequences of
the invention). Enzymes of the invention can also be used in
alginate dressings, antimicrobial barrier dressings, burn
dressings, compression bandages, diagnostic tools, gel dressings,
hydro-selective dressings, hydrocellular (foam) dressings,
hydrocolloid dressings, I.V dressings, incise drapes, low adherent
dressings, odor absorbing dressings, paste bandages, post operative
dressings, scar management, skin care, transparent film dressings
and/or wound closure. Enzymes of the invention can be used in wound
cleansing, wound bed preparation, to treat pressure ulcers, leg
ulcers, burns, diabetic foot ulcers, scars, IV fixation, surgical
wounds and minor wounds. Enzymes of the invention can be used to in
sterile enzymatic debriding compositions, e.g., ointments. In
various aspects, the cellulase is formulated as a tablet, gel,
pill, implant, liquid, spray, powder, food, feed pellet or as an
encapsulated formulation.
[0605] Biodefense Applications
[0606] In other aspects, cellulases of the invention (e.g., enzymes
having endoglucanase, cellobiohydrolase, mannanase and/or
beta-glucosidase activity) can be used in biodefense (e.g.,
destruction of spores or bacteria comprising a lignocellulosic
material). Use of cellulases of the invention in biodefense
applications offer a significant benefit, in that they can be very
rapidly developed against any currently unknown or biological
warfare agents of the future. In addition, cellulases of the
invention can be used for decontamination of affected environments.
In aspect, the invention provides a biodefense or bio-detoxifying
agent comprising a polypeptide having a cellulase activity, wherein
the polypeptide comprises a sequence of the invention (including,
e.g., exemplary sequences of the invention), or a polypeptide
encoded by a nucleic acid of the invention (including, e.g.,
exemplary sequences of the invention), wherein optionally the
polypeptide has activity comprising endoglucanase,
cellobiohydrolase, mannanase and/or beta-glucosidase activity.
REFERENCE LIST
[0607] 1. Sambrook, J. and Russell, D. W. 2001. Molecular Cloning:
A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory
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of phage and cell display. Biotechnology Advances 19, 1-13. 2001.
[0609] 3. Coutinho, P. M. and Henrissat, B. Carbohydrate-Active
Enzymes server at URL:
http://afmb.cnrs-mrs.fr/.about.cazy/CAZY/index.html. 1999. [0610]
4. Felix, C. R. and L. G. Ljungdahl. 1993. The cellulosome: the
exocellular organelle of Clostridium. Annu. Rev. Microbiol
47:791-819:791-819. [0611] 5. Gray, K. A., T. H. Richardson, K.
Kretz, J. M. Short, F. Bartnek, Knowles R., L. Kan, Swanson P. E.,
and Robertson D. E. 2001. Rapid evolution of reversible
denaturation and elevated melting temperature in a microbial
haloalkane dehalogenase. Advanced Synthesis and Catalysis
343:607-617. [0612] 6. Guttman, A., F. T. Chen, R. A. Evangelista,
and N. Cooke. 1996. High-resolution capillary gel electrophoresis
of reducing oligosaccharides labeled with
1-aminopyrene-3,6,8-trisulfonate. Anal. Biochem 233:234-242. [0613]
7. Harjunpaa, V., A. Teleman, A. Koivula, L. Ruohonen, T. T. Teeri,
O. Teleman, and T. Drakenberg. 1996. Cello-oligosaccharide
hydrolysis by cellobiohydrolase II from Trichoderma reesei.
Association and rate constants derived from an analysis of progress
curves. Eur. J Biochem 240:584-591. [0614] 8. Himmel, M. E., M. F.
Ruth, and C. E. Wyman. 1999. Cellulase for commodity products from
cellulosic biomass. Curr. Opin. Biotechnol 10:358-364. [0615] 9.
Kerr, R. A. 1998. GEOLOGY: The Next Oil Crisis Looms Large--and
Perhaps Close. Science 281:1128. [0616] 10. Kerr, R. A. 2000. OIL
OUTLOOK:USGS Optimistic on World Oil Prospects. Science 289:237.
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McGarry, and M. W. Kirschner. 1997. Expression cloning in the test
tube. Science 277:973-974. [0618] 12. Kuritz, T. 1999. An easy
colorimetric assay for screening and qualitative assessment of
deiodination and dehalogenation by bacterial cultures. Lett. Appl
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S. Provost, and J. M. Short. 1993. The use of selection in recovery
of transgenic targets for mutation analysis. Mutat. Res.
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T. A. Jones, H. F. Woldike, M. Schulein, S. G. Withers, and G. J.
Davies. 1998. Crystal structure of the family 7 endoglucanase I
(Cel7B) from Humicola insolens at 2.2 A resolution and
identification of the catalytic nucleophile by trapping of the
covalent glycosyl-enzyme intermediate. Biochem J 335:409-416.
[0621] 15. Richardson, T. H., X. Tan, G. Frey, W. Callen, M.
Cabell, D. Lam, J. Macomber, J. M. Short, D. E. Robertson, and C.
Miller. 2002. A novel, high performance enzyme for starch
liquefaction. Discovery and optimization of a low pH, thermostable
alpha-amylase. J Biol Chem 277:26501-26507. [0622] 16. Sakon, J.,
D. Irwin, D. B. Wilson, and P. A. Karplus. 1997. Structure and
mechanism of endo/exocellulase E4 from Thermomonospora fusca. Nat.
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J. A. Sorge, and W. D. Huse. 1988. Lambda ZAP: a bacteriophage
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Hunsperger, B. M. Chereskin, and J. Messing. 1988. Maize glutamine
synthetase cDNAs: isolation by direct genetic selection in
Escherichia coli. Genetics 120:1111-1123. [0625] 19. Varrot, A., S.
Hastrup, M. Schulein, and G. J. Davies. 1999. Crystal structure of
the catalytic core domain of the family 6 cellobiohydrolase II,
Ce16A, from Humicola insolens, at 1.92 A resolution. Biochem J
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Directed evolution of an aspartate aminotransferase with new
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[0628] The following examples are offered to illustrate, but not to
limit the claimed invention.
EXAMPLES
Example 1
GIGAMATRIX.TM. Screen
[0629] In one aspect, the methods of the invention use Diversa
Corporation's proprietary GIGAMATRIX.TM. platform; see PCT Patent
Publication No. WO 01/38583; U.S. patent application no.
20050046833; 20020080350; U.S. Pat. No. 6,918,738; Design Patent
No. D480,814. For example, in one aspect, GIGAMATRIX.TM. is used in
methods to determine if a polypeptide has cellulase activity and is
within the scope of the invention, or, to identify and isolate a
polypeptide having cellulase activity.
[0630] A GIGAMATRIX.TM. platform can include an ultra-high
throughput screen based on a 100,000 well microplate with the
dimensions of a conventional 96 well plate. In this example, the
GIGAMATRIX.TM. screen was implemented using 2 substrates based on
previously shown activity by CBHs. Methyl-umbelliferyl cellobioside
(MUC) and methylumbelliferyl lactoside (MUL) were tested. Phagemid
versions of the different clones were screened because the
substrate diffuses into cells and fluorescence was thought to be
more easily detectable. A host strain lacking, beta-galactosidase
was used in order to decrease activity on the lactoside substrate.
The lactoside substrate resulted in fewer hits and was deemed more
specific than the cellobiose substrate. In addition, the lactoside
substrate resulted in fewer beta-glucosidase hits. In order to test
the feasibility of using these substrates in a screen, 14 libraries
were chosen for screening based on the fact that these libraries
yielded endoglucanase hits from a previous screening program. Of
the libraries screened, there were a total of 50 primary hits from
11 of the libraries screened. Secondary screening consisted of
plating the clones on agar plates and then colony picking into 384
well plates containing media and MUL. Active clones against MUL are
differentiated from a background of inactive clones. Individual
clones were then grown overnight and fluorescence was measured and
the most active hits were picked for sequencing.
[0631] All genomic clone inserts from hits were sequenced. In
general, the hits were from several different glycosyl hydrolase
families including 1, 2, 5, 6, 10 and 16. Several other hits were
discovered where the open reading frame was not homologous to any
known glycosyl hydrolase families. In addition, some of the hits
encoded GTP cyclohydrolase genes.
TABLE-US-00002 TABLE 1 Summary of GIGAMATRIX .TM. hits Enzyme No.
Open Reading Frame SEQ ID NO: nearest relevant BLAST 1 SEQ ID NO:
22 (encoded by, e.g. SEQ ID NO: 21) ORF 001 - family 5 (cellulase)
1a SEQ ID NO: 24 (encoded by SEQ ID NO: 23) ORF 003 - Family 16 +
CBM 2 SEQ ID NO: 26 (encoded by, e.g. SEQ ID NO: 25) ORF 001 -
family 1 (.beta.-glucosidase) 3 SEQ ID NO: 92 (encoded by, e.g. SEQ
ID NO: 91) ORF 001 - family 3 3a SEQ ID NO: 94 (encoded by, e.g.
SEQ ID NO: 93) ORF 002 - alpha-rhamnosidase 4 SEQ ID NO: 96
(encoded by, e.g. SEQ ID NO: 95) ORF 001 - family 3 4a SEQ ID NO:
98 (encoded by, e.g. SEQ ID NO: 97) ORF 003 - beta-glucuronidase 5
SEQ ID NO: 128 (encoded by, e.g. SEQ ID NO: 127) ORF 004 - short
chain dehydrogenase 5a SEQ ID NO: 130 (encoded by, e.g. SEQ ID NO:
129) ORF 010 - short chain dehydrogenase 6 SEQ ID NO: 116 (encoded
by, e.g. SEQ ID NO: 115) ORF 004 - short chain dehydrogenase 6a SEQ
ID NO: 118 (encoded by, e.g. SEQ ID NO: 117) ORF 011 - short chain
dehydrogenase 7 SEQ ID NO: 40 (encoded by, e.g. SEQ ID NO: 39) ORF
004 - putative oxidoreductase 8 SEQ ID NO: 42 (encoded by, e.g. SEQ
ID NO: 41) ORF 004 - cysteinyl tRNA synthetase 8a SEQ ID NO: 44
(encoded by, e.g. SEQ ID NO: 43) ORF 011 - hypothetical protein 9
SEQ ID NO: 54 (encoded by, e.g. SEQ ID NO: 53) ORF 002 - Radical
SAM family 10 SEQ ID NO: 134 (encoded by, e.g. SEQ ID NO: 133) ORF
006 - family 1 (.beta.-glucosidase) 11 SEQ ID NO: 58 (encoded by,
e.g. SEQ ID NO: 57) ORF 001 - subtilisin like protease 12 SEQ ID
NO: 46 (encoded by, e.g. SEQ ID NO: 45) ORF 006 - family 1
(.beta.-glucosidase) 13 SEQ ID NO: 8 (encoded by, e.g. SEQ ID NO:
7) ORF 003 - Isocitrate dehydrogenase 13a SEQ ID NO: 10 (encoded
by, e.g. SEQ ID NO: 9) ORF 004 - family 10 (xylanase) 14 SEQ ID NO:
48 (encoded by, e.g. SEQ ID NO: 47) ORF 002 - family 1
(.beta.-glucosidase) 14a SEQ ID NO: 50 (encoded by, e.g. SEQ ID NO:
49) ORF 006 - fdhd/narq oxidoreductase 15 SEQ ID NO: 4 (encoded by,
e.g. SEQ ID NO: 3) ORF 008 - family 1 (.beta.-glucosidase) 15a SEQ
ID NO: 6 (encoded by, e.g. SEQ ID NO: 5) ORF 012 - family 6
(cellulase) 16 SEQ ID NO: 136 (encoded by, e.g. SEQ ID NO: 135) ORF
001 - cellulase (glycosyl hydrolase family 5) 17 SEQ ID NO: 56
(encoded by, e.g. SEQ ID NO: 55) ORF 004 - family 1
(.beta.-glucosidase) 18 SEQ ID NO: 126 (encoded by, e.g. SEQ ID NO:
125) ORF 009 - family 1 (.beta.-glucosidase) 19 SEQ ID NO: 120
(encoded by, e.g. SEQ ID NO: 119) ORF 002 - oxidoreductase 19a SEQ
ID NO: 122 (encoded by, e.g. SEQ ID NO: 121) ORF 004 - family 5
(cellulase) 20 SEQ ID NO: 124 (encoded by, e.g. SEQ ID NO: 123) ORF
006 - family 1 (.beta.-glucosidase) 21 SEQ ID NO: 132 (encoded by,
e.g. SEQ ID NO: 131) ORF 007 - family 5 (cellulase) 22 SEQ ID NO:
38 (encoded by, e.g. SEQ ID NO: 37) ORF 011 - family 1
(.beta.-glucosidase) 22a SEQ ID NO: 36 (encoded by, e.g. SEQ ID NO:
35) ORF 007 - family 5 (cellulase) 23 SEQ ID NO: 138 (encoded by,
e.g. SEQ ID NO: 137) ORF 001 - peptidase.sub.- M37 24 SEQ ID NO:
146 (encoded by, e.g. SEQ ID NO: 145) ORF 002 - family 1
(.beta.-glucosidase) 25 SEQ ID NO: 52 (encoded by, e.g. SEQ ID NO:
51) ORF 001 - family 5 (cellulase) 26 SEQ ID NO: 20 (encoded by,
e.g. SEQ ID NO: 19) ORF 008 - family 10 (xylanase) 26a SEQ ID NO:
18 (encoded by, e.g. SEQ ID NO: 17) ORF 005 - .beta.-lactamase 27
SEQ ID NO: 16 (encoded by, e.g. SEQ ID NO: 15) ORF 007 - family 1
(.beta.-glucosidase) 27a SEQ ID NO: 14 (encoded by, e.g. SEQ ID NO:
13) ORF 005 - NADH dependent dehydrogenase 27b SEQ ID NO: 12
(encoded by, e.g. SEQ ID NO: 11) ORF 003 - NAD binding
oxidoreductase 28 SEQ ID NO: 28 (encoded by, e.g. SEQ ID NO: 27)
ORF 002 - family 1 (.beta.-glucosidase) 29 SEQ ID NO: 114 (encoded
by, e.g. SEQ ID NO: 113) ORF 003 - family 10 30 SEQ ID NO: 34
(encoded by, e.g. SEQ ID NO: 33) ORF 006 - family 1
(.beta.-glucosidase) 30a SEQ ID NO: 32 (encoded by, e.g. SEQ ID NO:
31) ORF 002 - cellodextrin phosphorylase 31 SEQ ID NO: 30 (encoded
by, e.g. SEQ ID NO: 29) ORF 004 - family 1 (.beta.-glucosidase) 32
SEQ ID NO: 100 (encoded by, e.g. SEQ ID NO: 99) ORF 012 - family 1
(.beta.-glucosidase) 33 SEQ ID NO: 84 (encoded by, e.g. SEQ ID NO:
83) ORF 008 - dehydrogenase 34 SEQ ID NO: 102 (encoded by, e.g. SEQ
ID NO: 101) ORF 003 - family 5 (cellulase) 35 SEQ ID NO: 140
(encoded by, e.g. SEQ ID NO: 139) ORF 001 - threonine dehydrogenase
36 SEQ ID NO: 142 (encoded by, e.g. SEQ ID NO: 141) ORF 005 -
family 1 (.beta.-glucosidase) 37 SEQ ID NO: 144 (encoded by, e.g.
SEQ ID NO: 143) ORF 003 - family 1 (.beta.-glucosidase) 38 SEQ ID
NO: 2 (encoded by, e.g. SEQ ID NO: 1) ORF 001 - family 1
(.beta.-glucosidase) 39 SEQ ID NO: 86 (encoded by, e.g. SEQ ID NO:
85) ORF 008 - family 1 (.beta.-glucosidase) Abbreviations:
CBM--carbohydrate binding module
Characterization Enzyme and Substrate Activity
[0632] The 39 hits (see Table 1, above) discovered in the
GIGAMATRIX.TM. screen were first screened against cellohexaose to
determine action pattern on a cellulose oligomer. Genomic clones
are defined as clones that have an entire DNA insert potentially
containing multiple open reading frames. For example, in Table 1,
above, one such genomic clone contains two open reading frames
annoted as Enzymes No. 22 and 22a, with said open reading frames
having the sequences as depicted in SEQ ID NO:37 and SEQ ID NO:35,
respectively. Another such genomic clone is contains three open
reading frames, which are annotated as Enzymes 27, 27a and 27b.
Subclones are derived from genomic clones and can contain only a
single open reading frame. Genomic clones were grown overnight in
TB media containing antibiotic, cells were lysed and lysates were
clarified by centrifugation. Subclones are grown to an OD600=0.5
induced with an appropriate inducer and then grown an additional 3
h before lysing the cells and clarifying the lysate. Genomic clones
will generally have less activity than a subclone, but are a more
facile way of assessing activity in a large range of clones.
Initial studies were performed using thin layer chromatography
(TLC) for endpoint reactions usually run for 24 h. Enzymes were
also tested on phosphoric acid swollen cellulose (PASC), which is
crystalline cellulose that is made more amorphous through swelling
by acid treatment.
[0633] A number of cellulases which were cloned from environmental
libraries were active against PASC, but released cellobiose as well
as celltriose and/or glucose. The genomic clones from the
GIGAMATRIX.TM. discovery effort were also tested against PASC and
on cellulosic substrates such as cellohexaose (Seikagaku, Japan).
Thin layer chromatography (TLC) experiments showed that several
genomic clones were able to hydrolyze the cellohexaose, as
illustrated in FIGS. 6 and 7. Of these clones, many were able to
generate glucose as the final product which is consistent with the
fact that they have sequence identity to glycosyl hydrolase family
1, which includes beta-glucosidases. Several enzymes produced
cellobiose and/or larger fragments, but the exact nature of the
product pattern could not be discerned from the TLC experiments, so
a capillary electrophoresis (CE) method was developed.
Example 2
Capillary Electrophoresis
[0634] In some aspects, Capillary Electrophoresis (CE) is used in
assays to screen for enzyme activity, e.g., CE is used in methods
to determine if a polypeptide has cellulase activity and is within
the scope of the invention, or, to identify and isolate a
polypeptide having cellulase activity. Capillary Electrophoresis
(CE) offers the advantages of faster run times and greater assay
sensitivity. The CE method used 1-aminopyrene-3,6,8-trisulfonate
(APTS) as the fluorophore and was optimized for use with sugars and
sugar oligomers (Guttman (1996) High-resolution capillary gel
electrophoresis of reducing oligosaccharides labeled with
1-aminopyrene-3,6,8-trisulfonate. Anal. Biochem 233:234-242).
Enzymes that were shown to be active on cellohexaose were subjected
to tests on phosphoric acid swollen cellulose as well as
cellohexaose. Genes were subcloned, expressed and partially
purified using a nickel-chelating column. Enzymes were incubated
with substrate for 1 h and the products were analyzed using a 10 cm
or 48 cm capillary. Cellohexaose elutes at 2 and 9 minutes for the
10 and 48 cm capillaries respectively. The 48 cm capillary gives
better separation of products in case there are low amounts of
sugar or if there are contaminants in the mixture. The CE method
was implemented for studies on enzymes from the GIGAMATRIX.TM.
discovery that showed good activity on cellohexaose with TLC
detection.
[0635] Enzyme 22/22a (see Table, 1 above) showed good performance
on PASC (data summarized in graph form in FIG. 8), releasing mainly
cellobiose. In addition, enzyme 22/22a was able to release
cellobiose from AVICEL.RTM. Microcrystalline Cellulose (MCC) (FMC
Corporation, Philadelphia, Pa.) (data summarized in graph form in
FIG. 9). Sequence analysis showed that enzyme 22 and enzyme 21 are
.about.92% identical and belong to glycosyl hydrolase family 5.
Family 5 contains mainly endoglucanases, but there are examples of
cellobiohydrolases. CelO from Clostridium thermocellum has been
characterized as a cellobiohydrolase based on activity on release
of only cellobiose from amorphic and crystalline cellulose (Zverlov
(2002) A newly described cellulosomal cellobiohydrolase, CelO, from
Clostridium thermocellum: investigation of the exo-mode of
hydrolysis, and binding capacity to crystalline cellulose.
Microbiology 148:247-255).
[0636] All three of these enzymes, when compared to the
endoglucanase from Acidothermus cellulolyticus have an insertion
that is in close proximity to the substrate binding site. This
insertion could form a loop which encloses the substrate binding
site thus converting this enzyme from an endoglucanase to a
cellobiohydrolase. When these enzymes were tested on cellohexaose
they produced mainly cellobiose with a smaller amount of
cellotriose. These results are explained by the fact that
cellobiohydrolases have the capability to produce both cellobiose
and cellotriose from a cellohexaose substrate (Harjunpaa (1996)
Cello-oligosaccharide hydrolysis by cellobiohydrolase II from
Trichoderma reesei. Association and rate constants derived from an
analysis of progress curves. Eur. J Biochem 240:584-591).
Example 3
Sequence Based Discovery
[0637] The invention provides methods for identifying and isolating
cellulases, e.g., cellobiohydrolases, using sequences of the
invention. In one exemplary method, primers that were homologous to
conserved regions of three glycosyl hydrolase families that contain
cellobiohydrolases were used to screen either polynucleotide
libraries or DNA derived from fungal samples. Primers were designed
towards family 48 conserved regions and 96 libraries were screened
resulting in 1 confirmed hit. In addition, primers were designed
towards family 6 and family 7. Fungal libraries were screened with
these primers, resulting in 1 hit for family 6 and 56 hits for
family 7. One of the family 7 hits was chosen for studies to
extract the full length sequence. The full-length sequence was
successfully obtained and showed 73% identity to
exo-cellobiohydrolase I of Penicillium janthinellum.
Example 4
Genetic Engineering of an Enzyme with Cellobiohydrolase
Activity
[0638] This example described the genetic engineering of an
exemplary enzyme of the invention. This enzyme can be used in the
conversion of biomass to fuels and chemicals, and for making
effective and sustainable alternatives to petroleum-based products.
This enzyme can be expressed in organisms (e.g., microorganisms,
such as bacteria) for its participation in chemical cycles
involving natural biomass conversion. In one aspect, this enzyme is
used in "enzyme ensembles" for the efficient depolymerization of
cellulosic and hemicellulosic polymers to metabolizable carbon
moieties. As discussed above, the invention provides methods for
discovering and implementing the most effective of enzymes to
enable these important new "biomass conversion" and alternative
energy industrial processes.
[0639] Using metagenomic discovery and a non-stochastic method of
directed evolution (called "DIRECTEVOLUTION.RTM., as described,
e.g., in U.S. Pat. No. 6,939,689, which includes Gene Site
Saturation Mutagenesis (GSSM) (as discussed above, see also U.S.
Pat. Nos. 6,171,820 and 6,579,258) and Tunable GeneReassembly (TGR)
(see, e.g., U.S. Pat. No. 6,537,776) technologies. This effort
focused on the discovery and optimization of an important enzyme
component for cellulose reduction to glucose,
cellobiohydrolase.
[0640] An enzyme discovery screen was implemented using Diversa
Corporation's GIGAMATRIX.TM. high throughput expression screening
platform (discussed above) to identify cellobiohydrolases using
methylumbelliferyl cellobioside as substrate. A total of 100
complex environmental libraries were screened resulting in 25
confirmed cellobiohydrolase hits mainly from glycosyl hydrolase
families 5 and 10. These hits were characterized for activity
against AVICEL.RTM. Microcrystalline Cellulose (MCC) (FMC
Corporation, Philadelphia, Pa.). Based on its performance
characteristics, one enzyme, SEQ ID NO:162 (encoded by, e.g., SEQ
ID NO:161) was chosen as a candidate for optimization using Gene
Site Saturation Mutagenesis (GSSM) technology. However, before GSSM
evolution was performed, the signal sequence (amino acids 1 through
30) was removed from SEQ ID NO:162 and a starting methionine was
added. This signal-free sequence, hereinafter called the
"wild-type" and represented by SEQ ID NO:164 (encoded by, e.g., SEQ
ID NO:163), was the parental sequence that was optimized using GSSM
technology. As discussed above, GSSM technology can rapidly mutate
all amino acids in the protein to the 19 other amino acids in a
sequential fashion. Mutants were screened using a fiber-based assay
and potential upmutants representing single amino acid changes were
identified. These upmutants were combined into a new library
representing combinations of the upmutants. This library was
screened resulting in identification of several candidate enzymes
for commercialization.
Research Summary
[0641] GIGAMATRIX.TM. Screen
[0642] The GIGAMATRIX.TM. (GMx) screening platform is an ultra-high
throughput method based on a 100,000 well microplate with the
dimensions of a conventional 96 well plate (see Phase II
application for details). The screen works with fluorescent
substrates. The GMx screen was implemented using 2 substrates based
on previously shown activity by cellulases. Methylumbelliferyl
cellobioside (MUC) was used as the screening substrate. In
addition, resorufin-beta-glucopyranoside was also included in the
screen in order to eliminate clones that have activity on both
substrates and are presumed to be beta-glucosidases.
[0643] Amplified phage or phagemid versions of the target libraries
were screened. Two host strains (CEH6 & GAL631) lacking
beta-galactosidase genes were used in order to decrease endogenous
host activity on the substrates. 100 libraries were chosen for
screening based on the fact that these libraries yielded cellulase
hits from a previous screening program. Of the libraries screened,
there were a total of 355 primary hits from 69 of the libraries
screened.
[0644] Secondary screening consisted of plating the clones on agar
plates and then colony picking into 384 well plates containing
media and methylumbelliferyl cellobioside (MUC) termed a
"breakout". FIG. 10 illustrates in graphic form data showing a
typical GIGAMATRIX.TM. (GMx) breakout. To generate this data,
active clones against MUC (i.e., able to hydrolyze
methylumbelliferyl cellobioside) are differentiated from a
background of inactive clones. Individual clones were then grown
overnight and fluorescence was measured and the most active hits
were picked for sequencing. In FIG. 10, the X axis shows sample
name; Y axis is relative fluorescent units. Positive "hits" were
plated onto agar plates and then colony picked into 384 well plates
containing LB+antibiotic plus 50 .mu.M MUC and grown overnight.
TABLE-US-00003 TABLE 2 Summary of GIGAMATRIX .TM. (GMx) hits Enzyme
No. Open Reading Frame SEQ ID NO: Clone Family Characterization 40
SEQ ID NO: 104 (encoded by, e.g., SEQ ID NO: 103) family 5
(cellulase) 41 SEQ ID NO: 108 (encoded by, e.g., SEQ ID NO: 107)
family 5 (cellulase) 42 SEQ ID NO: 112 (encoded by, e.g., SEQ ID
NO: 111) family 5 (cellulase) H7 SEQ ID NO: 60 (encoded by, e.g.,
SEQ ID NO: 59) family 5 (cellulase) 43 SEQ ID NO: 82 (encoded by,
e.g., SEQ ID NO: 81) family 5 (cellulase) 44 SEQ ID NO: 78 (encoded
by, e.g., SEQ ID NO: 77) family 5 (cellulase) 45 SEQ ID NO: 68
(encoded by, e.g., SEQ ID NO: 67) family 5 (cellulase)-ORF 2 45a
SEQ ID NO: 70 (encoded by, e.g., SEQ ID NO: 69) family 26
(mannanase)- ORF4 46 SEQ ID NO: 74 (encoded by, e.g., SEQ ID NO:
73) family 10 (xylanase) 47 SEQ ID NO: 110 (encoded by, e.g., SEQ
ID NO: 109) family 10 (xylanase) 48 SEQ ID NO: 106 (encoded by,
e.g., SEQ ID NO: 105) family 5 (cellulase) 49 SEQ ID NO: 66
(encoded by, e.g., SEQ ID NO: 65) family 10 (xylanase) 50 SEQ ID
NO: 72 (encoded by, e.g., SEQ ID NO: 71) family 5 (cellulase) 51
SEQ ID NO: 80 (encoded by, e.g., SEQ ID NO: 79) family 5
(cellulase) H8 SEQ ID NO: 62 (encoded by, e.g., SEQ ID NO: 61)
family 5 (cellulase) ORF 1 H8a SEQ ID NO: 64 (encoded by, e.g., SEQ
ID NO: 63) family 5 (cellulase) ORF 4 52 SEQ ID NO: 76 (encoded by,
e.g., SEQ ID NO: 75) family 5 (cellulase) 53 SEQ ID NO: 160
(encoded by, e.g., SEQ ID NO: 159) family 10 (xylanase) 54 SEQ ID
NO: 88 (encoded by, e.g., SEQ ID NO: 87) family 5 (cellulase) 55
SEQ ID NO: 148 (encoded by, e.g., SEQ ID NO: 147) family 10
(xylanase) 56 SEQ ID NO: 90 (encoded by, e.g., SEQ ID NO: 89)
family 5 (cellulase) 57 SEQ ID NO: 152 (encoded by, e.g., SEQ ID
NO: 151) family 5 (cellulase) 58 SEQ ID NO: 150 (encoded by, e.g.,
SEQ ID NO: 149) family 5 (cellulase) 59 SEQ ID NO: 154 (encoded by,
e.g., SEQ ID NO: 153) family 5 (cellulase) H6 SEQ ID NO: 158
(encoded by, e.g., SEQ ID NO: 157) family 5 (cellulase) 60 SEQ ID
NO: 156 (encoded by, e.g., SEQ ID NO: 155) family 5 (cellulase)
[0645] All genomic clone inserts from hits were sequenced. As with
Table 1 above, some genomic clones contained more than one open
reading frame. For example, one such genomic clone contains two
open reading frames annoted as Enzymes No. H8 and H8a, with said
open reading frames having the sequences as depicted in SEQ ID
NO:67 and SEQ ID NO:69, respectively. There was a total of 25
glycosyl hydrolase hits from 17 of the libraries screened. In
general, the hits were from several different glycosyl hydrolase
families including 5 and 10. Table 2 (above) lists the hits and
their identities. Several other hits were discovered where the open
reading frame was not homologous to any known glycosyl hydrolase
families. In addition, some of the hits encoded GTP cyclohydrolase
genes that are known false positives in this system as they create
fluorescence regardless of substrate degradation. Overall the
screen was successful in identifying enzymes that were active on
MUC.
[0646] Characterization
[0647] Genes discovered in the GIGAMATRIX.TM. screen were sequenced
and the data were analyzed. Open reading frames (ORFs) were
annotated using a software system. The ORFs were subcloned into the
appropriate vector(s) with the introduction of DNA encoding
C-terminal His-tags. Construct DNA was transformed into the
appropriate E. coli host(s) and expressed for characterization
studies. The gene products were screened against phosphoric
acid-swollen cellulose (PASC). PASC is crystalline cellulose that
is made more amorphous through swelling by acid treatment. PASC was
prepared from AVICEL.RTM. Microcrystalline Cellulose (MCC).
Subclones were grown, expressed and lysed. Lysates were incubated
with PASC and the reaction products were analyzed using the
bicinchoninic acid (BCA) reducing sugar assay. The most active
subclones were selected for larger scale growth and purification.
The specific activity of these subclones was determined on
PASC.
[0648] The subclones were also analyzed by capillary
electrophoresis (CE). Lysates were incubated with substrate for 30
hours. The reaction products were derivatized with the fluorophore
1-aminopyrene-3,6,8-trisulfonate (APTS). The products were analyzed
using a 48 cm capillary. Cellobiose elutes at 6 minutes. FIG. 11
illustrates in graph form data showing the activity of selected
enzymes against PASC by capillary electrophoresis (CE) analysis.
Samples H9 through H1 are individual clones. In FIG. 11, a number
of samples had reaction product profiles representative of
processive enzymes. A processive enzyme is defined as having a
ratio of cellobiose/(glucose+cellotriose).gtoreq.10. Two potential
processive enzymes that were the most active had specific
activities on PASC of 0.35 and 0.04 U/mg, respectively.
[0649] Fungal CBHs in Pichia
[0650] Genes of newly discovered family 6 & 7 fungal
cellobiohydrolases were transformed into P. pastoris and the
transformations were spread onto solid agar plates. 160 colonies
were selected for each construct. The samples were grown and
induced and the supernatants were incubated with PASC in the
presence of a .beta.-glucosidase. The reaction products were
analyzed using the glucose-oxidase assay. A glycosyl hydrolase
family 6 cellobiohydrolase, was successfully heterologously
expressed in P. pastoris.
[0651] Exo-Endo Acting Cellulase
[0652] The wild-type enzyme, a family 9 glycosyl hydrolase
discovered in an enzyme screen, is a homolog of Thermomonospora
fusca E4. E4 has been shown to have both endo- and exo-activity.
Initial tests of the wild-type enzyme showed it to be active on
both PASC and AVICEL.RTM. Microcrystalline Cellulose (MCC). HPLC
analysis of the reaction products showed the primary products to be
glucose and cellobiose. The wild-type enzyme is a multi-domain
protein which includes a glycosyl hydrolase family 9 catalytic
domain, a family 3 cellulose binding domain, and three bacterial
Ig-like domains that are believed to be involved in cell adhesion.
Three additional subclone variants of the wild-type enzyme were
tested to determine the effects of the domains on activity. The
wild-type enzyme was subcloned with: 1) the catalytic domain alone
(CD); 2) the catalytic and carbohydrate domain (CCD); and 3) the
catalytic and carbohydrate binding domain plus the 11 downstream
amino acids (CCD+11). The full-length protein and the 3 subclone
variants were assayed on AVICEL.RTM. Microcrystalline Cellulose
(MCC) and the reaction products were analyzed by the BCA reducing
sugar assay, and the data is summarized in graphic form in FIG. 12.
The data illustrated in FIG. 12 was generated by BCA of the
wild-type enzyme and truncation mutants incubated with AVICEL.RTM.
Microcrystalline Cellulose (MCC) for 74 hours, 37.degree. C., pH 5.
CBH1 is a positive control. The negative control is the host
without insert.
[0653] The wild-type enzyme, the full-length protein (SEQ ID
NO:164, encoded by, e.g., SEQ ID NO:163), was the most active. The
full length protein was selected for GSSM evolution. The catalytic
and the carbohydrate binding domain were evolved.
[0654] GSSM Screening
[0655] GSSM technology (discussed above) was used to rapidly and
sequentially mutate the amino acids of the catalytic and
carbohydrate binding domain of the target protein into the 19 other
amino acids. The goal of the GSSM screen was to identify mutants
that increased the extent of hydrolysis on insoluble
microcrystalline cellulose. A robotic screening method was
developed to facilitate the GSSM screening process.
[0656] DNA from the mutation constructs was transformed into DH10b
host cells. Individual colonies were picked into 96 well (shallow)
plates containing 150 uL LB/Ampicillin using the automatic colony
picking system. The plates were incubated for 24 hours at
37.degree. C., 400 rpm. 15 uL of culture was transferred from each
well into an induction plate. Each well of the induction plate
contained 135 uL LB/Ampicillin with 1.1 mM IPTG. The induction
plates were incubated for 24 hours at 37.degree. C., 400 rpm. The
plates were centrifuged and the supernatant was discarded.
[0657] The automated portion of the assay began at this point. The
cells were lysed and resuspended by the robot. 150 uL of lysis
buffer (125 uL water plus 25 uL BPER containing 0.2 mg/ml lysozyme
and 20 unit/ml DNase I) was added to each well. 15 uL lysate was
transferred from each well to a reaction plate. Each well of the
reaction plate contained 185 uL of a reaction mix (1% AVICEL.RTM.
Microcrystalline Cellulose (MCC), 50 mM sodium acetate buffer
pH5.0). The reaction plates were incubated at 37.degree. C. for 30
hours with 95% humidity. After incubation, the plates were
centrifuged and 15 uL supernatant was transferred to BCA plates.
The BCA plates contained 50 uL reagent A, 50 uL reagent B, and 80
uL 400 mM Carbonate buffer, pH 10 per well. The plates were covered
with rubber seals and incubated at 80.degree. C. for 30 minutes,
then cooled by centrifugation and the absorbance read at A560.
[0658] Results
[0659] At least 80 random mutation colonies were screened for each
amino acid site. An example of the primary GSSM.TM. screening data
is graphically illustrated in FIG. 13. Column 6 contained the
wildtype samples and column 12 contained the host/vector negative
controls. After a 30 hour incubation with AVICEL.RTM.
Microcrystalline Cellulose (MCC), the signal produced from the
wildtype samples was around 0.53, with a standard deviation at
0.07. The negative control had an average signal at 0.29. Samples
with signal higher than the average of positive controls plus 2
times the standard deviation were deemed primary hits. From this
screening plate, about ten primary hits were selected for the
secondary confirmation screening.
[0660] Primary hits were reconfirmed in a secondary assay. This
assay was the same as the primary screen. Samples were run in
quadruplicate however. An example of the secondary GSSM screening
data is graphically illustrated in FIG. 14. Samples in wells E3-H3,
A4-D4, A7-D7 on average, had higher activity than the wildtype.
These 12 wells correspond to 3 hits since the samples were run in
quadruplicate. These samples were the primary hits shown in wells
E4, G2, and H3 in FIG. 13 (plate 29805-AA89 BCA plate).
[0661] There were 77 hits from the secondary screening. These
samples were sequenced. Thirty five of the samples had amino acid
changes, 22 had transposon insertions, and the rest were wildtype
or had deletions.
[0662] Hits from the secondary screen were further analyzed. The
GSSM upmutants were mapped onto the crystal structure of T. fusca
E4. Samples were prioritized based on amino acid location, amino
acid change and the fold improvement score. Eight upmutants were
selected from the GSSM screening and selected for gene reassembly
evolution, i.e., Tunable GeneReassembly (TGR), discussed above, and
also see, e.g., U.S. Pat. No. 6,537,776.
TABLE-US-00004 TABLE 2 Up-mutants selected for site directed
mutagenesis reassembly. Residue OLD AA NEW AA 89 M R 103 F G 110 P
G 114 Y L 157 A S 481 W F 550 P N 590 G R
Blending of Upmutants
[0663] Using gene reassembly (Tunable GeneReassembly (TGR))
technology, the upmutants shown in Table 2, above, were blended in
order to identify the candidate with the best activity. Activity
assays were the same as for the GSSM screening except reactions
were further diluted to account for increased activity of upmutants
over the wildtype enzyme. FIG. 15 illustrates in graph form data
from mixed, or "blended", GSSM.TM. screening assays.
[0664] In summary, the invention provides enzymes having cellulase
activity having the following sequences based on SEQ ID NO:164
(encoded by, e.g., SEQ ID NO:163):
TABLE-US-00005 Codons Encoding New Amino Original Original Acid
(after Codons Encoding Residue Amino Acid Amino Acid GSSM
Evolution) New Amino Acid 89 M ATG R CGT, CGC, CGA, CGG, AGA, AGG
103 F TTT, TTC G GGT, GGC, GGA, GGG 110 P CCA, CCC, CCG, CCT G GGT,
GGC, GGA, GGG 114 Y TAT, TAC L TTA, TTG, CTT, CTC, CTA, CTG 157 A
GCT, GCC, GCA, GCG S TCT, TCC, TCA, TCG, AGT, AGC 481 W TGG F TTT,
TTC 550 P CCA, CCC, CCG, CCT N AAT, AAC 590 G GGT, GGC, GGA, GGG R
CGT, CGC, CGA, CGG, AGA, AGG
[0665] A number of aspects of the invention have been described.
Nevertheless, it will be understood that various modifications may
be made without departing from the spirit and scope of the
invention. Accordingly, other aspects are within the scope of the
following claims.
Sequence CWU 1
1
16611323DNAUnknownObtained from environmental sample 1atgtcaacct
ataaatttcc gcacaacttt ttttggggag ccgcaaccgc gtcttatcag 60atcgaaggcg
catggaacga ggatggcaaa ggcgaatcca tttgggatcg cttcagccat
120acgcccggaa aggtcaccaa tgccgatacc ggtgacatcg cctgtgacca
ctatcaccgt 180tgggaggaag atatcgccct tatgcgccaa cttgggttga
aggcgtaccg cttttccact 240tcatggcccc gtgtgatccc ggcgggccgc
agacgggtga atgtcaaagg gctggatttc 300tacgatcgcc tggtggatgg
tctgtgcgcc gcgaacatcg aaccgttcct caccctgtat 360cactgggacc
tgccgcaggc tcttcaagac gaaggcggct gggataatcg caacaccgcc
420catgcctttg ccgattatgc cgcattgatg gtgaaacgac ttggcgaccg
tatccgctat 480tggacgacgt tcaacgaacc cagcgttgtg gcgttcaatg
gtcattactc aggctcgcac 540gccccgggca ttcaagatgc ccgtgttacc
cgccaggtgg tgcatcattt gctggtggcg 600catgggttgg ctgtgcaggc
gatccgcggc gcaaactcca aagtggatgt gggcatcgtg 660cttaatttat
ggcccgccga acccgattcg gactcccccg aagatgccgc cgccgccgaa
720gccgcctgga accggcacga gaccctgttc cttgacccca tctttaaggc
gcattatccc 780gtatctgccc ttgatgcgat tggggaggat atgccccgca
tccacgacgg cgatctggcg 840ttgatctctc aggaattgga ttttgtcggc
atcaactatt actcccgcca tgtggtcagt 900gccacaaaag aaataggcag
gcttcccgaa tcggaataca ctgaaatggg ctgggaagta 960tgcgcccccg
cactccgccg cctgctggtc aagatccata acgattaccg tttgccgccc
1020atctatatca ccgaaaacgg atcggcattc aaggacgaag ttaacgcaga
cggaaaggtt 1080catgacccgc ggcggttgga ttacctgaaa caacacctga
ttcaactttg ccttgccatg 1140caggacggcg tggatgtgcg cggctacatg
gcttggtccc tgctggataa tttcgagtgg 1200ggtcacggct tttccaagcg
ctttggcttg gtccatgtgg attacgagag ccagaagcgg 1260attattaaag
actcgggtga atggtatgca agtgtgatac ggaagaacga ggttgttgaa 1320taa
13232440PRTUnknownObtained from environmental sample 2Met Ser Thr
Tyr Lys Phe Pro His Asn Phe Phe Trp Gly Ala Ala Thr 1 5 10 15 Ala
Ser Tyr Gln Ile Glu Gly Ala Trp Asn Glu Asp Gly Lys Gly Glu 20 25
30 Ser Ile Trp Asp Arg Phe Ser His Thr Pro Gly Lys Val Thr Asn Ala
35 40 45 Asp Thr Gly Asp Ile Ala Cys Asp His Tyr His Arg Trp Glu
Glu Asp 50 55 60 Ile Ala Leu Met Arg Gln Leu Gly Leu Lys Ala Tyr
Arg Phe Ser Thr 65 70 75 80 Ser Trp Pro Arg Val Ile Pro Ala Gly Arg
Arg Arg Val Asn Val Lys 85 90 95 Gly Leu Asp Phe Tyr Asp Arg Leu
Val Asp Gly Leu Cys Ala Ala Asn 100 105 110 Ile Glu Pro Phe Leu Thr
Leu Tyr His Trp Asp Leu Pro Gln Ala Leu 115 120 125 Gln Asp Glu Gly
Gly Trp Asp Asn Arg Asn Thr Ala His Ala Phe Ala 130 135 140 Asp Tyr
Ala Ala Leu Met Val Lys Arg Leu Gly Asp Arg Ile Arg Tyr 145 150 155
160 Trp Thr Thr Phe Asn Glu Pro Ser Val Val Ala Phe Asn Gly His Tyr
165 170 175 Ser Gly Ser His Ala Pro Gly Ile Gln Asp Ala Arg Val Thr
Arg Gln 180 185 190 Val Val His His Leu Leu Val Ala His Gly Leu Ala
Val Gln Ala Ile 195 200 205 Arg Gly Ala Asn Ser Lys Val Asp Val Gly
Ile Val Leu Asn Leu Trp 210 215 220 Pro Ala Glu Pro Asp Ser Asp Ser
Pro Glu Asp Ala Ala Ala Ala Glu 225 230 235 240 Ala Ala Trp Asn Arg
His Glu Thr Leu Phe Leu Asp Pro Ile Phe Lys 245 250 255 Ala His Tyr
Pro Val Ser Ala Leu Asp Ala Ile Gly Glu Asp Met Pro 260 265 270 Arg
Ile His Asp Gly Asp Leu Ala Leu Ile Ser Gln Glu Leu Asp Phe 275 280
285 Val Gly Ile Asn Tyr Tyr Ser Arg His Val Val Ser Ala Thr Lys Glu
290 295 300 Ile Gly Arg Leu Pro Glu Ser Glu Tyr Thr Glu Met Gly Trp
Glu Val 305 310 315 320 Cys Ala Pro Ala Leu Arg Arg Leu Leu Val Lys
Ile His Asn Asp Tyr 325 330 335 Arg Leu Pro Pro Ile Tyr Ile Thr Glu
Asn Gly Ser Ala Phe Lys Asp 340 345 350 Glu Val Asn Ala Asp Gly Lys
Val His Asp Pro Arg Arg Leu Asp Tyr 355 360 365 Leu Lys Gln His Leu
Ile Gln Leu Cys Leu Ala Met Gln Asp Gly Val 370 375 380 Asp Val Arg
Gly Tyr Met Ala Trp Ser Leu Leu Asp Asn Phe Glu Trp 385 390 395 400
Gly His Gly Phe Ser Lys Arg Phe Gly Leu Val His Val Asp Tyr Glu 405
410 415 Ser Gln Lys Arg Ile Ile Lys Asp Ser Gly Glu Trp Tyr Ala Ser
Val 420 425 430 Ile Arg Lys Asn Glu Val Val Glu 435 440
31389DNAUnknownObtained from environmental sample 3atgagcgctc
cgagtcccgc ccgccccgtg tcctttcctc cccgcttcgt gtggggagcc 60gcggccgcat
cctatcaaat cgagggcgcc gtccgggagg acggcaaggg cccttcggtg
120tgggacatgt tctgcgagaa gccgggagcc gtcttcgagg ggcacgacgg
ggcggtggct 180tgcgatcact accaccgtta ccgggaagac gtggccctga
tgcggcagat tgggctccag 240gcttaccgcc tgagcgtgtg ctggcccagg
gtgctgcccg aggggaccgg gcagcccaac 300gagaaggggc tcgacttcta
ctcccggctc gtcgacgcct tgctcgaggc ggggatcacg 360ccttgggtca
ccctttttca ctgggactac ccactagccc tatatcaccg gggaggctgg
420ctcaatcggg atagctcaga ctggttcggc gagtacgcgg gtctgattgc
ggagcgcctc 480tccgatcggg tgagccactt cttcacccag aacgagcccc
aggtgtacat cggcttcggg 540cacctcgagg ggaaacacgc gccgggcgat
acccttcccc tgtcgcagat gctgctggcc 600ggtcaccaca gcctgctcgc
ccatggaaag gccgtgcagg cgctgcgcgc ccacggcaag 660cagcagctgc
gggttggata cgctccggtg gggatgccgc tgcatccggt cagcgagtcc
720gccgaagacg tggcggctgc acgcaccgcc actttccgcg tccgagagaa
gaattcctgg 780aacaacgctt ggtggatgga cccggtgtac ctcggtgagt
accccgccca agggctcgag 840ttctacgggc gagacgtccc cgcgatccgg
tccggagaca tggaactcat ccggcaaccc 900ttggactttt tcggcgtcaa
catctaccag agcacgcccg tgcgcgccgc gggggcgccc 960caggggttcg
aggtcgtccg gcatccgacg ggccacccca tcaccgcgtt caactggccg
1020gttacgccac aggccttgta ttgggggccg cggttcttct acgagcgcta
tggcaagccc 1080atcgtcatta cggaaaacgg gctttcctgc cgagacgtga
tcgcccttga cggcaaggtg 1140cacgatccgt cccgcatcga cttcaccacg
cgctacctgc gcgagctcca ccgcgccatc 1200gccgaaggca acgaggtgga
gggctacttc cactggtcca tcatggacaa cttcgaatgg 1260gctgccggat
accgagaacg cttcgggctc gttcacgtgg attacgagac cctggtgagg
1320acacccaagg actctgcggc gtggtaccgc caggtcatcc agagcaacgg
ggccgtgctg 1380ttcgattga 13894462PRTUnknownObtained from
environmental sample 4Met Ser Ala Pro Ser Pro Ala Arg Pro Val Ser
Phe Pro Pro Arg Phe 1 5 10 15 Val Trp Gly Ala Ala Ala Ala Ser Tyr
Gln Ile Glu Gly Ala Val Arg 20 25 30 Glu Asp Gly Lys Gly Pro Ser
Val Trp Asp Met Phe Cys Glu Lys Pro 35 40 45 Gly Ala Val Phe Glu
Gly His Asp Gly Ala Val Ala Cys Asp His Tyr 50 55 60 His Arg Tyr
Arg Glu Asp Val Ala Leu Met Arg Gln Ile Gly Leu Gln 65 70 75 80 Ala
Tyr Arg Leu Ser Val Cys Trp Pro Arg Val Leu Pro Glu Gly Thr 85 90
95 Gly Gln Pro Asn Glu Lys Gly Leu Asp Phe Tyr Ser Arg Leu Val Asp
100 105 110 Ala Leu Leu Glu Ala Gly Ile Thr Pro Trp Val Thr Leu Phe
His Trp 115 120 125 Asp Tyr Pro Leu Ala Leu Tyr His Arg Gly Gly Trp
Leu Asn Arg Asp 130 135 140 Ser Ser Asp Trp Phe Gly Glu Tyr Ala Gly
Leu Ile Ala Glu Arg Leu 145 150 155 160 Ser Asp Arg Val Ser His Phe
Phe Thr Gln Asn Glu Pro Gln Val Tyr 165 170 175 Ile Gly Phe Gly His
Leu Glu Gly Lys His Ala Pro Gly Asp Thr Leu 180 185 190 Pro Leu Ser
Gln Met Leu Leu Ala Gly His His Ser Leu Leu Ala His 195 200 205 Gly
Lys Ala Val Gln Ala Leu Arg Ala His Gly Lys Gln Gln Leu Arg 210 215
220 Val Gly Tyr Ala Pro Val Gly Met Pro Leu His Pro Val Ser Glu Ser
225 230 235 240 Ala Glu Asp Val Ala Ala Ala Arg Thr Ala Thr Phe Arg
Val Arg Glu 245 250 255 Lys Asn Ser Trp Asn Asn Ala Trp Trp Met Asp
Pro Val Tyr Leu Gly 260 265 270 Glu Tyr Pro Ala Gln Gly Leu Glu Phe
Tyr Gly Arg Asp Val Pro Ala 275 280 285 Ile Arg Ser Gly Asp Met Glu
Leu Ile Arg Gln Pro Leu Asp Phe Phe 290 295 300 Gly Val Asn Ile Tyr
Gln Ser Thr Pro Val Arg Ala Ala Gly Ala Pro 305 310 315 320 Gln Gly
Phe Glu Val Val Arg His Pro Thr Gly His Pro Ile Thr Ala 325 330 335
Phe Asn Trp Pro Val Thr Pro Gln Ala Leu Tyr Trp Gly Pro Arg Phe 340
345 350 Phe Tyr Glu Arg Tyr Gly Lys Pro Ile Val Ile Thr Glu Asn Gly
Leu 355 360 365 Ser Cys Arg Asp Val Ile Ala Leu Asp Gly Lys Val His
Asp Pro Ser 370 375 380 Arg Ile Asp Phe Thr Thr Arg Tyr Leu Arg Glu
Leu His Arg Ala Ile 385 390 395 400 Ala Glu Gly Asn Glu Val Glu Gly
Tyr Phe His Trp Ser Ile Met Asp 405 410 415 Asn Phe Glu Trp Ala Ala
Gly Tyr Arg Glu Arg Phe Gly Leu Val His 420 425 430 Val Asp Tyr Glu
Thr Leu Val Arg Thr Pro Lys Asp Ser Ala Ala Trp 435 440 445 Tyr Arg
Gln Val Ile Gln Ser Asn Gly Ala Val Leu Phe Asp 450 455 460
51098DNAUnknownObtained from environmental sample 5atgactcgga
ggtctatcgt gcgttcttct tccaacaagt ggcttgtcct tgccggtgcg 60gcgctgctcg
cctgcaccgc cctcgggtgc aagaaaaaag gcgagagcgg tgacgtcgcc
120tcggccccgg ggcaggccca ggcgggcggc aagcagccgt ttcccgacga
tgcgccgatc 180accgaaccgc ccgctccgcc ccctcgtagc ggcaatcctc
tggtgggcgc caagctcttc 240gtcgacccgg aatctttggc catgttgcag
gcgaacaagc tgcggcgcac cgacccggag 300aaggcggcga ttttggatcg
catcgcccag cagccccagg ctttgtggat gggcgagtgg 360aacacgaaca
tcttccgcgc ggtcgagcat ttcgtggctc gcgccaaggc ggagggcgcc
420gtgcccgtca tgatcgccta caacatcccc caccgcgact gcgggcagta
ctctcagggt 480gggctttcct ccaaggaggc ttaccagcgc tggattcgga
acgtcgccgc ggggattggc 540agcgatgcag cggtcgtcgt gctcgagccc
gacgcgctcg gccacttcca ggagtgtttg 600accgaggagc agagcgccga
gcgcatgttc ctgctcagcg acgccgtcaa ggtgctgcgc 660caaaatccga
agacggccgt gtacctggat gccgggcacg cgcgctgggt gccggtggag
720gagatggccg agcgcctcaa gctcgcgggc atcgagcacg cccatggctt
ttcgctcaac 780acctcgaact acgtgggcac cgaggagaac gccgcttacg
gccacaagct cgtcgaggcc 840ctgggtggga acgtgcgctt cgtcatcgac
acgagccgca atggggcggg cccctacgag 900gaggccaaga acgccgagga
gagctggtgc aacccgcccg gtcgcaagat cggcaagccg 960ccgaccaccg
agacggggga tcccctcatc gacggattcc tttggctgaa gcgcccgggc
1020gagtcggacg gtcagtgcaa cggcgggccc aaggccggtg tgttctggct
ggagcaggct 1080ctccagcagg cccagtaa 10986365PRTUnknownObtained from
environmental sample 6Met Thr Arg Arg Ser Ile Val Arg Ser Ser Ser
Asn Lys Trp Leu Val 1 5 10 15 Leu Ala Gly Ala Ala Leu Leu Ala Cys
Thr Ala Leu Gly Cys Lys Lys 20 25 30 Lys Gly Glu Ser Gly Asp Val
Ala Ser Ala Pro Gly Gln Ala Gln Ala 35 40 45 Gly Gly Lys Gln Pro
Phe Pro Asp Asp Ala Pro Ile Thr Glu Pro Pro 50 55 60 Ala Pro Pro
Pro Arg Ser Gly Asn Pro Leu Val Gly Ala Lys Leu Phe 65 70 75 80 Val
Asp Pro Glu Ser Leu Ala Met Leu Gln Ala Asn Lys Leu Arg Arg 85 90
95 Thr Asp Pro Glu Lys Ala Ala Ile Leu Asp Arg Ile Ala Gln Gln Pro
100 105 110 Gln Ala Leu Trp Met Gly Glu Trp Asn Thr Asn Ile Phe Arg
Ala Val 115 120 125 Glu His Phe Val Ala Arg Ala Lys Ala Glu Gly Ala
Val Pro Val Met 130 135 140 Ile Ala Tyr Asn Ile Pro His Arg Asp Cys
Gly Gln Tyr Ser Gln Gly 145 150 155 160 Gly Leu Ser Ser Lys Glu Ala
Tyr Gln Arg Trp Ile Arg Asn Val Ala 165 170 175 Ala Gly Ile Gly Ser
Asp Ala Ala Val Val Val Leu Glu Pro Asp Ala 180 185 190 Leu Gly His
Phe Gln Glu Cys Leu Thr Glu Glu Gln Ser Ala Glu Arg 195 200 205 Met
Phe Leu Leu Ser Asp Ala Val Lys Val Leu Arg Gln Asn Pro Lys 210 215
220 Thr Ala Val Tyr Leu Asp Ala Gly His Ala Arg Trp Val Pro Val Glu
225 230 235 240 Glu Met Ala Glu Arg Leu Lys Leu Ala Gly Ile Glu His
Ala His Gly 245 250 255 Phe Ser Leu Asn Thr Ser Asn Tyr Val Gly Thr
Glu Glu Asn Ala Ala 260 265 270 Tyr Gly His Lys Leu Val Glu Ala Leu
Gly Gly Asn Val Arg Phe Val 275 280 285 Ile Asp Thr Ser Arg Asn Gly
Ala Gly Pro Tyr Glu Glu Ala Lys Asn 290 295 300 Ala Glu Glu Ser Trp
Cys Asn Pro Pro Gly Arg Lys Ile Gly Lys Pro 305 310 315 320 Pro Thr
Thr Glu Thr Gly Asp Pro Leu Ile Asp Gly Phe Leu Trp Leu 325 330 335
Lys Arg Pro Gly Glu Ser Asp Gly Gln Cys Asn Gly Gly Pro Lys Ala 340
345 350 Gly Val Phe Trp Leu Glu Gln Ala Leu Gln Gln Ala Gln 355 360
365 72649DNAUnknownObtained from environmental sample 7atgcaaggaa
agaaaattga tttcattaac tcaaggttgt tagttcctga ttatccaatc 60gttcccttca
ttgagggaga tggtaccggc cctgatatct ggcgtgcttc agtcagggtg
120ctggatgttg ctgttgacag ggcatattcc ggcaagcgaa aacttctctg
gaaagaggtg 180ctggctggcg aaaaggcatt tacaaatacc gggtcctggc
ttccggagga aactcttaga 240gcatttcgtg aatatcatgt tggaattaaa
gggccactca ctacgccagt tggtggggga 300attcgttctc tcaatgtagc
cctcaggcaa gagcttgact tgtatgtttg cctgaggcca 360gtcaaatggt
ttaagggtgt accaagtcct ctaaaagatc cttccaaagt ggatatgcat
420attttccgcg aaaacactga agatatttat gcaggtattg aatttatgca
tggtgaaccg 480gaggccctga aagttaagaa atttcttacc gaagaaatgg
gaatcaagaa gtttcggttt 540cccgatacat cctccattgg tatcaagcct
atctcactcg aaggaacaga gcgtcttgta 600agagcttcca ttcaatatgc
acttgacagg aagttgcctt ccgtaacatt ggttcataaa 660ggcaatatca
tgaaattcac cgagggggca ttcaaaaaat ggggttatga acttgccgaa
720agagaatttg gcgacagggt ttttacatgg tcaatgtatg accgtatcgc
cgatgaacat 780ggaacggaag aagctggcaa agtgcaatcc gaagcgattg
caaaaggtaa actcctgata 840aaggatgtga ttgctgatgc ttttctgcag
caaatactac tcaggcctgc cgagtacagc 900gttatcgcaa ccatgaacct
gaatggcgat tatatcagcg atgcactggc agctatggtg 960gggggtatag
gaattgctcc cggagccaat attaaccatc aaactggcca tgcagtcttt
1020gaagcaacac acggcacggc tcccaaatat gccaaccttg atcaggtaaa
ccctggctca 1080gtaatactaa gtggcgcgct gatgctcgaa tacatgggct
ggaacgaagc cgctcagctc 1140attaccaatg gattggaggc taccattcaa
cagaaactgg taacctatga tttccatcgc 1200ttaatggaag gtgctacaaa
gttgaagact tcagaatttg gcgatgctgt gatccggccg 1260gcacgttccg
cctgggcgga cacggctgcc gatgccctct ccgggcggcg gcgtcgtgcg
1320cggaacggcg ggcttgttgc cccgcccgcg gcctgtcgcc gggggcgggt
acgggactca 1380gcgcttgcgc gcctccttca gggtggactg cagggcgaag
aaggccggct tgcggacgaa 1440cttctccgtc atgaccgtgg cgctgccctc
accctcgaag aagaccggca cccacgagta 1500cttgtcggtg aagccccaga
tggtgaagga gttgcagtcg ttcacggcca ggcaggccga 1560cagtgcctgc
tggtagtagt cggcctgctg ccgcagctgc tccttggtgg gcttgccgct
1620cgccgggagg tccatgcgga cgtcgatctc ggtgatggcg gtctccagac
cgaggtcggc 1680gaaccgctgc aggttctgct gcaggtcgcc cgggaagccg
tagcgggtgc tcaggtggcc 1740ctgggcgccg aatccgtgga gcggcacgcc
ctgctccagc atctcctggg cgagctcgta 1800gtaggcgtcg ctcttggcgt
tgatgccctc gacgttgtag tcgttgagga acagcttggc 1860ctcggggtcg
gcctcgtggg cccagcggaa ggcgtccgcg acgatctccg ggccgagctc
1920acgtatccag atgttctcgt cggtgcgcag ctcggcctgg tcgttgaaga
tctcgttggc 1980cacgtcccac tgctggatct tgccggcgta gcggccgacg
accgtgtcga tgtggtcctt 2040gaggatggcg cgcagttcct ccttggtgaa
gtcgccctcc tccagccatt cggggttctg 2100gctgtgccac aggagggtgt
gcccgcgcac ggcctggcgg ttccgctggg cgaactcgac 2160gatggcgtcg
gcctcctcga agcggtactg gtcgcgctcg gggtggatga actcccactt
2220catctggttc tcggcggaga ccgagttgaa ctgctggccc aggatcttcc
ggtacttctt 2280gtcgaaggtg aaggggtccg ggtagtcctg ttcgaggtgg
tggccgccgc cggccgccgc 2340ggagcctatg aagaaccctt cgggggcggc
ccagcgcagg cggtcgaact tggcgttgga 2400gtggggcgcg gcctcgtggt
cggcggacgg cttggccgtg gccgtcgacg tcaccagcgg 2460gacggccagc
gcggcggcga gagcaaaggt gacgatgcgg acggatctca tcagaggtcc
2520ctcattcgat cgcggctccg aaagttttcg gaggattacc ggaatgtttc
agggacctta 2580aggcgcccgg agccgggtcg tcaacggttt ggcccggccc
ggtcgaagct tctcccgacc 2640aggcgttga 26498882PRTUnknownObtained from
environmental sample 8Met Gln Gly Lys Lys Ile Asp Phe Ile Asn Ser
Arg Leu Leu Val Pro 1 5 10 15 Asp Tyr Pro Ile Val Pro Phe Ile Glu
Gly Asp Gly Thr Gly Pro Asp 20 25 30 Ile Trp Arg Ala Ser Val Arg
Val Leu Asp Val Ala Val Asp Arg Ala 35 40 45 Tyr Ser Gly Lys Arg
Lys Leu Leu Trp Lys Glu Val Leu Ala Gly Glu 50 55 60 Lys Ala Phe
Thr Asn Thr Gly Ser Trp Leu Pro Glu Glu Thr Leu Arg 65 70 75 80 Ala
Phe Arg Glu Tyr His Val Gly Ile Lys Gly Pro Leu Thr Thr Pro 85 90
95 Val Gly Gly Gly Ile Arg Ser Leu Asn Val Ala Leu Arg Gln Glu Leu
100 105 110 Asp Leu Tyr Val Cys Leu Arg Pro Val Lys Trp Phe Lys Gly
Val Pro 115 120 125 Ser Pro Leu Lys Asp Pro Ser Lys Val Asp Met His
Ile Phe Arg Glu 130 135 140 Asn Thr Glu Asp Ile Tyr Ala Gly Ile Glu
Phe Met His Gly Glu Pro 145 150 155 160 Glu Ala Leu Lys Val Lys Lys
Phe Leu Thr Glu Glu Met Gly Ile Lys 165 170 175 Lys Phe Arg Phe Pro
Asp Thr Ser Ser Ile Gly Ile Lys Pro Ile Ser 180 185 190 Leu Glu Gly
Thr Glu Arg Leu Val Arg Ala Ser Ile Gln Tyr Ala Leu 195 200 205 Asp
Arg Lys Leu Pro Ser Val Thr Leu Val His Lys Gly Asn Ile Met 210 215
220 Lys Phe Thr Glu Gly Ala Phe Lys Lys Trp Gly Tyr Glu Leu Ala Glu
225 230 235 240 Arg Glu Phe Gly Asp Arg Val Phe Thr Trp Ser Met Tyr
Asp Arg Ile 245 250 255 Ala Asp Glu His Gly Thr Glu Glu Ala Gly Lys
Val Gln Ser Glu Ala 260 265 270 Ile Ala Lys Gly Lys Leu Leu Ile Lys
Asp Val Ile Ala Asp Ala Phe 275 280 285 Leu Gln Gln Ile Leu Leu Arg
Pro Ala Glu Tyr Ser Val Ile Ala Thr 290 295 300 Met Asn Leu Asn Gly
Asp Tyr Ile Ser Asp Ala Leu Ala Ala Met Val 305 310 315 320 Gly Gly
Ile Gly Ile Ala Pro Gly Ala Asn Ile Asn His Gln Thr Gly 325 330 335
His Ala Val Phe Glu Ala Thr His Gly Thr Ala Pro Lys Tyr Ala Asn 340
345 350 Leu Asp Gln Val Asn Pro Gly Ser Val Ile Leu Ser Gly Ala Leu
Met 355 360 365 Leu Glu Tyr Met Gly Trp Asn Glu Ala Ala Gln Leu Ile
Thr Asn Gly 370 375 380 Leu Glu Ala Thr Ile Gln Gln Lys Leu Val Thr
Tyr Asp Phe His Arg 385 390 395 400 Leu Met Glu Gly Ala Thr Lys Leu
Lys Thr Ser Glu Phe Gly Asp Ala 405 410 415 Val Ile Arg Pro Ala Arg
Ser Ala Trp Ala Asp Thr Ala Ala Asp Ala 420 425 430 Leu Ser Gly Arg
Arg Arg Arg Ala Arg Asn Gly Gly Leu Val Ala Pro 435 440 445 Pro Ala
Ala Cys Arg Arg Gly Arg Val Arg Asp Ser Ala Leu Ala Arg 450 455 460
Leu Leu Gln Gly Gly Leu Gln Gly Glu Glu Gly Arg Leu Ala Asp Glu 465
470 475 480 Leu Leu Arg His Asp Arg Gly Ala Ala Leu Thr Leu Glu Glu
Asp Arg 485 490 495 His Pro Arg Val Leu Val Gly Glu Ala Pro Asp Gly
Glu Gly Val Ala 500 505 510 Val Val His Gly Gln Ala Gly Arg Gln Cys
Leu Leu Val Val Val Gly 515 520 525 Leu Leu Pro Gln Leu Leu Leu Gly
Gly Leu Ala Ala Arg Arg Glu Val 530 535 540 His Ala Asp Val Asp Leu
Gly Asp Gly Gly Leu Gln Thr Glu Val Gly 545 550 555 560 Glu Pro Leu
Gln Val Leu Leu Gln Val Ala Arg Glu Ala Val Ala Gly 565 570 575 Ala
Gln Val Ala Leu Gly Ala Glu Ser Val Glu Arg His Ala Leu Leu 580 585
590 Gln His Leu Leu Gly Glu Leu Val Val Gly Val Ala Leu Gly Val Asp
595 600 605 Ala Leu Asp Val Val Val Val Glu Glu Gln Leu Gly Leu Gly
Val Gly 610 615 620 Leu Val Gly Pro Ala Glu Gly Val Arg Asp Asp Leu
Arg Ala Glu Leu 625 630 635 640 Thr Tyr Pro Asp Val Leu Val Gly Ala
Gln Leu Gly Leu Val Val Glu 645 650 655 Asp Leu Val Gly His Val Pro
Leu Leu Asp Leu Ala Gly Val Ala Ala 660 665 670 Asp Asp Arg Val Asp
Val Val Leu Glu Asp Gly Ala Gln Phe Leu Leu 675 680 685 Gly Glu Val
Ala Leu Leu Gln Pro Phe Gly Val Leu Ala Val Pro Gln 690 695 700 Glu
Gly Val Pro Ala His Gly Leu Ala Val Pro Leu Gly Glu Leu Asp 705 710
715 720 Asp Gly Val Gly Leu Leu Glu Ala Val Leu Val Ala Leu Gly Val
Asp 725 730 735 Glu Leu Pro Leu His Leu Val Leu Gly Gly Asp Arg Val
Glu Leu Leu 740 745 750 Ala Gln Asp Leu Pro Val Leu Leu Val Glu Gly
Glu Gly Val Arg Val 755 760 765 Val Leu Phe Glu Val Val Ala Ala Ala
Gly Arg Arg Gly Ala Tyr Glu 770 775 780 Glu Pro Phe Gly Gly Gly Pro
Ala Gln Ala Val Glu Leu Gly Val Gly 785 790 795 800 Val Gly Arg Gly
Leu Val Val Gly Gly Arg Leu Gly Arg Gly Arg Arg 805 810 815 Arg His
Gln Arg Asp Gly Gln Arg Gly Gly Glu Ser Lys Gly Asp Asp 820 825 830
Ala Asp Gly Ser His Gln Arg Ser Leu Ile Arg Ser Arg Leu Arg Lys 835
840 845 Phe Ser Glu Asp Tyr Arg Asn Val Ser Gly Thr Leu Arg Arg Pro
Glu 850 855 860 Pro Gly Arg Gln Arg Phe Gly Pro Ala Arg Ser Lys Leu
Leu Pro Thr 865 870 875 880 Arg Arg 91134DNAUnknownObtained from
environmental sample 9atgagatccg tccgcatcgt cacctttgct ctcgccgccg
cgctggccgt cccgctggtg 60acgtcgacgg ccacggccaa gccgtccgcc gaccacgagg
ccgcgcccca ctccaacgcc 120aagttcgacc gcctgcgctg ggccgccccc
gaagggttct tcataggctc cgcggcggcc 180ggcggcggcc accacctcga
acaggactac ccggacccct tcaccttcga caagaagtac 240cggaagatcc
tgggccagca gttcaactcg gtctccgccg agaaccagat gaagtgggag
300ttcatccacc ccgagcgcga ccagtaccgc ttcgaggagg ccgacgccat
cgtcgagttc 360gcccagcgga accgccaggc cgtgcgcggg cacaccctcc
tgtggcacag ccagaacccc 420gaatggctgg aggagggcga cttcaccaag
gaggaactgc gcgccatcct caaggaccac 480atcgacacgg tcgtcggccg
ctacgccggc aagatccagc agtgggacgt ggccaacgag 540atcttcaacg
accaggccga gctgcgcacc gacgagaaca tctggatacg tgagctcggc
600ccggagatcg tcgcggacgc cttccgctgg gcccacgagg ccgaccccga
ggccaagctg 660ttcctcaacg actacaacgt cgagggcatc aacgccaaga
gcgacgccta ctacgagctc 720gcccaggaga tgctggagca gggcgtgccg
ctccacggat tcggcgccca gggccacctg 780agcacccgct acggcttccc
gggcgacctg cagcagaacc tgcagcggtt cgccgacctc 840ggtctggaga
ccgccatcac cgagatcgac gtccgcatgg acctcccggc gagcggcaag
900cccaccaagg agcagctgcg gcagcaggcc gactactacc agcaggcact
gtcggcctgc 960ctggccgtga acgactgcaa ctccttcacc atctggggct
tcaccgacaa gtactcgtgg 1020gtgccggtct tcttcgaggg tgagggcagc
gccacggtca tgacggagaa gttcgtccgc 1080aagccggcct tcttcgccct
gcagtccacc ctgaaggagg cgcgcaagcg ctga 113410377PRTUnknownObtained
from environmental sample 10Met Arg Ser Val Arg Ile Val Thr Phe Ala
Leu Ala Ala Ala Leu Ala 1 5 10 15 Val Pro Leu Val Thr Ser Thr Ala
Thr Ala Lys Pro Ser Ala Asp His 20 25 30 Glu Ala Ala Pro His Ser
Asn Ala Lys Phe Asp Arg Leu Arg Trp Ala 35 40 45 Ala Pro Glu Gly
Phe Phe Ile Gly Ser Ala Ala Ala Gly Gly Gly His 50 55 60 His Leu
Glu Gln Asp Tyr Pro Asp Pro Phe Thr Phe Asp Lys Lys Tyr 65 70 75 80
Arg Lys Ile Leu Gly Gln Gln Phe Asn Ser Val Ser Ala Glu Asn Gln 85
90 95 Met Lys Trp Glu Phe Ile His Pro Glu Arg Asp Gln Tyr Arg Phe
Glu 100 105 110 Glu Ala Asp Ala Ile Val Glu Phe Ala Gln Arg Asn Arg
Gln Ala Val 115 120 125 Arg Gly His Thr Leu Leu Trp His Ser Gln Asn
Pro Glu Trp Leu Glu 130 135 140 Glu Gly Asp Phe Thr Lys Glu Glu Leu
Arg Ala Ile Leu Lys Asp His 145 150 155 160 Ile Asp Thr Val Val Gly
Arg Tyr Ala Gly Lys Ile Gln Gln Trp Asp 165 170 175 Val Ala Asn Glu
Ile Phe Asn Asp Gln Ala Glu Leu Arg Thr Asp Glu 180 185 190 Asn Ile
Trp Ile Arg Glu Leu Gly Pro Glu Ile Val Ala Asp Ala Phe 195 200 205
Arg Trp Ala His Glu Ala Asp Pro Glu Ala Lys Leu Phe Leu Asn Asp 210
215 220 Tyr Asn Val Glu Gly Ile Asn Ala Lys Ser Asp Ala Tyr Tyr Glu
Leu 225 230 235 240 Ala Gln Glu Met Leu Glu Gln Gly Val Pro Leu His
Gly Phe Gly Ala 245 250 255 Gln Gly His Leu Ser Thr Arg Tyr Gly Phe
Pro Gly Asp Leu Gln Gln 260 265 270 Asn Leu Gln Arg Phe Ala Asp Leu
Gly Leu Glu Thr Ala Ile Thr Glu 275 280 285 Ile Asp Val Arg Met Asp
Leu Pro Ala Ser Gly Lys Pro Thr Lys Glu 290 295 300 Gln Leu Arg Gln
Gln Ala Asp Tyr Tyr Gln Gln Ala Leu Ser Ala Cys 305 310 315 320 Leu
Ala Val Asn Asp Cys Asn Ser Phe Thr Ile Trp Gly Phe Thr Asp 325 330
335 Lys Tyr Ser Trp Val Pro Val Phe Phe Glu Gly Glu Gly Ser Ala Thr
340 345 350 Val Met Thr Glu Lys Phe Val Arg Lys Pro Ala Phe Phe Ala
Leu Gln 355 360 365 Ser Thr Leu Lys Glu Ala Arg Lys Arg 370 375
111080DNAUnknownObtained from environmental sample 11atgccctgga
gctcatcaac gggacctgca cctatgacga gtaacccgcc cctcaaacgc 60cccctgcgta
tcggtctggt cggcacgggc atcggctcac tgcacgccgc cggaatttcc
120cggatgcctc agcttgccac gctgggggcc atctgtgggc ttgataccca
cgccgtgaat 180gccctagcca cacgctacgg ggtagaaaaa accacatctc
gctatgagga tttactgaac 240gatcccggcc ttgatgtcat cgatctgtgc
gttcctcacg atgaacacat gcccatggcc 300attgccgccg cccgggccgg
aaaacatctc ctcatcgaaa aacctttggc ccgcaccctg 360gaagaggccg
atgcaatcct cgaggccgtg aaaagcgccg gtgtaacgct gatgatggga
420cacaaccagc gttactacgc ccatcacgcc agggctaaag cattggtcga
cgccggggtc 480atcggaaaac cctacatgat cgtagcttcg gttcatgtgc
acgggcagat tgatggtttt 540cgccgctttc ttaagcacgc cgggggtggc
acgttgatcg attcgggagt gcaccgcttc 600gacctcattc gctggatcat
gggtgaagtc gagaccgtct tcgctcaaac gggtcgcttc 660ctccagatgc
aaatggaagg agaagactgc gcggtggtca ccctccgctt ccgcagcgga
720gccatcggga gcttctcatg cagctggagc gccaaaggcc ctgttccaga
agaaacattg 780caaattttcg gcccctatgg ttcgatttat accgaagacc
acacccgcac cttacgcctt 840tacaccgaaa gacccacccc cgaactggaa
gacgtaaggc agtttgtctt cccggtcgat 900caggctgagt ccatccgccg
catgattgaa gcgcacttca ccagcctgca acaggggtta 960ccccctccga
tcaccggtat ggacggacgc gcttcccttg agctcagcat ggcctcctat
1020cgctcggctc aaaccggcca gcctgttcat cttccccttc agagaggaaa
ccagaaatga 108012359PRTUnknownObtained from environmental sample
12Met Pro Trp Ser Ser Ser Thr Gly Pro Ala Pro Met Thr Ser Asn Pro 1
5 10 15 Pro Leu Lys Arg Pro Leu Arg Ile Gly Leu Val Gly Thr Gly Ile
Gly 20 25 30 Ser Leu His Ala Ala Gly Ile Ser Arg Met Pro Gln Leu
Ala Thr Leu 35 40 45 Gly Ala Ile Cys Gly Leu Asp Thr His Ala Val
Asn Ala Leu Ala Thr 50 55 60 Arg Tyr Gly Val Glu Lys Thr Thr Ser
Arg Tyr Glu Asp Leu Leu Asn 65 70 75 80 Asp Pro Gly Leu Asp Val Ile
Asp Leu Cys Val Pro His Asp Glu His 85 90 95 Met Pro Met Ala Ile
Ala Ala Ala Arg Ala Gly Lys His Leu Leu Ile 100 105 110 Glu Lys Pro
Leu Ala Arg Thr Leu Glu Glu Ala Asp Ala Ile Leu Glu 115 120 125 Ala
Val Lys Ser Ala Gly Val Thr Leu Met Met Gly His Asn Gln Arg 130 135
140 Tyr Tyr Ala His His Ala Arg Ala Lys Ala Leu Val Asp Ala Gly Val
145 150 155 160 Ile Gly Lys Pro Tyr Met Ile Val Ala Ser Val His Val
His Gly Gln 165 170 175 Ile Asp Gly Phe Arg Arg Phe Leu Lys His Ala
Gly Gly Gly Thr Leu 180 185 190 Ile Asp Ser Gly Val His Arg Phe Asp
Leu Ile Arg Trp Ile Met Gly 195 200 205 Glu Val Glu Thr Val Phe Ala
Gln Thr Gly Arg Phe Leu Gln Met Gln 210 215 220 Met Glu Gly Glu Asp
Cys Ala Val Val Thr Leu Arg Phe Arg Ser Gly 225 230 235 240 Ala Ile
Gly Ser Phe Ser Cys Ser Trp Ser Ala Lys Gly Pro Val Pro 245 250 255
Glu Glu Thr Leu Gln Ile Phe Gly Pro Tyr Gly Ser Ile Tyr Thr Glu 260
265 270 Asp His Thr Arg Thr Leu Arg Leu Tyr Thr Glu Arg Pro Thr Pro
Glu 275 280 285 Leu Glu Asp Val Arg Gln Phe Val Phe Pro Val Asp Gln
Ala Glu Ser 290 295 300 Ile Arg Arg Met Ile Glu Ala His Phe Thr Ser
Leu Gln Gln Gly Leu 305 310 315 320 Pro Pro Pro Ile Thr Gly Met Asp
Gly Arg Ala Ser Leu Glu Leu Ser 325 330 335 Met Ala Ser Tyr Arg Ser
Ala Gln Thr Gly Gln Pro Val His Leu Pro 340 345 350 Leu Gln Arg Gly
Asn Gln Lys 355 131038DNAUnknownObtained from environmental sample
13atgagcccgg tgcgcgttgc tgtcatcggc gccgggcaaa ttgcccagcg cgggcattta
60cccgggcttc tggaagctgg cgccgaaatt accgttctgt gcgataattc ccttcctcag
120cttgaagaaa ttggggccaa atttcacgtt caccgggtct accgcgactg
gcacgccatg 180ctggatgccg gcggattcga agccgtcacc atttgtaccc
cgcccttcct ccatgccgag 240atggccatcg aatgtgcccg cagagggttg
catgtactgg tagaaaaacc catggctgta 300aatctccaac aatgcgatca
aatgatcgcc gcgtctgaac aggccggaac catcttaatg 360gtctcgcata
accagcgctt tatggaggca catcgtctgg ccaaagaaat ccttgatgcc
420ggcctcctcg gcaggctcta cctggcgcac ggggtctttg gccacggcgg
cccggaggtt 480tggagcccaa cccagcaatg gtacttccga cctgaccgcg
ccggcgctgg cgtgatcgct 540gacctggggt atcataaact tgacctgatc
cgctggctca ccgggcaaga aattaccgcg 600gtgggagcac tgggcgccac
ctttgaaaag caaacctcgc ttgaagactc tgctgtgatg 660ctggttcacc
tttcggaggg tactctcgcc accatccagg taagctgggt gttcaggcct
720gactgggaaa acagcctggt ccttcgagga gaacgggggg tgctcgccat
ccccactgat 780gcctcgcaac ccctgcgggt ctcttacata tcttcttcgg
gtcaggtcat tgaaagtacg 840catcgttgcg actccggcga tacctccggc
tggttcggag cgatccgggc atttctcacc 900gcgatcgaaa aaagcgctcc
cgctcccatt gacggaaaag aagggcgtgc tgtcatggcg 960gcagttctgg
cggccacacg ctccattcaa aaacatacga tcatttctat aaccgaggta
1020gaaaccatcc atgactga 103814345PRTUnknownObtained from
environmental sample 14Met Ser Pro Val Arg Val Ala Val Ile Gly Ala
Gly Gln Ile Ala Gln 1 5 10 15 Arg Gly His Leu Pro Gly Leu Leu Glu
Ala Gly Ala Glu Ile Thr Val 20 25 30 Leu Cys Asp Asn Ser Leu Pro
Gln Leu Glu Glu Ile Gly Ala Lys Phe 35 40 45 His Val His Arg Val
Tyr Arg Asp Trp His Ala Met Leu Asp Ala Gly 50 55 60 Gly Phe Glu
Ala Val Thr Ile Cys Thr Pro Pro Phe Leu His Ala Glu 65 70 75 80 Met
Ala Ile Glu Cys Ala Arg Arg Gly Leu His Val Leu Val Glu Lys 85 90
95 Pro Met Ala Val Asn Leu Gln Gln Cys Asp Gln Met Ile Ala Ala
Ser 100 105 110 Glu Gln Ala Gly Thr Ile Leu Met Val Ser His Asn Gln
Arg Phe Met 115 120 125 Glu Ala His Arg Leu Ala Lys Glu Ile Leu Asp
Ala Gly Leu Leu Gly 130 135 140 Arg Leu Tyr Leu Ala His Gly Val Phe
Gly His Gly Gly Pro Glu Val 145 150 155 160 Trp Ser Pro Thr Gln Gln
Trp Tyr Phe Arg Pro Asp Arg Ala Gly Ala 165 170 175 Gly Val Ile Ala
Asp Leu Gly Tyr His Lys Leu Asp Leu Ile Arg Trp 180 185 190 Leu Thr
Gly Gln Glu Ile Thr Ala Val Gly Ala Leu Gly Ala Thr Phe 195 200 205
Glu Lys Gln Thr Ser Leu Glu Asp Ser Ala Val Met Leu Val His Leu 210
215 220 Ser Glu Gly Thr Leu Ala Thr Ile Gln Val Ser Trp Val Phe Arg
Pro 225 230 235 240 Asp Trp Glu Asn Ser Leu Val Leu Arg Gly Glu Arg
Gly Val Leu Ala 245 250 255 Ile Pro Thr Asp Ala Ser Gln Pro Leu Arg
Val Ser Tyr Ile Ser Ser 260 265 270 Ser Gly Gln Val Ile Glu Ser Thr
His Arg Cys Asp Ser Gly Asp Thr 275 280 285 Ser Gly Trp Phe Gly Ala
Ile Arg Ala Phe Leu Thr Ala Ile Glu Lys 290 295 300 Ser Ala Pro Ala
Pro Ile Asp Gly Lys Glu Gly Arg Ala Val Met Ala 305 310 315 320 Ala
Val Leu Ala Ala Thr Arg Ser Ile Gln Lys His Thr Ile Ile Ser 325 330
335 Ile Thr Glu Val Glu Thr Ile His Asp 340 345
151347DNAUnknownObtained from environmental sample 15atgactgacc
atcgttttcc aaaaggattc atctggggaa ccgctacggc gtctttccag 60attgaaggcg
ccacccgcga agatggccgg ggcgaatcca tctgggaccg cttctgcgcc
120acgccgggga aaattgtcac gggcgaaacc ggcgatcctg cctgcgactc
ctatcatcgt 180taccctgaag acatcgccct gatgaaggct atgtcgctca
atggttaccg cttttcaatc 240gcctggcctc gcgtcattcc tgacggagac
ggtaaagtct gtcaggccgg gctcgactac 300tacgatcgtg tggtagatgc
tctcctggcg gagaatatcc aaccttttat caccctgtac 360cactgggacc
tgccccaggc attacaggat cggggtggct ggggcaaccg tgccacggtt
420gaggcgttca ctcgctacgt agatattgtg gtttctcgcc tgggtgaccg
cgtaaagtac 480tggatgacac acaacgaacc ctggtgtgta tccattttga
gccatgagct tggtgaacat 540gcccccgggt tgaaggaccg aaaactggcc
ctccaggtgg cgcaccatgt cctcgtttct 600cacggcctgg ccgtgcccat
catccgccag cgttgtaaag aggcgcaggt tggcatcgtg 660ttgaattttt
cacctgctta cccggccacc gatagcctgg ccgaccagat ggccacccgt
720cagcaccacg cccggtttaa cctctggttc ctcgatccca tcgccgggcg
cggctacccg 780caggatgcct gggaagggta cggagccgat gttcccgcca
tgaggcctga tgacatgcag 840atcatcgccg cccccatcga cttcctgggc
gtcaatttct acagtcgggc ggtctgccac 900gatccggccg ggggcgaagg
ttcccgggtg ctcaatgtgc gcagtaaaac cgaggccacc 960gatcgagact
gggagattta ccctcaggcg ctctacgatt tactcatctg gatccacaat
1020ggataccagt tcagagatat ttacattacc gagaatggcg cctcatacaa
cgatgtggtc 1080tccccggatg ggaaagtgca cgatcctaaa cgtctggact
atctgaaacg ccatctggcc 1140atggctctgc gggccatcga agcgggcgtt
ccactgcgtg gttatttctg ctggagcttg 1200atggacaact tcgaatgggc
catgggcacc agcagccgat tcgggttggc ctacaccgac 1260ttcactaccc
agaagcgtat tctcaaagac agtgggctct ggtttggcga agtggcacgg
1320gcaaacgcct taatcgacct tccctga 134716448PRTUnknownObtained from
environmental sample 16Met Thr Asp His Arg Phe Pro Lys Gly Phe Ile
Trp Gly Thr Ala Thr 1 5 10 15 Ala Ser Phe Gln Ile Glu Gly Ala Thr
Arg Glu Asp Gly Arg Gly Glu 20 25 30 Ser Ile Trp Asp Arg Phe Cys
Ala Thr Pro Gly Lys Ile Val Thr Gly 35 40 45 Glu Thr Gly Asp Pro
Ala Cys Asp Ser Tyr His Arg Tyr Pro Glu Asp 50 55 60 Ile Ala Leu
Met Lys Ala Met Ser Leu Asn Gly Tyr Arg Phe Ser Ile 65 70 75 80 Ala
Trp Pro Arg Val Ile Pro Asp Gly Asp Gly Lys Val Cys Gln Ala 85 90
95 Gly Leu Asp Tyr Tyr Asp Arg Val Val Asp Ala Leu Leu Ala Glu Asn
100 105 110 Ile Gln Pro Phe Ile Thr Leu Tyr His Trp Asp Leu Pro Gln
Ala Leu 115 120 125 Gln Asp Arg Gly Gly Trp Gly Asn Arg Ala Thr Val
Glu Ala Phe Thr 130 135 140 Arg Tyr Val Asp Ile Val Val Ser Arg Leu
Gly Asp Arg Val Lys Tyr 145 150 155 160 Trp Met Thr His Asn Glu Pro
Trp Cys Val Ser Ile Leu Ser His Glu 165 170 175 Leu Gly Glu His Ala
Pro Gly Leu Lys Asp Arg Lys Leu Ala Leu Gln 180 185 190 Val Ala His
His Val Leu Val Ser His Gly Leu Ala Val Pro Ile Ile 195 200 205 Arg
Gln Arg Cys Lys Glu Ala Gln Val Gly Ile Val Leu Asn Phe Ser 210 215
220 Pro Ala Tyr Pro Ala Thr Asp Ser Leu Ala Asp Gln Met Ala Thr Arg
225 230 235 240 Gln His His Ala Arg Phe Asn Leu Trp Phe Leu Asp Pro
Ile Ala Gly 245 250 255 Arg Gly Tyr Pro Gln Asp Ala Trp Glu Gly Tyr
Gly Ala Asp Val Pro 260 265 270 Ala Met Arg Pro Asp Asp Met Gln Ile
Ile Ala Ala Pro Ile Asp Phe 275 280 285 Leu Gly Val Asn Phe Tyr Ser
Arg Ala Val Cys His Asp Pro Ala Gly 290 295 300 Gly Glu Gly Ser Arg
Val Leu Asn Val Arg Ser Lys Thr Glu Ala Thr 305 310 315 320 Asp Arg
Asp Trp Glu Ile Tyr Pro Gln Ala Leu Tyr Asp Leu Leu Ile 325 330 335
Trp Ile His Asn Gly Tyr Gln Phe Arg Asp Ile Tyr Ile Thr Glu Asn 340
345 350 Gly Ala Ser Tyr Asn Asp Val Val Ser Pro Asp Gly Lys Val His
Asp 355 360 365 Pro Lys Arg Leu Asp Tyr Leu Lys Arg His Leu Ala Met
Ala Leu Arg 370 375 380 Ala Ile Glu Ala Gly Val Pro Leu Arg Gly Tyr
Phe Cys Trp Ser Leu 385 390 395 400 Met Asp Asn Phe Glu Trp Ala Met
Gly Thr Ser Ser Arg Phe Gly Leu 405 410 415 Ala Tyr Thr Asp Phe Thr
Thr Gln Lys Arg Ile Leu Lys Asp Ser Gly 420 425 430 Leu Trp Phe Gly
Glu Val Ala Arg Ala Asn Ala Leu Ile Asp Leu Pro 435 440 445
171215DNAUnknownObtained from environmental sample 17atgcggtacg
tgctgatttc ctgccttgcg ctggcttccc tgtgcgcgca gcctcttcct 60gtttccacgc
ctgaaaaaga gggcttctcg gcggagcgcc tcgggcggat gcaccggtat
120ttcgagaacc tgacgaaaac cggagagcgg cctggcgcga tcacgctgat
cgtgcgcaac 180gggcgcatcg tggactggcg cacgttcggg ctgcgcgacg
tcgagaacaa tctgccgatg 240gagaaggaca cgatcgtcca catctactcg
atgacgaagc cggtgacgtc cgtggccgtg 300atgatgctgg tggaggaggg
caggctggcg ctggacgacc gggtggacaa gttcattccc 360gagttcaagg
ggatgaaggt gtacaagggc ggcacggtgg agcggccgga gctggaggac
420gcggcgcggc cgatcacggt gaagcatctg ctgacgcaca cgagcgggct
gagctacggc 480tggggcaacg acaacgtctc cgcgatgtac cgcaaggccg
acccgctcgg cgcgccgagc 540ctgaaagagt ttatcgacag gctggtgaaa
ctgccgctgg cattccaccc gggcgagcgt 600tacgagtatt cgatgtcgat
cgacgtgctg ggctacctgg tggaggctgt ctccggcgag 660ccgttcgatc
agttcgtgga gaagcggatc acggggccgc tgaagatgaa cgacacgcat
720ttcagactgc cggaggcgaa gcgggcgcgg ctggcgaaga tctactcgcg
gcgcgagggg 780aagctgacgg cgcagcgcgg cctgcagacg ggaggcgttc
cgtacggcgg catggggctg 840tactcgacga tcggcgacta tgcgcggttc
gcgcagatgc tgttgaacgg cggccatctc 900gacggagtgc gcctgctggg
gcggaagacg gtggatctga tgatgatgaa ccatctgggc 960ggactgtcga
agccgacgat cggcggcgat gattcagcgg gattcggact gggcggagcg
1020gtgcggatcg atccggcgaa atcgggccgt ccgggcacgg aaggactctt
cggctgggac 1080ggggcggctt cgacgtattt ccgggtggac cggaaagaga
agctggcgat gctgctgttc 1140ctgcaatgga tgccgtttga tcaggggacg
ctgaacctgt acgagacgct ggtctaccaa 1200gctctggtgg actga
121518404PRTUnknownObtained from environmental sample 18Met Arg Tyr
Val Leu Ile Ser Cys Leu Ala Leu Ala Ser Leu Cys Ala 1 5 10 15 Gln
Pro Leu Pro Val Ser Thr Pro Glu Lys Glu Gly Phe Ser Ala Glu 20 25
30 Arg Leu Gly Arg Met His Arg Tyr Phe Glu Asn Leu Thr Lys Thr Gly
35 40 45 Glu Arg Pro Gly Ala Ile Thr Leu Ile Val Arg Asn Gly Arg
Ile Val 50 55 60 Asp Trp Arg Thr Phe Gly Leu Arg Asp Val Glu Asn
Asn Leu Pro Met 65 70 75 80 Glu Lys Asp Thr Ile Val His Ile Tyr Ser
Met Thr Lys Pro Val Thr 85 90 95 Ser Val Ala Val Met Met Leu Val
Glu Glu Gly Arg Leu Ala Leu Asp 100 105 110 Asp Arg Val Asp Lys Phe
Ile Pro Glu Phe Lys Gly Met Lys Val Tyr 115 120 125 Lys Gly Gly Thr
Val Glu Arg Pro Glu Leu Glu Asp Ala Ala Arg Pro 130 135 140 Ile Thr
Val Lys His Leu Leu Thr His Thr Ser Gly Leu Ser Tyr Gly 145 150 155
160 Trp Gly Asn Asp Asn Val Ser Ala Met Tyr Arg Lys Ala Asp Pro Leu
165 170 175 Gly Ala Pro Ser Leu Lys Glu Phe Ile Asp Arg Leu Val Lys
Leu Pro 180 185 190 Leu Ala Phe His Pro Gly Glu Arg Tyr Glu Tyr Ser
Met Ser Ile Asp 195 200 205 Val Leu Gly Tyr Leu Val Glu Ala Val Ser
Gly Glu Pro Phe Asp Gln 210 215 220 Phe Val Glu Lys Arg Ile Thr Gly
Pro Leu Lys Met Asn Asp Thr His 225 230 235 240 Phe Arg Leu Pro Glu
Ala Lys Arg Ala Arg Leu Ala Lys Ile Tyr Ser 245 250 255 Arg Arg Glu
Gly Lys Leu Thr Ala Gln Arg Gly Leu Gln Thr Gly Gly 260 265 270 Val
Pro Tyr Gly Gly Met Gly Leu Tyr Ser Thr Ile Gly Asp Tyr Ala 275 280
285 Arg Phe Ala Gln Met Leu Leu Asn Gly Gly His Leu Asp Gly Val Arg
290 295 300 Leu Leu Gly Arg Lys Thr Val Asp Leu Met Met Met Asn His
Leu Gly 305 310 315 320 Gly Leu Ser Lys Pro Thr Ile Gly Gly Asp Asp
Ser Ala Gly Phe Gly 325 330 335 Leu Gly Gly Ala Val Arg Ile Asp Pro
Ala Lys Ser Gly Arg Pro Gly 340 345 350 Thr Glu Gly Leu Phe Gly Trp
Asp Gly Ala Ala Ser Thr Tyr Phe Arg 355 360 365 Val Asp Arg Lys Glu
Lys Leu Ala Met Leu Leu Phe Leu Gln Trp Met 370 375 380 Pro Phe Asp
Gln Gly Thr Leu Asn Leu Tyr Glu Thr Leu Val Tyr Gln 385 390 395 400
Ala Leu Val Asp 191794DNAUnknownObtained from environmental sample
19atgcccgttt tgttcgccct gtttcttgtt gcctcgtcct gcgcggcgca gtcgctggcc
60gggccggttt ccctgcttgg cggagatgcg ggcgcggcgt tccgctatac cgggccatcg
120gcgggcgcgg cgagcggctc ggccgaatgg gtggcggtgg agaacatgcc
gttcacgcac 180gcctggcggc tgcgcacgaa tccgctgccg gagagcggcg
gcaacgaatg ggacctgcgc 240atccgcgccc gcggagcggc ggctgtttcg
gcaggggaca agatcctggc cgagttctgg 300atgcgctgcg tggagcccga
aaacggcgac tgcattctgc gcctgaacgt ggagcgcgac 360gggtcgccgt
ggaccaaatc catcagcaac ccctacccgg tgggccggga gtggcggcgg
420ttccgcgtgc tgttcgagat gcgggagagc tacgccgccg gcggctacat
gatcgatttc 480tggatgggcc agcaggtgca gacggcggaa gtgggcggga
tttccctgct gaattacggt 540ccgcaggcca cggccgagca gcttggcctg
gaccggtttt atgagggcgc ggcggcggac 600gccgcgtggc ggcaggcggc
cgagcagcgg atcgaggaga tccggaaagc gggcatgatc 660atcgtggcgg
tgacgccgga cggcgagccg atcgagggcg ctgaaatccg ggcgaagctg
720aagcggcacg cgttcgggtg gggcacggct gtggcggcat cacggcttct
ggggacggga 780acggacagcg agcgctaccg caacttcatc cgcgagaact
tcaacatggc ggtgctcgag 840aacgacctga aatggggccc gttcgaagag
aaccgcaacc gcgcgatgaa cgcgctgcgc 900tggctgcatg agaacgggat
cacgtggatc cgcgggcaca atctcgtctg gccgggctgg 960cggtggatgc
cgaacgacgt gcgcaacctg gcgaacaatc ccgaggcgct gcggcagcgg
1020attctggacc gcatccggga cacggccacg gccacgcgcg ggctggtggt
gcactgggac 1080gtcgtcaacg agccggtggc cgagcgcgac gtgctgaaca
ttctgggcga cgaggtgatg 1140gcggactggt tccgcgccgc gaaggagtgc
gatcccgagg cgaggatgtt catcaatgag 1200tacgacattc tggcggcgaa
cggggccaat ctgcggaagc agaacgcgta ttaccgcatg 1260atcgagatgc
tgttgaagct cgaggcgccg gtggagggca tcggcttcca gggccacttc
1320gacacggcca cgccgccgga gcggatgctg gagatcatga accggtacgc
ccggctcggg 1380ctgccgatcg ccatcaccga gtacgatttc gccacggcgg
acgaggagct gcaggcgcag 1440ttcacgcgcg acctgatgat tctcgccttc
agccatccgg cggtttcgga cttcctgatg 1500tggggcttct gggaagggag
ccactggaag ccgctgggcg ccatgatccg gcgcgactgg 1560agcgagaagc
cgatgtaccg cgtctggcgc gagctgatct tcgagcgctg gcagacggat
1620gaaacaggcg tgacgccgga gcacggtgcc atctacgtgc ggggcttcaa
gggcgactac 1680gagatcacgg tgaaggcggg cgggcaggaa gtccgggtgc
cgtacacgct gaaagaagac 1740ggccaggtgc tgtgggtgac ggtgggcggg
gcttctgaag agcgcgtgca gtaa 179420597PRTUnknownObtained from
environmental sample 20Met Pro Val Leu Phe Ala Leu Phe Leu Val Ala
Ser Ser Cys Ala Ala 1 5 10 15 Gln Ser Leu Ala Gly Pro Val Ser Leu
Leu Gly Gly Asp Ala Gly Ala 20 25 30 Ala Phe Arg Tyr Thr Gly Pro
Ser Ala Gly Ala Ala Ser Gly Ser Ala 35 40 45 Glu Trp Val Ala Val
Glu Asn Met Pro Phe Thr His Ala Trp Arg Leu 50 55 60 Arg Thr Asn
Pro Leu Pro Glu Ser Gly Gly Asn Glu Trp Asp Leu Arg 65 70 75 80 Ile
Arg Ala Arg Gly Ala Ala Ala Val Ser Ala Gly Asp Lys Ile Leu 85 90
95 Ala Glu Phe Trp Met Arg Cys Val Glu Pro Glu Asn Gly Asp Cys Ile
100 105 110 Leu Arg Leu Asn Val Glu Arg Asp Gly Ser Pro Trp Thr Lys
Ser Ile 115 120 125 Ser Asn Pro Tyr Pro Val Gly Arg Glu Trp Arg Arg
Phe Arg Val Leu 130 135 140 Phe Glu Met Arg Glu Ser Tyr Ala Ala Gly
Gly Tyr Met Ile Asp Phe 145 150 155 160 Trp Met Gly Gln Gln Val Gln
Thr Ala Glu Val Gly Gly Ile Ser Leu 165 170 175 Leu Asn Tyr Gly Pro
Gln Ala Thr Ala Glu Gln Leu Gly Leu Asp Arg 180 185 190 Phe Tyr Glu
Gly Ala Ala Ala Asp Ala Ala Trp Arg Gln Ala Ala Glu 195 200 205 Gln
Arg Ile Glu Glu Ile Arg Lys Ala Gly Met Ile Ile Val Ala Val 210 215
220 Thr Pro Asp Gly Glu Pro Ile Glu Gly Ala Glu Ile Arg Ala Lys Leu
225 230 235 240 Lys Arg His Ala Phe Gly Trp Gly Thr Ala Val Ala Ala
Ser Arg Leu 245 250 255 Leu Gly Thr Gly Thr Asp Ser Glu Arg Tyr Arg
Asn Phe Ile Arg Glu 260 265 270 Asn Phe Asn Met Ala Val Leu Glu Asn
Asp Leu Lys Trp Gly Pro Phe 275 280 285 Glu Glu Asn Arg Asn Arg Ala
Met Asn Ala Leu Arg Trp Leu His Glu 290 295 300 Asn Gly Ile Thr Trp
Ile Arg Gly His Asn Leu Val Trp Pro Gly Trp 305 310 315 320 Arg Trp
Met Pro Asn Asp Val Arg Asn Leu Ala Asn Asn Pro Glu Ala 325 330 335
Leu Arg Gln Arg Ile Leu Asp Arg Ile Arg Asp Thr Ala Thr Ala Thr 340
345 350 Arg Gly Leu Val Val His Trp Asp Val Val Asn Glu Pro Val Ala
Glu 355 360 365 Arg Asp Val Leu Asn Ile Leu Gly Asp Glu Val Met Ala
Asp Trp Phe 370 375 380 Arg Ala Ala Lys Glu Cys Asp Pro Glu Ala Arg
Met Phe Ile Asn Glu 385 390 395 400 Tyr Asp Ile Leu Ala Ala Asn Gly
Ala Asn Leu Arg Lys Gln Asn Ala 405 410 415 Tyr Tyr Arg Met Ile Glu
Met Leu Leu Lys Leu Glu Ala Pro Val Glu 420 425 430 Gly Ile Gly Phe
Gln Gly His Phe Asp Thr Ala Thr Pro Pro Glu Arg 435 440 445 Met Leu
Glu Ile Met Asn Arg Tyr Ala Arg Leu Gly Leu Pro Ile Ala 450 455 460
Ile Thr Glu Tyr Asp Phe Ala Thr Ala Asp Glu Glu Leu Gln Ala Gln 465
470 475 480 Phe Thr Arg Asp Leu Met Ile Leu Ala Phe Ser His Pro Ala
Val Ser
485 490 495 Asp Phe Leu Met Trp Gly Phe Trp Glu Gly Ser His Trp Lys
Pro Leu 500 505 510 Gly Ala Met Ile Arg Arg Asp Trp Ser Glu Lys Pro
Met Tyr Arg Val 515 520 525 Trp Arg Glu Leu Ile Phe Glu Arg Trp Gln
Thr Asp Glu Thr Gly Val 530 535 540 Thr Pro Glu His Gly Ala Ile Tyr
Val Arg Gly Phe Lys Gly Asp Tyr 545 550 555 560 Glu Ile Thr Val Lys
Ala Gly Gly Gln Glu Val Arg Val Pro Tyr Thr 565 570 575 Leu Lys Glu
Asp Gly Gln Val Leu Trp Val Thr Val Gly Gly Ala Ser 580 585 590 Glu
Glu Arg Val Gln 595 211032DNAClostridium thermocellum 21atggtgagtt
ttaaagcagg tataaattta ggcggatgga tatcacaata tcaagttttc 60agcaaagagc
atttcgatac attcattacg gagaaggaca ttgaaactat tgcagaagca
120gggtttgacc atgtcagact gccttttgat tatccaatta tcgagtctga
tgacaatgtg 180ggagaatata aagaagatgg gctttcttat attgaccggt
gccttgagtg gtgtaaaaaa 240tacaatttgg ggcttgtgtt ggatatgcat
cacgctcccg ggtaccgctt tcaagatttt 300aagacaagca ccttgtttga
agatccgaac cagcaaaaga gatttgttga catatggaga 360tttttagcca
agcgttacat aaatgaacgg gaacatattg cctttgaact gttaaatgaa
420gttgttgagc ctgacagtac ccgctggaac aagttgatgc ttgagtgtgt
aaaagcaatc 480agggaaattg attccaccag gtggctttac attgggggca
ataactataa cagtcctgat 540gagcttaaaa accttgcaga tattgatgat
gattacatag tttacaattt ccatttttac 600aatccttttt tctttacgca
tcagaaagcc cactggtcgg aaagtgccat ggcgtacaac 660aggactgtaa
aatatccggg acaatatgag ggaattgaag agtttgtgaa aaataatcct
720aagtacagtt ttatgatgga attgaataac ctgaagctga ataaagagct
tttgcgcaaa 780gatttaaaac cagcaattga gttcagggaa aagaaaaaat
gcaaactata ttgcggggag 840tttggcgtaa ttgccattgc tgacctggag
tccaggataa aatggcatga agattatata 900agtcttctag aggagtatga
tatcggcggc gcggtgtgga actacaaaaa aatggatttt 960gaaatttata
atgaggatag aaaacctgtc tcgcaagaat tggtaaatat actggcgaga
1020agaaaaactt ga 103222343PRTClostridium
thermocellumDOMAIN(1)...(323)Cellulase (glycosyl hydrolase family
5) 22Met Val Ser Phe Lys Ala Gly Ile Asn Leu Gly Gly Trp Ile Ser
Gln 1 5 10 15 Tyr Gln Val Phe Ser Lys Glu His Phe Asp Thr Phe Ile
Thr Glu Lys 20 25 30 Asp Ile Glu Thr Ile Ala Glu Ala Gly Phe Asp
His Val Arg Leu Pro 35 40 45 Phe Asp Tyr Pro Ile Ile Glu Ser Asp
Asp Asn Val Gly Glu Tyr Lys 50 55 60 Glu Asp Gly Leu Ser Tyr Ile
Asp Arg Cys Leu Glu Trp Cys Lys Lys 65 70 75 80 Tyr Asn Leu Gly Leu
Val Leu Asp Met His His Ala Pro Gly Tyr Arg 85 90 95 Phe Gln Asp
Phe Lys Thr Ser Thr Leu Phe Glu Asp Pro Asn Gln Gln 100 105 110 Lys
Arg Phe Val Asp Ile Trp Arg Phe Leu Ala Lys Arg Tyr Ile Asn 115 120
125 Glu Arg Glu His Ile Ala Phe Glu Leu Leu Asn Glu Val Val Glu Pro
130 135 140 Asp Ser Thr Arg Trp Asn Lys Leu Met Leu Glu Cys Val Lys
Ala Ile 145 150 155 160 Arg Glu Ile Asp Ser Thr Arg Trp Leu Tyr Ile
Gly Gly Asn Asn Tyr 165 170 175 Asn Ser Pro Asp Glu Leu Lys Asn Leu
Ala Asp Ile Asp Asp Asp Tyr 180 185 190 Ile Val Tyr Asn Phe His Phe
Tyr Asn Pro Phe Phe Phe Thr His Gln 195 200 205 Lys Ala His Trp Ser
Glu Ser Ala Met Ala Tyr Asn Arg Thr Val Lys 210 215 220 Tyr Pro Gly
Gln Tyr Glu Gly Ile Glu Glu Phe Val Lys Asn Asn Pro 225 230 235 240
Lys Tyr Ser Phe Met Met Glu Leu Asn Asn Leu Lys Leu Asn Lys Glu 245
250 255 Leu Leu Arg Lys Asp Leu Lys Pro Ala Ile Glu Phe Arg Glu Lys
Lys 260 265 270 Lys Cys Lys Leu Tyr Cys Gly Glu Phe Gly Val Ile Ala
Ile Ala Asp 275 280 285 Leu Glu Ser Arg Ile Lys Trp His Glu Asp Tyr
Ile Ser Leu Leu Glu 290 295 300 Glu Tyr Asp Ile Gly Gly Ala Val Trp
Asn Tyr Lys Lys Met Asp Phe 305 310 315 320 Glu Ile Tyr Asn Glu Asp
Arg Lys Pro Val Ser Gln Glu Leu Val Asn 325 330 335 Ile Leu Ala Arg
Arg Lys Thr 340 233966DNAClostridium thermocellum 23atgtataaaa
gattattgtc gtcagtactg ataattatgc tgttattatc agcctggtcg 60ccaatatccg
tacaagcttc tgatggaatc aatgacatta gaggtcattg ggctgaagaa
120gacttgaaca aatggatgga aaaaggtatt ttggtgggct accaggatgg
gacgataagg 180cccgataata atatcacaag agccgaattt gtcacattaa
ttaacaaggt tttcgggctt 240tatgaattaa gccgggagca attcgcagat
gttgaagact caaaatggta ttcccgtgaa 300atattaaaag ccagggctgc
gggatatatt gcaggttatg gaagcaatgt tttcaaacct 360gacaattata
ttacaagaca agaagccgtt gttataatcg cgaaagtttt tgaacttcaa
420agcggcagca attatacaag caagtttaaa gatggaagtc tggtaaagga
atacgcaaaa 480gattccgtta gcgcgttggt tgaaaaaggc tacatagcag
gttatgaaga tggcactttc 540aggccggaca actacattac ccgtgcagaa
acaataaaaa ttctgaataa aattattcct 600tccttgtata acgagaaagg
agattataaa aatgaagaag tagccggaaa cgctctgatt 660aacaccgaag
gagttatttt aaaagatacc gtaataaacg gggatttgta tcttgctcag
720ggaattcaga acggcgatgt tacccttgac ggtgtgaatg taaaaggaac
ggttttcgta 780aatggtggag gaagcgacag catacatttt ataaatacga
aaataaacag ggttgttgtc 840aataaaacag gagttagaat tgtaacttcc
ggcaatacct cggttgaaag tgttgtcgtt 900aaatccggtg caaaacttga
agaaaaagaa ttgacgggcg acggctttaa aaacgttaca 960gtcgattctc
aactttcagc cggcaatgaa ataatatttg tcggggattt tgaacaggtc
1020gatgttctgg cggatgatgc cttgctggaa accaaagagg caaaaatgaa
actgagaata 1080ttcggccaaa ggattaaagt aaatggaaag gcaatagaaa
aatcatcaaa gaactatatt 1140gtaaacgggg aacttatatc aactgaggaa
gaacccggtc cttccgacgc acccggtgcg 1200gaagacgatc aaaattcagg
tagtccgggc tcatcgacta atcctgcacc aaccaagaat 1260ccgaatgaag
agtggcgtct ggtttggagc gatgagttta acggttctga aataaatatg
1320gctaattgga gctatgacga cccgaccaac ggaagatgga acggggaagt
acaatcctac 1380acacaaaaca atgcctatat caaagacggc gcgttggtta
ttgaagcaag aaaagaagac 1440attacggaac caagcggtga gacttatcat
tatacatcgt caaagctgat taccaaaggc 1500aaaaagtcat ggaagtacgg
aaaatttgaa ataagggcaa aaatgccaca gggacaaggt 1560atatggcctg
caatctggat gatgccggaa gacgaaccct tctacggaac atggccaaag
1620tgcggcgaaa tagatattat ggagcttttg ggccacgagc ctgataaaat
ttatggaacg 1680atccattttg gagagcctca taaagaatcc cagggaacgt
ataccttgcc ggaaggccag 1740acttttgctg atgatttcca cgtttattcg
attgaatggg aaccgggaga aatacgctgg 1800tatatagacg gcaagctgta
tcatgtcgct aatgactggt actcgaggga cccgtacctt 1860gccgatgact
acacttatcc cgcacctttt gaccagaatt tcttcttgat tctcaatata
1920tccgttggtg gcggctggcc gggatatcct gacgaaacga cagttttccc
gcagcaaatg 1980gttgtggact atgtgagagt atatcaaaaa gataaatatc
ctcacaggga aaaaccggca 2040aaggaagaag tgaagccaag agagcctctt
gaggacggca attatatcta taacggcggt 2100tttgatgtgg atgattctgc
agcagttggt gtggacggtg ttccctatac gtcttactgg 2160acattcttaa
cagcatccgg tggagctgcg acagtcaatg tagaggaagg tgttatgcac
2220gtacagatag aaaacggagg gacaaccgac tacggcgtac aattgcttca
agctccgatt 2280catcttgaaa aaggcgcaaa atataaagca tcttttgaca
tgaaagctga aaatccaagg 2340caggtaaaac tgaaaatagg cggagacggc
gacaggggat ggaaagatta tgcggctatt 2400ccaccgttta cggtctcaac
agagatgacc aactatgagt ttgagtttac tatgaaagat 2460gataccgatg
ttaaggcacg gtttgagttt aatatgggtt tggacgataa tgatgtctgg
2520attgacaatg ttaaactgat taaaacagaa gatgcgccgg ttatagatcc
ttccgaaata 2580gcaagacctc cgcttctttc cggcaactat atatacaacg
gtacctttga ccaaggtccg 2640aacagaatgg gattctggaa ttttgttgtg
gatagcactg caaaggctac atactatatt 2700ggaagcgatg ttaatgagcg
caggtttgaa acaagaatag aaaaaggcgg aacatcgagg 2760ggagccataa
gattggttca gccgggaatt aacattgaaa acggcaaaac atacaaggtt
2820agcttcgaag ccagtgcggc aaatacaaga actattgagg tggaaattgc
aagcaatctt 2880cacaacagca gcatttttgc gacaactttt gaaataagca
aagagagcaa gatatacgaa 2940tttgagttta caatggacaa agattcggac
aagaacggag aacttaggtt caatctgggc 3000ggaagcaacg tgaacgtcta
tattgataat gtcgttatga aaagagtaag taccgatgaa 3060gttgaaggaa
acctgatttt aaacggcgta tttaacggcc tggcaggctg gggatatgga
3120gcgtatgaac ctggatcggc agattttgaa agtcatgagg aacaatttag
ggcaattatt 3180agctctgtcg gtaatgaagg ttggaatgta cagttgtatc
aggataatgt tccgctggaa 3240caagggcaaa cctacgaagt ttcttttgat
gcaaaatcaa cgattgacag aaagataatt 3300gttcagctgc aaaggaacgg
tacttcggat aataattggg actcctattt ctatcaagaa 3360gttgaactta
ctaatgaact taaaacattc aaatatgaat ttacaatgag taaacctaca
3420gattcggcgt caagatttaa ttttgctttg ggtaatactg aaaacaaaac
ttatgctcct 3480catgaaataa taattgacaa tgttgtagta agaaaagttg
cgactccttc tgcgctgata 3540ttgaacggaa cctttgacga tggaatggat
cattggctgc tatactgggg agacggtgaa 3600ggcaattgcg atgtaactga
cggagagctt gaaattaaca ttaccaaggt aggtaccgcg 3660gattacatgc
cgcagattaa acaggaaaac atagcgttgc aagagggtgt gacgtatact
3720ttgtctctta aagcgagagc gcttgaggca agaagtatta aagtggacat
attggattct 3780tcttataact ggtatggcgg aactattttc gatttaacaa
cggaagatgc cgtatacacg 3840tttacattta cccaaagcaa gtcgataaat
aacggtgtct taactataaa tttaggtacc 3900atagaaggta agacatccgc
cgcaactact gtctatcttg atgatatttt gctggaacaa 3960cagtaa
3966241321PRTClostridium
thermocellumSIGNAL(1)...(26)DOMAIN(30)...(71)S-layer homology
domain 24Met Tyr Lys Arg Leu Leu Ser Ser Val Leu Ile Ile Met Leu
Leu Leu 1 5 10 15 Ser Ala Trp Ser Pro Ile Ser Val Gln Ala Ser Asp
Gly Ile Asn Asp 20 25 30 Ile Arg Gly His Trp Ala Glu Glu Asp Leu
Asn Lys Trp Met Glu Lys 35 40 45 Gly Ile Leu Val Gly Tyr Gln Asp
Gly Thr Ile Arg Pro Asp Asn Asn 50 55 60 Ile Thr Arg Ala Glu Phe
Val Thr Leu Ile Asn Lys Val Phe Gly Leu 65 70 75 80 Tyr Glu Leu Ser
Arg Glu Gln Phe Ala Asp Val Glu Asp Ser Lys Trp 85 90 95 Tyr Ser
Arg Glu Ile Leu Lys Ala Arg Ala Ala Gly Tyr Ile Ala Gly 100 105 110
Tyr Gly Ser Asn Val Phe Lys Pro Asp Asn Tyr Ile Thr Arg Gln Glu 115
120 125 Ala Val Val Ile Ile Ala Lys Val Phe Glu Leu Gln Ser Gly Ser
Asn 130 135 140 Tyr Thr Ser Lys Phe Lys Asp Gly Ser Leu Val Lys Glu
Tyr Ala Lys 145 150 155 160 Asp Ser Val Ser Ala Leu Val Glu Lys Gly
Tyr Ile Ala Gly Tyr Glu 165 170 175 Asp Gly Thr Phe Arg Pro Asp Asn
Tyr Ile Thr Arg Ala Glu Thr Ile 180 185 190 Lys Ile Leu Asn Lys Ile
Ile Pro Ser Leu Tyr Asn Glu Lys Gly Asp 195 200 205 Tyr Lys Asn Glu
Glu Val Ala Gly Asn Ala Leu Ile Asn Thr Glu Gly 210 215 220 Val Ile
Leu Lys Asp Thr Val Ile Asn Gly Asp Leu Tyr Leu Ala Gln 225 230 235
240 Gly Ile Gln Asn Gly Asp Val Thr Leu Asp Gly Val Asn Val Lys Gly
245 250 255 Thr Val Phe Val Asn Gly Gly Gly Ser Asp Ser Ile His Phe
Ile Asn 260 265 270 Thr Lys Ile Asn Arg Val Val Val Asn Lys Thr Gly
Val Arg Ile Val 275 280 285 Thr Ser Gly Asn Thr Ser Val Glu Ser Val
Val Val Lys Ser Gly Ala 290 295 300 Lys Leu Glu Glu Lys Glu Leu Thr
Gly Asp Gly Phe Lys Asn Val Thr 305 310 315 320 Val Asp Ser Gln Leu
Ser Ala Gly Asn Glu Ile Ile Phe Val Gly Asp 325 330 335 Phe Glu Gln
Val Asp Val Leu Ala Asp Asp Ala Leu Leu Glu Thr Lys 340 345 350 Glu
Ala Lys Met Lys Leu Arg Ile Phe Gly Gln Arg Ile Lys Val Asn 355 360
365 Gly Lys Ala Ile Glu Lys Ser Ser Lys Asn Tyr Ile Val Asn Gly Glu
370 375 380 Leu Ile Ser Thr Glu Glu Glu Pro Gly Pro Ser Asp Ala Pro
Gly Ala 385 390 395 400 Glu Asp Asp Gln Asn Ser Gly Ser Pro Gly Ser
Ser Thr Asn Pro Ala 405 410 415 Pro Thr Lys Asn Pro Asn Glu Glu Trp
Arg Leu Val Trp Ser Asp Glu 420 425 430 Phe Asn Gly Ser Glu Ile Asn
Met Ala Asn Trp Ser Tyr Asp Asp Pro 435 440 445 Thr Asn Gly Arg Trp
Asn Gly Glu Val Gln Ser Tyr Thr Gln Asn Asn 450 455 460 Ala Tyr Ile
Lys Asp Gly Ala Leu Val Ile Glu Ala Arg Lys Glu Asp 465 470 475 480
Ile Thr Glu Pro Ser Gly Glu Thr Tyr His Tyr Thr Ser Ser Lys Leu 485
490 495 Ile Thr Lys Gly Lys Lys Ser Trp Lys Tyr Gly Lys Phe Glu Ile
Arg 500 505 510 Ala Lys Met Pro Gln Gly Gln Gly Ile Trp Pro Ala Ile
Trp Met Met 515 520 525 Pro Glu Asp Glu Pro Phe Tyr Gly Thr Trp Pro
Lys Cys Gly Glu Ile 530 535 540 Asp Ile Met Glu Leu Leu Gly His Glu
Pro Asp Lys Ile Tyr Gly Thr 545 550 555 560 Ile His Phe Gly Glu Pro
His Lys Glu Ser Gln Gly Thr Tyr Thr Leu 565 570 575 Pro Glu Gly Gln
Thr Phe Ala Asp Asp Phe His Val Tyr Ser Ile Glu 580 585 590 Trp Glu
Pro Gly Glu Ile Arg Trp Tyr Ile Asp Gly Lys Leu Tyr His 595 600 605
Val Ala Asn Asp Trp Tyr Ser Arg Asp Pro Tyr Leu Ala Asp Asp Tyr 610
615 620 Thr Tyr Pro Ala Pro Phe Asp Gln Asn Phe Phe Leu Ile Leu Asn
Ile 625 630 635 640 Ser Val Gly Gly Gly Trp Pro Gly Tyr Pro Asp Glu
Thr Thr Val Phe 645 650 655 Pro Gln Gln Met Val Val Asp Tyr Val Arg
Val Tyr Gln Lys Asp Lys 660 665 670 Tyr Pro His Arg Glu Lys Pro Ala
Lys Glu Glu Val Lys Pro Arg Glu 675 680 685 Pro Leu Glu Asp Gly Asn
Tyr Ile Tyr Asn Gly Gly Phe Asp Val Asp 690 695 700 Asp Ser Ala Ala
Val Gly Val Asp Gly Val Pro Tyr Thr Ser Tyr Trp 705 710 715 720 Thr
Phe Leu Thr Ala Ser Gly Gly Ala Ala Thr Val Asn Val Glu Glu 725 730
735 Gly Val Met His Val Gln Ile Glu Asn Gly Gly Thr Thr Asp Tyr Gly
740 745 750 Val Gln Leu Leu Gln Ala Pro Ile His Leu Glu Lys Gly Ala
Lys Tyr 755 760 765 Lys Ala Ser Phe Asp Met Lys Ala Glu Asn Pro Arg
Gln Val Lys Leu 770 775 780 Lys Ile Gly Gly Asp Gly Asp Arg Gly Trp
Lys Asp Tyr Ala Ala Ile 785 790 795 800 Pro Pro Phe Thr Val Ser Thr
Glu Met Thr Asn Tyr Glu Phe Glu Phe 805 810 815 Thr Met Lys Asp Asp
Thr Asp Val Lys Ala Arg Phe Glu Phe Asn Met 820 825 830 Gly Leu Asp
Asp Asn Asp Val Trp Ile Asp Asn Val Lys Leu Ile Lys 835 840 845 Thr
Glu Asp Ala Pro Val Ile Asp Pro Ser Glu Ile Ala Arg Pro Pro 850 855
860 Leu Leu Ser Gly Asn Tyr Ile Tyr Asn Gly Thr Phe Asp Gln Gly Pro
865 870 875 880 Asn Arg Met Gly Phe Trp Asn Phe Val Val Asp Ser Thr
Ala Lys Ala 885 890 895 Thr Tyr Tyr Ile Gly Ser Asp Val Asn Glu Arg
Arg Phe Glu Thr Arg 900 905 910 Ile Glu Lys Gly Gly Thr Ser Arg Gly
Ala Ile Arg Leu Val Gln Pro 915 920 925 Gly Ile Asn Ile Glu Asn Gly
Lys Thr Tyr Lys Val Ser Phe Glu Ala 930 935 940 Ser Ala Ala Asn Thr
Arg Thr Ile Glu Val Glu Ile Ala Ser Asn Leu 945 950 955 960 His Asn
Ser Ser Ile Phe Ala Thr Thr Phe Glu Ile Ser Lys Glu Ser 965 970 975
Lys Ile Tyr Glu Phe Glu Phe Thr Met Asp Lys Asp Ser Asp Lys Asn 980
985 990 Gly Glu Leu Arg Phe Asn Leu Gly Gly Ser Asn Val Asn Val Tyr
Ile 995 1000 1005 Asp Asn Val Val Met Lys Arg Val Ser Thr Asp Glu
Val Glu Gly 1010 1015 1020 Asn Leu Ile Leu Asn Gly Val Phe Asn
Gly
Leu Ala Gly Trp Gly 1025 1030 1035 Tyr Gly Ala Tyr Glu Pro Gly Ser
Ala Asp Phe Glu Ser His Glu 1040 1045 1050 Glu Gln Phe Arg Ala Ile
Ile Ser Ser Val Gly Asn Glu Gly Trp 1055 1060 1065 Asn Val Gln Leu
Tyr Gln Asp Asn Val Pro Leu Glu Gln Gly Gln 1070 1075 1080 Thr Tyr
Glu Val Ser Phe Asp Ala Lys Ser Thr Ile Asp Arg Lys 1085 1090 1095
Ile Ile Val Gln Leu Gln Arg Asn Gly Thr Ser Asp Asn Asn Trp 1100
1105 1110 Asp Ser Tyr Phe Tyr Gln Glu Val Glu Leu Thr Asn Glu Leu
Lys 1115 1120 1125 Thr Phe Lys Tyr Glu Phe Thr Met Ser Lys Pro Thr
Asp Ser Ala 1130 1135 1140 Ser Arg Phe Asn Phe Ala Leu Gly Asn Thr
Glu Asn Lys Thr Tyr 1145 1150 1155 Ala Pro His Glu Ile Ile Ile Asp
Asn Val Val Val Arg Lys Val 1160 1165 1170 Ala Thr Pro Ser Ala Leu
Ile Leu Asn Gly Thr Phe Asp Asp Gly 1175 1180 1185 Met Asp His Trp
Leu Leu Tyr Trp Gly Asp Gly Glu Gly Asn Cys 1190 1195 1200 Asp Val
Thr Asp Gly Glu Leu Glu Ile Asn Ile Thr Lys Val Gly 1205 1210 1215
Thr Ala Asp Tyr Met Pro Gln Ile Lys Gln Glu Asn Ile Ala Leu 1220
1225 1230 Gln Glu Gly Val Thr Tyr Thr Leu Ser Leu Lys Ala Arg Ala
Leu 1235 1240 1245 Glu Ala Arg Ser Ile Lys Val Asp Ile Leu Asp Ser
Ser Tyr Asn 1250 1255 1260 Trp Tyr Gly Gly Thr Ile Phe Asp Leu Thr
Thr Glu Asp Ala Val 1265 1270 1275 Tyr Thr Phe Thr Phe Thr Gln Ser
Lys Ser Ile Asn Asn Gly Val 1280 1285 1290 Leu Thr Ile Asn Leu Gly
Thr Ile Glu Gly Lys Thr Ser Ala Ala 1295 1300 1305 Thr Thr Val Tyr
Leu Asp Asp Ile Leu Leu Glu Gln Gln 1310 1315 1320
251347DNAClostridium thermocellum 25atgtcaaaga taactttccc
aaaagatttc atatggggtt ctgcaacagc agcatatcag 60attgaaggtg catacaacga
agacggcaaa ggtgaatcta tatgggaccg tttttcccac 120acgccaggaa
atatagcaga cggacatacc ggcgatgttg catgcgacca ctatcatcgt
180tatgaagaag atatcaaaat aatgaaagaa atcggtatta aatcatacag
gttttccatc 240tcatggccca gaatctttcc tgaaggaaca ggtaaattaa
atcaaaaggg actggatttt 300tacaaaaggc tcacaaatct gcttctggaa
aacggaatta tgcctgcaat cactctttat 360cactgggacc ttccccaaaa
gcttcaggat aaaggcggat ggaaaaaccg ggacaccacc 420gattatttta
cagaatactc tgaagtaata tttaaaaatc tcggagatat cgttccaata
480tggtttactc acaatgaacc cggtgttgtt tctttgcttg gccacttttt
aggaattcat 540gcccctggga taaaagacct ccgcacttca ttggaagtct
cgcacaatct tcttttgtcc 600cacggcaagg ccgtgaaact gtttagagaa
atgaatattg acgcccaaat tggaatagct 660ctcaatttat cttaccatta
tcccgcatcc gaaaaagctg aggatattga agcagcggaa 720ttgtcatttt
ctctggcggg aaggtggtat ctggatcctg tgctaaaagg ccggtatcct
780gaaaacgcat tgaaacttta taaaaagaag ggtattgagc tttctttccc
tgaagatgac 840ctgaaactta tcagtcagcc aatagacttc atagcattca
acaattattc ttcggaattt 900ataaaatatg atccgtccag tgagtcaggt
ttttcacctg caaactccat attagaaaag 960ttcgaaaaaa cagatatggg
ctggatcata tatcctgaag gcttgtatga tctgcttatg 1020ctccttgaca
gggattatgg aaagccaaac attgttatca gcgaaaacgg agccgccttc
1080aaagatgaaa taggtagcaa cggaaagata gaagacacaa agagaatcca
atatcttaaa 1140gattatctga cccaggctca cagggcaatt caggacggtg
taaacttaaa agcatactac 1200ttgtggtcgc ttttggacaa ctttgaatgg
gcttacgggt acaacaagag attcggaatc 1260gttcacgtaa attttgatac
gttggaaaga aaaataaagg atagcggcta ctggtacaaa 1320gaagtaatca
aaaacaacgg tttttaa 134726448PRTClostridium
thermocellumDOMAIN(2)...(448)Glycosyl hydrolase family 1 26Met Ser
Lys Ile Thr Phe Pro Lys Asp Phe Ile Trp Gly Ser Ala Thr 1 5 10 15
Ala Ala Tyr Gln Ile Glu Gly Ala Tyr Asn Glu Asp Gly Lys Gly Glu 20
25 30 Ser Ile Trp Asp Arg Phe Ser His Thr Pro Gly Asn Ile Ala Asp
Gly 35 40 45 His Thr Gly Asp Val Ala Cys Asp His Tyr His Arg Tyr
Glu Glu Asp 50 55 60 Ile Lys Ile Met Lys Glu Ile Gly Ile Lys Ser
Tyr Arg Phe Ser Ile 65 70 75 80 Ser Trp Pro Arg Ile Phe Pro Glu Gly
Thr Gly Lys Leu Asn Gln Lys 85 90 95 Gly Leu Asp Phe Tyr Lys Arg
Leu Thr Asn Leu Leu Leu Glu Asn Gly 100 105 110 Ile Met Pro Ala Ile
Thr Leu Tyr His Trp Asp Leu Pro Gln Lys Leu 115 120 125 Gln Asp Lys
Gly Gly Trp Lys Asn Arg Asp Thr Thr Asp Tyr Phe Thr 130 135 140 Glu
Tyr Ser Glu Val Ile Phe Lys Asn Leu Gly Asp Ile Val Pro Ile 145 150
155 160 Trp Phe Thr His Asn Glu Pro Gly Val Val Ser Leu Leu Gly His
Phe 165 170 175 Leu Gly Ile His Ala Pro Gly Ile Lys Asp Leu Arg Thr
Ser Leu Glu 180 185 190 Val Ser His Asn Leu Leu Leu Ser His Gly Lys
Ala Val Lys Leu Phe 195 200 205 Arg Glu Met Asn Ile Asp Ala Gln Ile
Gly Ile Ala Leu Asn Leu Ser 210 215 220 Tyr His Tyr Pro Ala Ser Glu
Lys Ala Glu Asp Ile Glu Ala Ala Glu 225 230 235 240 Leu Ser Phe Ser
Leu Ala Gly Arg Trp Tyr Leu Asp Pro Val Leu Lys 245 250 255 Gly Arg
Tyr Pro Glu Asn Ala Leu Lys Leu Tyr Lys Lys Lys Gly Ile 260 265 270
Glu Leu Ser Phe Pro Glu Asp Asp Leu Lys Leu Ile Ser Gln Pro Ile 275
280 285 Asp Phe Ile Ala Phe Asn Asn Tyr Ser Ser Glu Phe Ile Lys Tyr
Asp 290 295 300 Pro Ser Ser Glu Ser Gly Phe Ser Pro Ala Asn Ser Ile
Leu Glu Lys 305 310 315 320 Phe Glu Lys Thr Asp Met Gly Trp Ile Ile
Tyr Pro Glu Gly Leu Tyr 325 330 335 Asp Leu Leu Met Leu Leu Asp Arg
Asp Tyr Gly Lys Pro Asn Ile Val 340 345 350 Ile Ser Glu Asn Gly Ala
Ala Phe Lys Asp Glu Ile Gly Ser Asn Gly 355 360 365 Lys Ile Glu Asp
Thr Lys Arg Ile Gln Tyr Leu Lys Asp Tyr Leu Thr 370 375 380 Gln Ala
His Arg Ala Ile Gln Asp Gly Val Asn Leu Lys Ala Tyr Tyr 385 390 395
400 Leu Trp Ser Leu Leu Asp Asn Phe Glu Trp Ala Tyr Gly Tyr Asn Lys
405 410 415 Arg Phe Gly Ile Val His Val Asn Phe Asp Thr Leu Glu Arg
Lys Ile 420 425 430 Lys Asp Ser Gly Tyr Trp Tyr Lys Glu Val Ile Lys
Asn Asn Gly Phe 435 440 445 271362DNAUnknownObtained from
environmental sample 27atggcaaaca agataacctt tcctgaaaat tttctgtggg
gcgcggcaac ggcttcgtac 60cagatcgaag gcgcctggaa caaacatggt aaaggcgaat
ccacctggga tcgcttttca 120cacacgcccg gtaagatcag gaacaacgat
acgggcgatg tagcaaatga ccattatcgc 180ctctggaaaa aagacattgg
cttgatgaag aagatcgggt tgaaggctta tcgattttcc 240atttcgtggc
cgcgtattct tcctgctgga agaggcaagg tcaatcaaag agggctggat
300ttttacaaca agatcgtaga tgagctgctg aaagcagata tcatcccatt
tgttactctc 360aatcactggg acctgcccca aaaactggaa gatgagggcg
gctggccggc ccgttctact 420gccgatgctt ttattgaata cacagatgtg
atcacccgct cccttggcga ccgcgcaaag 480aattggatca ctcacaatga
acctgccgtc gttgcctgga tgggatactc cactggccaa 540cacgcacccg
gactgaagga ctatgggctt ggtgcccgcg ccgcgcatca cctgttgctc
600tcacatggac aggctgtacc ggtcattcgc agcaatagcg cgggggcaga
agtgggaatt 660acgctcgata ttagctggcg gatcgctgcc tcaaacagcc
gcgccgaccg ggagctggtc 720cgtgaggatg atgggaggtg gttccgctgg
tttgccgacc cgctttacgg gcgcggatat 780ccctccgata aggtgtctga
tttcactaag ttgggagcac tgcccaacgg acttgatttt 840gtgcaggcag
gcgacatgga cacgatcgcg acaccgactg attttatggg gctaaactac
900tactcccgaa atgtctaccg cgcggacggt gcagataatg atccgcaaac
tgttttccca 960caaccgaaga tgcccgaaca ctggaccgag atgggctggg
aaatttaccc ggatgggctg 1020accaacattc tgggacgcgt ctatttcaac
tatcagccgc gcaaactata cgtcacagaa 1080aacggcgcca gttactccac
gcctcctgat gataagggga atgtcgcgga tgaactccgc 1140atccattatc
tgaggacaca ttttgcagct gcctatcggg ccattcaaat gggcgtgcct
1200ctggcaggat acttcgtctg gtccctcatg gacaactttg agtggtcatg
gggctatatg 1260caacgctttg gactcatctg ggtggattat gagacccaaa
aacgcacttt aaaggatagc 1320gcaaaatggt ataagcgcgt gatcaagaag
aatgggctct aa 136228453PRTUnknownObtained from environmental sample
28Met Ala Asn Lys Ile Thr Phe Pro Glu Asn Phe Leu Trp Gly Ala Ala 1
5 10 15 Thr Ala Ser Tyr Gln Ile Glu Gly Ala Trp Asn Lys His Gly Lys
Gly 20 25 30 Glu Ser Thr Trp Asp Arg Phe Ser His Thr Pro Gly Lys
Ile Arg Asn 35 40 45 Asn Asp Thr Gly Asp Val Ala Asn Asp His Tyr
Arg Leu Trp Lys Lys 50 55 60 Asp Ile Gly Leu Met Lys Lys Ile Gly
Leu Lys Ala Tyr Arg Phe Ser 65 70 75 80 Ile Ser Trp Pro Arg Ile Leu
Pro Ala Gly Arg Gly Lys Val Asn Gln 85 90 95 Arg Gly Leu Asp Phe
Tyr Asn Lys Ile Val Asp Glu Leu Leu Lys Ala 100 105 110 Asp Ile Ile
Pro Phe Val Thr Leu Asn His Trp Asp Leu Pro Gln Lys 115 120 125 Leu
Glu Asp Glu Gly Gly Trp Pro Ala Arg Ser Thr Ala Asp Ala Phe 130 135
140 Ile Glu Tyr Thr Asp Val Ile Thr Arg Ser Leu Gly Asp Arg Ala Lys
145 150 155 160 Asn Trp Ile Thr His Asn Glu Pro Ala Val Val Ala Trp
Met Gly Tyr 165 170 175 Ser Thr Gly Gln His Ala Pro Gly Leu Lys Asp
Tyr Gly Leu Gly Ala 180 185 190 Arg Ala Ala His His Leu Leu Leu Ser
His Gly Gln Ala Val Pro Val 195 200 205 Ile Arg Ser Asn Ser Ala Gly
Ala Glu Val Gly Ile Thr Leu Asp Ile 210 215 220 Ser Trp Arg Ile Ala
Ala Ser Asn Ser Arg Ala Asp Arg Glu Leu Val 225 230 235 240 Arg Glu
Asp Asp Gly Arg Trp Phe Arg Trp Phe Ala Asp Pro Leu Tyr 245 250 255
Gly Arg Gly Tyr Pro Ser Asp Lys Val Ser Asp Phe Thr Lys Leu Gly 260
265 270 Ala Leu Pro Asn Gly Leu Asp Phe Val Gln Ala Gly Asp Met Asp
Thr 275 280 285 Ile Ala Thr Pro Thr Asp Phe Met Gly Leu Asn Tyr Tyr
Ser Arg Asn 290 295 300 Val Tyr Arg Ala Asp Gly Ala Asp Asn Asp Pro
Gln Thr Val Phe Pro 305 310 315 320 Gln Pro Lys Met Pro Glu His Trp
Thr Glu Met Gly Trp Glu Ile Tyr 325 330 335 Pro Asp Gly Leu Thr Asn
Ile Leu Gly Arg Val Tyr Phe Asn Tyr Gln 340 345 350 Pro Arg Lys Leu
Tyr Val Thr Glu Asn Gly Ala Ser Tyr Ser Thr Pro 355 360 365 Pro Asp
Asp Lys Gly Asn Val Ala Asp Glu Leu Arg Ile His Tyr Leu 370 375 380
Arg Thr His Phe Ala Ala Ala Tyr Arg Ala Ile Gln Met Gly Val Pro 385
390 395 400 Leu Ala Gly Tyr Phe Val Trp Ser Leu Met Asp Asn Phe Glu
Trp Ser 405 410 415 Trp Gly Tyr Met Gln Arg Phe Gly Leu Ile Trp Val
Asp Tyr Glu Thr 420 425 430 Gln Lys Arg Thr Leu Lys Asp Ser Ala Lys
Trp Tyr Lys Arg Val Ile 435 440 445 Lys Lys Asn Gly Leu 450
291362DNAUnknownObtained from environmental sample 29atggcgaaca
aaattacctt tcccgaaaat tttctttggg gcgcggcaac agcctcctac 60cagatcgaag
gtgcgtggga caaacatggc aagggtgaat ccatctggga tcgcttttcg
120catacccctg gcaagatcag aaataatgat acgggcgatg ttgccaatga
tcattatcgt 180ctctggaaaa aagacattgg cttgatgaag aagatcggct
tgaaggcata tcgtttttcc 240atttcgtggc cgcgtgttct tcccgccgga
cgcggcaaag tcaatcagaa gggactggat 300ttctataaca ggctggtaga
tgctctgttg aaagaagata tcatcccatt tgtgactctc 360aatcactggg
acctgcccca aaagctggag gaggaaggcg gttggccggt tcgctccacc
420gcagatgcct ttgtggaata cacagacgtg gtcacacgtt ccctcggcga
ccgcgtaaag 480aattggatca cgcataatga gcctgccgtc gttgcctgga
tgggatattc cacaggtcaa 540cacgcacccg gtttgaagga ctatgggctt
ggtgtgcgcg ccgcgcatca tctgctgctc 600tcccacgggc aggcggtgcc
agtcatccgc agtaacagcg ccgatgcaga agtgggcatt 660acgctggata
ttagctggcg gattcctgcc tccaatagcc gagcagaccg ggaattggtc
720cgtaaagatg acggactatg gttccgctgg ttcgccgatc cgctttatgg
gcgcggatac 780ccctcggata aagtcaccga ttttacaaag atcggcgcgc
tgcccaatgg tctggacttt 840atgcaagccg gtgatatgga tgcgatcgcc
acgccaaccg atttcatggg gctgaactat 900tatttccgaa atgtctaccg
cgcgaatggc gaagacaatg atccgcaggt cgttttccca 960caaccaaaga
tgcccgaaca ctggacggag atgggctggg aaatctatcc ggatggactg
1020acgaacatcc tgggacgcgt ttatttcaat taccagccac ataaactgta
tatcacagag 1080aacggcgcga gctactccac cccgcccgat gaaaagggga
atgtcgccga tgagctccgc 1140actcattatt tacggacaca cttcgcggct
gcctaccggg cgattcagat gggcgtgcct 1200ctggcaggat actttgtctg
gtccctcatg gacaactttg agtggtcctg gggatatatg 1260cagcgctttg
ggctcatctg ggtggactac gagacacaga aacgcaccct gaaggatagc
1320gccaagtggt acaaacgtgt gatcaggaag aatgggtttt ag
136230453PRTUnknownObtained from environmental sample 30Met Ala Asn
Lys Ile Thr Phe Pro Glu Asn Phe Leu Trp Gly Ala Ala 1 5 10 15 Thr
Ala Ser Tyr Gln Ile Glu Gly Ala Trp Asp Lys His Gly Lys Gly 20 25
30 Glu Ser Ile Trp Asp Arg Phe Ser His Thr Pro Gly Lys Ile Arg Asn
35 40 45 Asn Asp Thr Gly Asp Val Ala Asn Asp His Tyr Arg Leu Trp
Lys Lys 50 55 60 Asp Ile Gly Leu Met Lys Lys Ile Gly Leu Lys Ala
Tyr Arg Phe Ser 65 70 75 80 Ile Ser Trp Pro Arg Val Leu Pro Ala Gly
Arg Gly Lys Val Asn Gln 85 90 95 Lys Gly Leu Asp Phe Tyr Asn Arg
Leu Val Asp Ala Leu Leu Lys Glu 100 105 110 Asp Ile Ile Pro Phe Val
Thr Leu Asn His Trp Asp Leu Pro Gln Lys 115 120 125 Leu Glu Glu Glu
Gly Gly Trp Pro Val Arg Ser Thr Ala Asp Ala Phe 130 135 140 Val Glu
Tyr Thr Asp Val Val Thr Arg Ser Leu Gly Asp Arg Val Lys 145 150 155
160 Asn Trp Ile Thr His Asn Glu Pro Ala Val Val Ala Trp Met Gly Tyr
165 170 175 Ser Thr Gly Gln His Ala Pro Gly Leu Lys Asp Tyr Gly Leu
Gly Val 180 185 190 Arg Ala Ala His His Leu Leu Leu Ser His Gly Gln
Ala Val Pro Val 195 200 205 Ile Arg Ser Asn Ser Ala Asp Ala Glu Val
Gly Ile Thr Leu Asp Ile 210 215 220 Ser Trp Arg Ile Pro Ala Ser Asn
Ser Arg Ala Asp Arg Glu Leu Val 225 230 235 240 Arg Lys Asp Asp Gly
Leu Trp Phe Arg Trp Phe Ala Asp Pro Leu Tyr 245 250 255 Gly Arg Gly
Tyr Pro Ser Asp Lys Val Thr Asp Phe Thr Lys Ile Gly 260 265 270 Ala
Leu Pro Asn Gly Leu Asp Phe Met Gln Ala Gly Asp Met Asp Ala 275 280
285 Ile Ala Thr Pro Thr Asp Phe Met Gly Leu Asn Tyr Tyr Phe Arg Asn
290 295 300 Val Tyr Arg Ala Asn Gly Glu Asp Asn Asp Pro Gln Val Val
Phe Pro 305 310 315 320 Gln Pro Lys Met Pro Glu His Trp Thr Glu Met
Gly Trp Glu Ile Tyr 325 330 335 Pro Asp Gly Leu Thr Asn Ile Leu Gly
Arg Val Tyr Phe Asn Tyr Gln 340 345 350 Pro His Lys Leu Tyr Ile Thr
Glu Asn Gly Ala Ser Tyr Ser Thr Pro 355 360 365 Pro Asp Glu Lys Gly
Asn Val Ala Asp Glu Leu Arg Thr His Tyr Leu 370 375 380 Arg Thr His
Phe Ala Ala Ala Tyr Arg Ala Ile Gln Met Gly Val Pro 385 390 395 400
Leu Ala Gly Tyr Phe Val Trp Ser Leu Met Asp Asn Phe Glu Trp Ser 405
410
415 Trp Gly Tyr Met Gln Arg Phe Gly Leu Ile Trp Val Asp Tyr Glu Thr
420 425 430 Gln Lys Arg Thr Leu Lys Asp Ser Ala Lys Trp Tyr Lys Arg
Val Ile 435 440 445 Arg Lys Asn Gly Phe 450
311167DNAUnknownObtained from environmental sample 31atggaagacc
gcccgcacta ttacagcgac gaccatctct ggggtgtact gtgcgtgacc 60gcctacatca
aggaaactgg ggactttgca ttcctggacg agaaagttca cttttacgag
120aaggacccgg tcgagggcgt gtctgtgctg gatcacgtta aacgggcctt
gacctttacc 180cgcaacaaca tcgggaaaca tggtctgcct ctcctcggct
ttgcggattg gaacgacacg 240atcaatctgg cgaagggcgc cgagtctctt
ttcacgtcgc atctatatgg acgcgcgctg 300ctggagttta ttgatctgct
cacatatctt ggcaagaacg atgaagccga tgaatggcag 360cgagcccacg
ttgagatgca gtcccgcgtc gaaaaacatg cctgggatgg cgaatggtat
420ttcatgtact ttgaccacga cggcagcccg gttgggtctc acacgaatca
gtatggaaag 480atccatctca acggacagag ctgggctgtg ctttcgggct
ttgcctctcc gcagcgcgcc 540cgccaggcca tggactcggt ttacaagcat
ctcaacacaa agcacggcat caagctctcc 600acgccgggct acaatggcta
tgaccccaac tacggcggcg tgaccaccta cccaccggga 660gcaaaggaaa
acggcggcat cttcctgcac ccgaatccct gggccatgat cgcagagacc
720atgctcgggg atggcgatcg cgcctacgag tattactcgc agatcaaccc
ggccggcaag 780aacgatgaca tcgacctgta cgaggtcgag ccatatgttt
acgctcaaaa catcctgggc 840gatgagcatc cgcagttcgg gctgggacgc
aactcgtggc tctcgggtac ggcatcctgg 900tgctatcagg ctgccacaca
gtggatcctc ggaatccgcg ccgactatga agggctgcgc 960atcgacccgt
gcattccgtc caagtgggat gggttcaagg caacgcgcct gtatcgcggc
1020gtgaagtaca acattacggt caccaacccg aagcacatct gcaaaggcgt
ggaaaaagtt 1080ctggtcaacg gcaaaccggt tgaggggaat gtggtccggg
cagacgtggg tttgcgcgaa 1140gtgaacgtgg aagttacctt aggataa
116732388PRTUnknownObtained from environmental sample 32Met Glu Asp
Arg Pro His Tyr Tyr Ser Asp Asp His Leu Trp Gly Val 1 5 10 15 Leu
Cys Val Thr Ala Tyr Ile Lys Glu Thr Gly Asp Phe Ala Phe Leu 20 25
30 Asp Glu Lys Val His Phe Tyr Glu Lys Asp Pro Val Glu Gly Val Ser
35 40 45 Val Leu Asp His Val Lys Arg Ala Leu Thr Phe Thr Arg Asn
Asn Ile 50 55 60 Gly Lys His Gly Leu Pro Leu Leu Gly Phe Ala Asp
Trp Asn Asp Thr 65 70 75 80 Ile Asn Leu Ala Lys Gly Ala Glu Ser Leu
Phe Thr Ser His Leu Tyr 85 90 95 Gly Arg Ala Leu Leu Glu Phe Ile
Asp Leu Leu Thr Tyr Leu Gly Lys 100 105 110 Asn Asp Glu Ala Asp Glu
Trp Gln Arg Ala His Val Glu Met Gln Ser 115 120 125 Arg Val Glu Lys
His Ala Trp Asp Gly Glu Trp Tyr Phe Met Tyr Phe 130 135 140 Asp His
Asp Gly Ser Pro Val Gly Ser His Thr Asn Gln Tyr Gly Lys 145 150 155
160 Ile His Leu Asn Gly Gln Ser Trp Ala Val Leu Ser Gly Phe Ala Ser
165 170 175 Pro Gln Arg Ala Arg Gln Ala Met Asp Ser Val Tyr Lys His
Leu Asn 180 185 190 Thr Lys His Gly Ile Lys Leu Ser Thr Pro Gly Tyr
Asn Gly Tyr Asp 195 200 205 Pro Asn Tyr Gly Gly Val Thr Thr Tyr Pro
Pro Gly Ala Lys Glu Asn 210 215 220 Gly Gly Ile Phe Leu His Pro Asn
Pro Trp Ala Met Ile Ala Glu Thr 225 230 235 240 Met Leu Gly Asp Gly
Asp Arg Ala Tyr Glu Tyr Tyr Ser Gln Ile Asn 245 250 255 Pro Ala Gly
Lys Asn Asp Asp Ile Asp Leu Tyr Glu Val Glu Pro Tyr 260 265 270 Val
Tyr Ala Gln Asn Ile Leu Gly Asp Glu His Pro Gln Phe Gly Leu 275 280
285 Gly Arg Asn Ser Trp Leu Ser Gly Thr Ala Ser Trp Cys Tyr Gln Ala
290 295 300 Ala Thr Gln Trp Ile Leu Gly Ile Arg Ala Asp Tyr Glu Gly
Leu Arg 305 310 315 320 Ile Asp Pro Cys Ile Pro Ser Lys Trp Asp Gly
Phe Lys Ala Thr Arg 325 330 335 Leu Tyr Arg Gly Val Lys Tyr Asn Ile
Thr Val Thr Asn Pro Lys His 340 345 350 Ile Cys Lys Gly Val Glu Lys
Val Leu Val Asn Gly Lys Pro Val Glu 355 360 365 Gly Asn Val Val Arg
Ala Asp Val Gly Leu Arg Glu Val Asn Val Glu 370 375 380 Val Thr Leu
Gly 385 331362DNAUnknownObtained from environmental sample
33atggcaaata aaattctctt ccccgagaac tttctctggg gcacggcgac cgcatcctac
60cagatcgagg gggcttggga taaacatggt aagggcgagt cgacctggga ccgttttacg
120catacacctg gaaagatcaa aaacaatgat acgggcgatg tagcagatga
ccattatcga 180ttatggaaaa aagatatcgg cttgatgaag aagctcggct
tgaaggctta tcgtttttcg 240acttcctggc cgcgggtgct gccggccggg
cgcggtaaga gcaatcaaaa aggactcgat 300ttctacagca agctggttga
tgagttgcta aaagcaaata tcatcccatt cgtgacattg 360aatcactggg
acatcccaca aaagttggag gacgagggtg gctgggccgt gcgctcaacg
420gctgaggcat ttgtggaata tgccgatctc atgtcgcgca cgcttggaga
ccgcgtcaag 480aactggatca cgcacaacga accggccgtc gtcgcctgga
tgggatacgg gatgggcatc 540cacgcgccgg gcttaacgga tttctcgatt
gcggtgccgg tctcgcatca tctgctcctt 600tcgcacggat gggccgtgcc
tgtgattcgc ggtaacagcc cggatgccga ggtgggcatt 660accctcaaca
ttcaatgggg cgaagcagca tccaacagcc gggccgacct aaacgccctg
720cgcctgaacg atggacagtg gttccgctgg tttgccgatc cggtttatgg
ccgcggctat 780ccttccgacg tggtggctga tttcgagaaa atgggcgcgc
tgccgaacgg catgaatttc 840gtgcaacctg gcgatatgga tgtcatcgcc
acgccaaccg atttcctcgg gctcaattat 900tattcccgcc atgtgcatcg
cgtcaacaca ccggataacg atcaacaggt tgtgtttgcc 960aaacagcagg
gtcccgagaa ctggaccgag atgggctggg agatccatcc tgatggattg
1020gccggaattt tatccagagc gtatttcaat taccagccgc gcaaagtata
tgtgactgaa 1080aacggtgcca gctattccac cgcgcccgat gagaatggta
ttgtcaacga cattcaccgc 1140gtcaattatc tacggacgca cttcgcggct
gcccatcgcg ccctgcaggc gggcgtgcca 1200ttggcaggat acttcgtctg
gtcaatgctc gataacttcg aatggagtca cgggtacagc 1260cagcgctttg
gcatcgttta tgtggactat caaacccaga agcgttactt gaaagacagc
1320gccaagtggt acaaaggtgt catcaaaaag aatgggttct aa
136234453PRTUnknownObtained from environmental sample 34Met Ala Asn
Lys Ile Leu Phe Pro Glu Asn Phe Leu Trp Gly Thr Ala 1 5 10 15 Thr
Ala Ser Tyr Gln Ile Glu Gly Ala Trp Asp Lys His Gly Lys Gly 20 25
30 Glu Ser Thr Trp Asp Arg Phe Thr His Thr Pro Gly Lys Ile Lys Asn
35 40 45 Asn Asp Thr Gly Asp Val Ala Asp Asp His Tyr Arg Leu Trp
Lys Lys 50 55 60 Asp Ile Gly Leu Met Lys Lys Leu Gly Leu Lys Ala
Tyr Arg Phe Ser 65 70 75 80 Thr Ser Trp Pro Arg Val Leu Pro Ala Gly
Arg Gly Lys Ser Asn Gln 85 90 95 Lys Gly Leu Asp Phe Tyr Ser Lys
Leu Val Asp Glu Leu Leu Lys Ala 100 105 110 Asn Ile Ile Pro Phe Val
Thr Leu Asn His Trp Asp Ile Pro Gln Lys 115 120 125 Leu Glu Asp Glu
Gly Gly Trp Ala Val Arg Ser Thr Ala Glu Ala Phe 130 135 140 Val Glu
Tyr Ala Asp Leu Met Ser Arg Thr Leu Gly Asp Arg Val Lys 145 150 155
160 Asn Trp Ile Thr His Asn Glu Pro Ala Val Val Ala Trp Met Gly Tyr
165 170 175 Gly Met Gly Ile His Ala Pro Gly Leu Thr Asp Phe Ser Ile
Ala Val 180 185 190 Pro Val Ser His His Leu Leu Leu Ser His Gly Trp
Ala Val Pro Val 195 200 205 Ile Arg Gly Asn Ser Pro Asp Ala Glu Val
Gly Ile Thr Leu Asn Ile 210 215 220 Gln Trp Gly Glu Ala Ala Ser Asn
Ser Arg Ala Asp Leu Asn Ala Leu 225 230 235 240 Arg Leu Asn Asp Gly
Gln Trp Phe Arg Trp Phe Ala Asp Pro Val Tyr 245 250 255 Gly Arg Gly
Tyr Pro Ser Asp Val Val Ala Asp Phe Glu Lys Met Gly 260 265 270 Ala
Leu Pro Asn Gly Met Asn Phe Val Gln Pro Gly Asp Met Asp Val 275 280
285 Ile Ala Thr Pro Thr Asp Phe Leu Gly Leu Asn Tyr Tyr Ser Arg His
290 295 300 Val His Arg Val Asn Thr Pro Asp Asn Asp Gln Gln Val Val
Phe Ala 305 310 315 320 Lys Gln Gln Gly Pro Glu Asn Trp Thr Glu Met
Gly Trp Glu Ile His 325 330 335 Pro Asp Gly Leu Ala Gly Ile Leu Ser
Arg Ala Tyr Phe Asn Tyr Gln 340 345 350 Pro Arg Lys Val Tyr Val Thr
Glu Asn Gly Ala Ser Tyr Ser Thr Ala 355 360 365 Pro Asp Glu Asn Gly
Ile Val Asn Asp Ile His Arg Val Asn Tyr Leu 370 375 380 Arg Thr His
Phe Ala Ala Ala His Arg Ala Leu Gln Ala Gly Val Pro 385 390 395 400
Leu Ala Gly Tyr Phe Val Trp Ser Met Leu Asp Asn Phe Glu Trp Ser 405
410 415 His Gly Tyr Ser Gln Arg Phe Gly Ile Val Tyr Val Asp Tyr Gln
Thr 420 425 430 Gln Lys Arg Tyr Leu Lys Asp Ser Ala Lys Trp Tyr Lys
Gly Val Ile 435 440 445 Lys Lys Asn Gly Phe 450
351116DNAUnknownObtained from environmental sample 35atgaataaaa
tcctcaaact cttcagcagc ctgctgcttt ttgcaggcat ctgtcccgcg 60cttcaggcag
agccagtaga aacctacttt cccctgtccc gcgggatcaa catgagccac
120tggctctctc aagtgaatga aaacattccc gaccgttcca cctatgtgac
ggagcgggat 180ttgcaatttc tgcgggcagc cggtttcgac catgtgcgtc
tgccaatcga tgaggtcgaa 240ctctgggatg aagagggcaa tcagatcgag
gaggcctggc aatacatgca taactttctc 300cgttggagcc gaaagaacga
tctccgggtc attctcgacc tgcacacggt attgtcccac 360cacttcaacg
cggtaaatat gggagaggtc aatacactct tcaatgatcc cagggaacag
420gaaaagttcc tcaacctatg ggaacaaatc atggatgccg tgggtcacca
tccgaatgag 480tttctcgcct atgaaatgct caatgaggcg gtcgcggaag
atgatgaaga ctggaatctg 540ctcctcaacc gcgccattgt ccgcatccgg
gaccgtgagc cttatcgggt gctgattgcg 600gggtcgaact ggtggcagca
tgccgaccgg gtccccaacc tgaggctccc gaaaggagac 660cccaatatca
tcatcagttt tcatttttat tccccttttc tcttcaccca ctaccgcagt
720agctggactg cgatgcaggc gtaccagggc ttcgtccaat accctggcaa
aaccatacct 780tccatacatc tcgaaggcat gaactacccg gagtccttcg
ttcatatgtg ggaagcgcac 840aatcggtact atgacatcca ttccatgtat
gccgaaatgg tcccggcggt gcgttttgcc 900gaaaagttgg gacttcggct
ctattgcgga gaattcgggg ccatgaagac cgttgatcgc 960gcccagatgc
tgcagtggta tcgggatgtt gtcactgtat ttaataaatt gggtattccc
1020tatactgcct gggattatca gggaaccttc ggaatccgcg atgagctgac
cggtgagccc 1080gatcatgaaa tgatcgatat tctcctcggg cgctga
111636371PRTUnknownObtained from environmental sample 36Met Asn Lys
Ile Leu Lys Leu Phe Ser Ser Leu Leu Leu Phe Ala Gly 1 5 10 15 Ile
Cys Pro Ala Leu Gln Ala Glu Pro Val Glu Thr Tyr Phe Pro Leu 20 25
30 Ser Arg Gly Ile Asn Met Ser His Trp Leu Ser Gln Val Asn Glu Asn
35 40 45 Ile Pro Asp Arg Ser Thr Tyr Val Thr Glu Arg Asp Leu Gln
Phe Leu 50 55 60 Arg Ala Ala Gly Phe Asp His Val Arg Leu Pro Ile
Asp Glu Val Glu 65 70 75 80 Leu Trp Asp Glu Glu Gly Asn Gln Ile Glu
Glu Ala Trp Gln Tyr Met 85 90 95 His Asn Phe Leu Arg Trp Ser Arg
Lys Asn Asp Leu Arg Val Ile Leu 100 105 110 Asp Leu His Thr Val Leu
Ser His His Phe Asn Ala Val Asn Met Gly 115 120 125 Glu Val Asn Thr
Leu Phe Asn Asp Pro Arg Glu Gln Glu Lys Phe Leu 130 135 140 Asn Leu
Trp Glu Gln Ile Met Asp Ala Val Gly His His Pro Asn Glu 145 150 155
160 Phe Leu Ala Tyr Glu Met Leu Asn Glu Ala Val Ala Glu Asp Asp Glu
165 170 175 Asp Trp Asn Leu Leu Leu Asn Arg Ala Ile Val Arg Ile Arg
Asp Arg 180 185 190 Glu Pro Tyr Arg Val Leu Ile Ala Gly Ser Asn Trp
Trp Gln His Ala 195 200 205 Asp Arg Val Pro Asn Leu Arg Leu Pro Lys
Gly Asp Pro Asn Ile Ile 210 215 220 Ile Ser Phe His Phe Tyr Ser Pro
Phe Leu Phe Thr His Tyr Arg Ser 225 230 235 240 Ser Trp Thr Ala Met
Gln Ala Tyr Gln Gly Phe Val Gln Tyr Pro Gly 245 250 255 Lys Thr Ile
Pro Ser Ile His Leu Glu Gly Met Asn Tyr Pro Glu Ser 260 265 270 Phe
Val His Met Trp Glu Ala His Asn Arg Tyr Tyr Asp Ile His Ser 275 280
285 Met Tyr Ala Glu Met Val Pro Ala Val Arg Phe Ala Glu Lys Leu Gly
290 295 300 Leu Arg Leu Tyr Cys Gly Glu Phe Gly Ala Met Lys Thr Val
Asp Arg 305 310 315 320 Ala Gln Met Leu Gln Trp Tyr Arg Asp Val Val
Thr Val Phe Asn Lys 325 330 335 Leu Gly Ile Pro Tyr Thr Ala Trp Asp
Tyr Gln Gly Thr Phe Gly Ile 340 345 350 Arg Asp Glu Leu Thr Gly Glu
Pro Asp His Glu Met Ile Asp Ile Leu 355 360 365 Leu Gly Arg 370
371383DNAUnknownObtained from environmental sample 37atgagcaaac
tccccaaatt cctctttgga gccggcacct caagttatca gatcgaaggt 60gcctggaata
tagatggcaa aggtccctcc atttgggatt tccacactcg ccatcccggc
120gcggtttatc ggatgcacaa cggggatatg gcctgcgatc attatcatcg
gtatcgaacg 180gatatcgagc tgatgcagaa gatcggccta gaggcttacc
gcttttccat aaactggccc 240cgggttctgc cggaagggac cggtgccgcc
aatgaagcag gtctggactt ttacgaccgg 300ctggtggacg cactgttgga
agcgggaatt cagccttgga tcacccttta tcactgggaa 360ctcccctggg
ctctccacct gcgcgggggt tggctcaatc gggacatgcc cgaccacatt
420gagaactacg ccgccttggt cgccaggtgc ctcggtgacc gggtgaaaaa
ctggattact 480ttgaatgagc ctcaggtttt catcgggctt ggctatgcca
gcggggttca tgcccccggc 540tataagttgt ccttgcggga gtgcctggtc
ggttcccacc atgccgtgct ttcccaccac 600cgggcagtca aggcgatccg
ggccaactgc gaaggcagcg tccagatcgg ctcagccccg 660gtgggtgttg
tctgccgacc ggaaacggag tcggcagcag acattgaggc tgcccgccag
720gccacctacc atatcaacac tcccagcacc cacactcccg acaatctgat
cggctgcctc 780tggaacagca cttggtggat agatccaatg gttctgggga
agtatccgga acacgggctg 840aaagcctttg aaagctatct gccggacaac
attcaggccg aactggatgc cgtattcgaa 900ccgacggact ttgtcggttc
caacatctac cacggccgca cggtgcgggc caagcaggat 960ggtggttttg
agtttatcga ccttccgccc ggcagccccc gcaccaccat gggctgggac
1020atcaccccgg acatcctcta ctggggagga aagtatcttt acgaacgcta
tggcaagccg 1080atgtttatca cggaaaacgg cattgccgtc ccggaactgg
tgaatgatga aggccaggtc 1140gaggataccg tccgtgagca atacatgaag
ctgcacctgc gtgggctgca gcgggcccgc 1200gatgaaggca tcccctatgc
cggatacttc cactggtccc tgctcgacaa cttcgagtgg 1260gaacaaggct
actcccagcg ctttggcatg gtctacgtcg actaccagac ccaggaacgt
1320atcctcaaac gttcgggcca gcatttcgct gccatcgtcc gggaaatcac
cggaaccgcc 1380taa 138338460PRTUnknownObtained from environmental
sample 38Met Ser Lys Leu Pro Lys Phe Leu Phe Gly Ala Gly Thr Ser
Ser Tyr 1 5 10 15 Gln Ile Glu Gly Ala Trp Asn Ile Asp Gly Lys Gly
Pro Ser Ile Trp 20 25 30 Asp Phe His Thr Arg His Pro Gly Ala Val
Tyr Arg Met His Asn Gly 35 40 45 Asp Met Ala Cys Asp His Tyr His
Arg Tyr Arg Thr Asp Ile Glu Leu 50 55 60 Met Gln Lys Ile Gly Leu
Glu Ala Tyr Arg Phe Ser Ile Asn Trp Pro 65 70 75 80 Arg Val Leu Pro
Glu Gly Thr Gly Ala Ala Asn Glu Ala Gly Leu Asp 85 90 95 Phe Tyr
Asp Arg Leu Val Asp Ala Leu Leu Glu Ala Gly Ile Gln Pro 100 105 110
Trp Ile Thr Leu Tyr His Trp Glu Leu Pro Trp Ala Leu His Leu Arg 115
120 125 Gly Gly Trp Leu Asn Arg Asp Met Pro Asp His Ile Glu Asn Tyr
Ala 130 135 140 Ala Leu Val Ala Arg Cys Leu Gly Asp Arg Val Lys Asn
Trp Ile Thr 145 150 155 160 Leu Asn Glu Pro Gln Val Phe Ile Gly Leu
Gly Tyr Ala Ser Gly Val 165 170 175 His Ala Pro Gly Tyr Lys Leu Ser
Leu Arg Glu Cys Leu Val Gly Ser 180 185 190 His His Ala Val Leu Ser
His His Arg Ala Val Lys Ala Ile Arg Ala 195 200
205 Asn Cys Glu Gly Ser Val Gln Ile Gly Ser Ala Pro Val Gly Val Val
210 215 220 Cys Arg Pro Glu Thr Glu Ser Ala Ala Asp Ile Glu Ala Ala
Arg Gln 225 230 235 240 Ala Thr Tyr His Ile Asn Thr Pro Ser Thr His
Thr Pro Asp Asn Leu 245 250 255 Ile Gly Cys Leu Trp Asn Ser Thr Trp
Trp Ile Asp Pro Met Val Leu 260 265 270 Gly Lys Tyr Pro Glu His Gly
Leu Lys Ala Phe Glu Ser Tyr Leu Pro 275 280 285 Asp Asn Ile Gln Ala
Glu Leu Asp Ala Val Phe Glu Pro Thr Asp Phe 290 295 300 Val Gly Ser
Asn Ile Tyr His Gly Arg Thr Val Arg Ala Lys Gln Asp 305 310 315 320
Gly Gly Phe Glu Phe Ile Asp Leu Pro Pro Gly Ser Pro Arg Thr Thr 325
330 335 Met Gly Trp Asp Ile Thr Pro Asp Ile Leu Tyr Trp Gly Gly Lys
Tyr 340 345 350 Leu Tyr Glu Arg Tyr Gly Lys Pro Met Phe Ile Thr Glu
Asn Gly Ile 355 360 365 Ala Val Pro Glu Leu Val Asn Asp Glu Gly Gln
Val Glu Asp Thr Val 370 375 380 Arg Glu Gln Tyr Met Lys Leu His Leu
Arg Gly Leu Gln Arg Ala Arg 385 390 395 400 Asp Glu Gly Ile Pro Tyr
Ala Gly Tyr Phe His Trp Ser Leu Leu Asp 405 410 415 Asn Phe Glu Trp
Glu Gln Gly Tyr Ser Gln Arg Phe Gly Met Val Tyr 420 425 430 Val Asp
Tyr Gln Thr Gln Glu Arg Ile Leu Lys Arg Ser Gly Gln His 435 440 445
Phe Ala Ala Ile Val Arg Glu Ile Thr Gly Thr Ala 450 455 460
391521DNAUnknownObtained from environmental sample 39gtgctcgccc
ataaccgctc gcaccgtgaa gaactcctca atcgccggcc ggttgaattc 60atcagcgccc
tggaggcccg gggcgagctc cagcgcatca ccgccgaggt ggacccctac
120ctcgagatca ccgagatctg cgatcgcacc ctgcgcgccg gcggcccggc
gctgctgttc 180gagaacgtca aggggcacga catgcctctg ctcggcaacc
tcttcggcac gccgaagcgg 240gttgccctcg gcatgggcca ggactccgtg
gccgccctgc gcgaagtggg cgagctgctc 300gccttcctca aggagccgga
gcctcccaag ggctttcgcg acgcctggga caagctgccg 360atcttcaagc
aggtgatgag catggggccg aagaaggtcc gctcggcgcc ggtgcaggaa
420aaggtgtacg agggcgacga ggtcgacctc gaccgcctgc cgatccagca
ctgctggccc 480ggcgacgccg cgcccctggt cacctggccg ctggtgatca
cccgcgggcc ccacaagaag 540cgccagaacc tcggcatcta ccgccagcag
aagctgtcga agaaccggct gatcatgcgc 600tggctctccc accgcggcgg
ggcgctggac ttcctggagt tccagaaggc ccaccccggc 660gagcccttcc
cggtggcggt ggcgctgggc gccgacccgg cgaccatcct cggcgcggtg
720accccggtgc cggattcgct ctccgagtac gccttcgccg ggctgctgcg
cggctcgcgc 780accgagctgg tcaagtgcgg ccacgccgac ctggacgtgc
cggcctcggc ggagatcatc 840ctggaggggt tcatctaccc ggatgacatg
gcccccgagg gcccctacgg cgaccatacc 900ggctactaca acgaggtgga
taccttcccg gtcttcacgg tgacgcgtat gaccatgcgc 960cgcgatgcca
tctatcactc cacctacacc ggccggccgc ccgacgagcc ggcgatcctt
1020gggctggcgc tcaacgaggt gttcgtgccg atcctgcgcc gccagttccc
ggagatcgtc 1080gacttctacc tgccgccgga gggctgctcc taccgcatgg
cggtggtgac catgaagaag 1140cagtacccgg gccacgccaa gcgggtgatg
atgggcgtgt ggagcttcct gcgccagttc 1200atgtacacca agttcgtggt
ggtgctcgac gacgacgtca gcgcccggga ctgggaggac 1260gtgatctggg
ccatcaccac ccgcatggac ccggcccggg acaccgtggt ggtggagaac
1320acccccatcg actacctgga cttcgcctcg ccggtctccg gcctcggttc
caagatgggc 1380ctggatgcca ccagcaagtg gcccggcgag accgaccgcg
agtggggggt gcccatcgtc 1440atggacgagg ccgtcaaggc ccgcgtcagc
gagcgctgga acgagctggg catcgagctc 1500cccgacaaca cgaccccctg a
152140506PRTUnknownObtained from environmental sample 40Met Leu Ala
His Asn Arg Ser His Arg Glu Glu Leu Leu Asn Arg Arg 1 5 10 15 Pro
Val Glu Phe Ile Ser Ala Leu Glu Ala Arg Gly Glu Leu Gln Arg 20 25
30 Ile Thr Ala Glu Val Asp Pro Tyr Leu Glu Ile Thr Glu Ile Cys Asp
35 40 45 Arg Thr Leu Arg Ala Gly Gly Pro Ala Leu Leu Phe Glu Asn
Val Lys 50 55 60 Gly His Asp Met Pro Leu Leu Gly Asn Leu Phe Gly
Thr Pro Lys Arg 65 70 75 80 Val Ala Leu Gly Met Gly Gln Asp Ser Val
Ala Ala Leu Arg Glu Val 85 90 95 Gly Glu Leu Leu Ala Phe Leu Lys
Glu Pro Glu Pro Pro Lys Gly Phe 100 105 110 Arg Asp Ala Trp Asp Lys
Leu Pro Ile Phe Lys Gln Val Met Ser Met 115 120 125 Gly Pro Lys Lys
Val Arg Ser Ala Pro Val Gln Glu Lys Val Tyr Glu 130 135 140 Gly Asp
Glu Val Asp Leu Asp Arg Leu Pro Ile Gln His Cys Trp Pro 145 150 155
160 Gly Asp Ala Ala Pro Leu Val Thr Trp Pro Leu Val Ile Thr Arg Gly
165 170 175 Pro His Lys Lys Arg Gln Asn Leu Gly Ile Tyr Arg Gln Gln
Lys Leu 180 185 190 Ser Lys Asn Arg Leu Ile Met Arg Trp Leu Ser His
Arg Gly Gly Ala 195 200 205 Leu Asp Phe Leu Glu Phe Gln Lys Ala His
Pro Gly Glu Pro Phe Pro 210 215 220 Val Ala Val Ala Leu Gly Ala Asp
Pro Ala Thr Ile Leu Gly Ala Val 225 230 235 240 Thr Pro Val Pro Asp
Ser Leu Ser Glu Tyr Ala Phe Ala Gly Leu Leu 245 250 255 Arg Gly Ser
Arg Thr Glu Leu Val Lys Cys Gly His Ala Asp Leu Asp 260 265 270 Val
Pro Ala Ser Ala Glu Ile Ile Leu Glu Gly Phe Ile Tyr Pro Asp 275 280
285 Asp Met Ala Pro Glu Gly Pro Tyr Gly Asp His Thr Gly Tyr Tyr Asn
290 295 300 Glu Val Asp Thr Phe Pro Val Phe Thr Val Thr Arg Met Thr
Met Arg 305 310 315 320 Arg Asp Ala Ile Tyr His Ser Thr Tyr Thr Gly
Arg Pro Pro Asp Glu 325 330 335 Pro Ala Ile Leu Gly Leu Ala Leu Asn
Glu Val Phe Val Pro Ile Leu 340 345 350 Arg Arg Gln Phe Pro Glu Ile
Val Asp Phe Tyr Leu Pro Pro Glu Gly 355 360 365 Cys Ser Tyr Arg Met
Ala Val Val Thr Met Lys Lys Gln Tyr Pro Gly 370 375 380 His Ala Lys
Arg Val Met Met Gly Val Trp Ser Phe Leu Arg Gln Phe 385 390 395 400
Met Tyr Thr Lys Phe Val Val Val Leu Asp Asp Asp Val Ser Ala Arg 405
410 415 Asp Trp Glu Asp Val Ile Trp Ala Ile Thr Thr Arg Met Asp Pro
Ala 420 425 430 Arg Asp Thr Val Val Val Glu Asn Thr Pro Ile Asp Tyr
Leu Asp Phe 435 440 445 Ala Ser Pro Val Ser Gly Leu Gly Ser Lys Met
Gly Leu Asp Ala Thr 450 455 460 Ser Lys Trp Pro Gly Glu Thr Asp Arg
Glu Trp Gly Val Pro Ile Val 465 470 475 480 Met Asp Glu Ala Val Lys
Ala Arg Val Ser Glu Arg Trp Asn Glu Leu 485 490 495 Gly Ile Glu Leu
Pro Asp Asn Thr Thr Pro 500 505 411410DNAUnknownObtained from
environmental sample 41atgaagacgc cttcgatcta cgataccatg acgcggtcgg
tgcagccgtt gacacccgcc 60gacggcgaca ccttccgctt ttattgctgc ggccccaccg
tctacgggcc ggcgcatgtc 120ggcaatttcc gcaccttcat cattcaggac
gtgctgcgac gcgttatcga agggtcgggc 180ctcaaaacga gacacgtacg
caacatcacc gatgtggacg acaaaaccat ccgccaatcg 240caagcggaag
gaaaatctct gaaaatcttc acagggtact ggctggaacg gttccacgcc
300gattgcgacg cgctgaatct gctgcgcccg cacgtcgagc ccggcgccgt
tgaccatatc 360ccggcgcaaa tccggatgat cgaacaactg atcgaaaaag
gccacgccta cgtggcggac 420gacaactcgg tctattatcg cgttgcttcg
ttcgaagcgt acggccggtt gtcacgcctg 480caagaacgac acatcaccac
cggctgcgcc gaacacgcgc ataccgacga tgaatacgag 540cgcgaatcgg
ccgccgactt cgccttgtgg aaagcgcata aatccgagga cggcccgaac
600gcgtggccga gcccgtgggg cgacggacga cccggctggc acatcgagtg
cagcgccatg 660tccgtcgagt atctgggcga gacattcgat ctgcacggcg
gcggcgtgga cctgaccttc 720ccccaccacg aaaacgaaat cgcgcaaagc
gaagccgcca ccggcaagcc cttcgcgcgt 780atctggttcc attccgcgca
tctcatggtc gaaggccaca agatgtccaa gagcctcggc 840aacctgttta
cgctcgacga tatccgcgcg cgcggattcg acgccatgac cctgcgctat
900gtcctgcttt cgggcaatta ccgccaaccc ctcaatttca cgtgggactc
ccttaacgcc 960gcgcaaagcg ccttacgccg cctgcgtcag ctcaaccacg
atctccagca ggcggcgggc 1020aagacggtcg cgcccgctga tacttcgtgg
gggccgttcg aaccggtgta cgacgcgctt 1080gccgacaacc tgaacacgcc
cgacgccctc ggccgcttat tctccgccct gcacagcatc 1140gagcgcgcgc
ttaacggcaa ggaaaggacg gccgaagagg ccgccctcgc ccgtgcgcag
1200ttcctgcggg tcatggacct tttcggtttc agcctggacg cgccgccgac
cgccgaagcg 1260cccgaagaag tgcgtgcgct ggcgcagcag cgatgggacg
ctaaacaagc gcgcgatttc 1320gtccgcgccg acgccttgcg caaacaggtc
accgacctcg gctggaccat ccgcgacgcc 1380aaagacggct acgaactcgt
ccaagagtaa 141042469PRTUnknownObtained from environmental sample
42Met Lys Thr Pro Ser Ile Tyr Asp Thr Met Thr Arg Ser Val Gln Pro 1
5 10 15 Leu Thr Pro Ala Asp Gly Asp Thr Phe Arg Phe Tyr Cys Cys Gly
Pro 20 25 30 Thr Val Tyr Gly Pro Ala His Val Gly Asn Phe Arg Thr
Phe Ile Ile 35 40 45 Gln Asp Val Leu Arg Arg Val Ile Glu Gly Ser
Gly Leu Lys Thr Arg 50 55 60 His Val Arg Asn Ile Thr Asp Val Asp
Asp Lys Thr Ile Arg Gln Ser 65 70 75 80 Gln Ala Glu Gly Lys Ser Leu
Lys Ile Phe Thr Gly Tyr Trp Leu Glu 85 90 95 Arg Phe His Ala Asp
Cys Asp Ala Leu Asn Leu Leu Arg Pro His Val 100 105 110 Glu Pro Gly
Ala Val Asp His Ile Pro Ala Gln Ile Arg Met Ile Glu 115 120 125 Gln
Leu Ile Glu Lys Gly His Ala Tyr Val Ala Asp Asp Asn Ser Val 130 135
140 Tyr Tyr Arg Val Ala Ser Phe Glu Ala Tyr Gly Arg Leu Ser Arg Leu
145 150 155 160 Gln Glu Arg His Ile Thr Thr Gly Cys Ala Glu His Ala
His Thr Asp 165 170 175 Asp Glu Tyr Glu Arg Glu Ser Ala Ala Asp Phe
Ala Leu Trp Lys Ala 180 185 190 His Lys Ser Glu Asp Gly Pro Asn Ala
Trp Pro Ser Pro Trp Gly Asp 195 200 205 Gly Arg Pro Gly Trp His Ile
Glu Cys Ser Ala Met Ser Val Glu Tyr 210 215 220 Leu Gly Glu Thr Phe
Asp Leu His Gly Gly Gly Val Asp Leu Thr Phe 225 230 235 240 Pro His
His Glu Asn Glu Ile Ala Gln Ser Glu Ala Ala Thr Gly Lys 245 250 255
Pro Phe Ala Arg Ile Trp Phe His Ser Ala His Leu Met Val Glu Gly 260
265 270 His Lys Met Ser Lys Ser Leu Gly Asn Leu Phe Thr Leu Asp Asp
Ile 275 280 285 Arg Ala Arg Gly Phe Asp Ala Met Thr Leu Arg Tyr Val
Leu Leu Ser 290 295 300 Gly Asn Tyr Arg Gln Pro Leu Asn Phe Thr Trp
Asp Ser Leu Asn Ala 305 310 315 320 Ala Gln Ser Ala Leu Arg Arg Leu
Arg Gln Leu Asn His Asp Leu Gln 325 330 335 Gln Ala Ala Gly Lys Thr
Val Ala Pro Ala Asp Thr Ser Trp Gly Pro 340 345 350 Phe Glu Pro Val
Tyr Asp Ala Leu Ala Asp Asn Leu Asn Thr Pro Asp 355 360 365 Ala Leu
Gly Arg Leu Phe Ser Ala Leu His Ser Ile Glu Arg Ala Leu 370 375 380
Asn Gly Lys Glu Arg Thr Ala Glu Glu Ala Ala Leu Ala Arg Ala Gln 385
390 395 400 Phe Leu Arg Val Met Asp Leu Phe Gly Phe Ser Leu Asp Ala
Pro Pro 405 410 415 Thr Ala Glu Ala Pro Glu Glu Val Arg Ala Leu Ala
Gln Gln Arg Trp 420 425 430 Asp Ala Lys Gln Ala Arg Asp Phe Val Arg
Ala Asp Ala Leu Arg Lys 435 440 445 Gln Val Thr Asp Leu Gly Trp Thr
Ile Arg Asp Ala Lys Asp Gly Tyr 450 455 460 Glu Leu Val Gln Glu 465
43984DNAUnknownObtained from environmental sample 43atgacgactg
aaaccaaatc caaactgtac ttgcataaag tgaacggcca gaaaggactg 60gacctgcgcc
agacctatca gcgcgacttc accgtgaccg aggcgtatcg cgatacgctg
120ccggatatgc agaacgcttc cgaggcgttg cagggggcca atgtcgccat
ccagaaagtc 180ggcgtatcca atttcaagct gccactcaag taccgcaccc
acacgggcga accgaccacg 240ctggaaacca gcgtaaccgg cagcgtatcc
ctgaagccgg gcctgaaggg catcaacatg 300tcccgcgtca tgcggacctt
ctacgacttc caggacgacg tgttcacgct cgacacgctg 360gcccgtatac
tggaagcgta caaacgggat gtcgacagca acgacgcaca tcttcggctg
420agtttctcct acccgctgct tcaaaaaagt ctgcgcagcg aattattcgg
ctggcaatat 480taccaggtcg cattcgaggg acggatcgat gccgaaaatc
gagtccgcac gttcattcat 540tttgacttcg tgtattcctc cgcctgtccc
tgttcggctg aactggccga acacgcgcgg 600gaagtgcgcg gcctatacag
catcccccac tcgcaacgca gcaaggcgcg cgtcttcgtg 660gaagttcagc
ccggcgccga actcaccatc gaagacgtgc acatgcactg cctgaacgcg
720ctccaaacgg aaacgcaagt gatggtcaaa cgcgaagacg agcaggcgtt
cgctgaaatg 780aacggcgccg ccatcaaatt cgtcgaagac gccgcccgtc
tgatctatga gcagttcgac 840caggatccgc gcatcaagga tttcgaaatc
gcctgcgcgc atctggaatc cttgcactcg 900cacgacgccg tatcggtcat
cgccaaaggc gtgcccggcg gcttccgcgc cgacttctcg 960gacttcaaga
gtctgatctg ctaa 98444327PRTUnknownObtained from environmental
sample 44Met Thr Thr Glu Thr Lys Ser Lys Leu Tyr Leu His Lys Val
Asn Gly 1 5 10 15 Gln Lys Gly Leu Asp Leu Arg Gln Thr Tyr Gln Arg
Asp Phe Thr Val 20 25 30 Thr Glu Ala Tyr Arg Asp Thr Leu Pro Asp
Met Gln Asn Ala Ser Glu 35 40 45 Ala Leu Gln Gly Ala Asn Val Ala
Ile Gln Lys Val Gly Val Ser Asn 50 55 60 Phe Lys Leu Pro Leu Lys
Tyr Arg Thr His Thr Gly Glu Pro Thr Thr 65 70 75 80 Leu Glu Thr Ser
Val Thr Gly Ser Val Ser Leu Lys Pro Gly Leu Lys 85 90 95 Gly Ile
Asn Met Ser Arg Val Met Arg Thr Phe Tyr Asp Phe Gln Asp 100 105 110
Asp Val Phe Thr Leu Asp Thr Leu Ala Arg Ile Leu Glu Ala Tyr Lys 115
120 125 Arg Asp Val Asp Ser Asn Asp Ala His Leu Arg Leu Ser Phe Ser
Tyr 130 135 140 Pro Leu Leu Gln Lys Ser Leu Arg Ser Glu Leu Phe Gly
Trp Gln Tyr 145 150 155 160 Tyr Gln Val Ala Phe Glu Gly Arg Ile Asp
Ala Glu Asn Arg Val Arg 165 170 175 Thr Phe Ile His Phe Asp Phe Val
Tyr Ser Ser Ala Cys Pro Cys Ser 180 185 190 Ala Glu Leu Ala Glu His
Ala Arg Glu Val Arg Gly Leu Tyr Ser Ile 195 200 205 Pro His Ser Gln
Arg Ser Lys Ala Arg Val Phe Val Glu Val Gln Pro 210 215 220 Gly Ala
Glu Leu Thr Ile Glu Asp Val His Met His Cys Leu Asn Ala 225 230 235
240 Leu Gln Thr Glu Thr Gln Val Met Val Lys Arg Glu Asp Glu Gln Ala
245 250 255 Phe Ala Glu Met Asn Gly Ala Ala Ile Lys Phe Val Glu Asp
Ala Ala 260 265 270 Arg Leu Ile Tyr Glu Gln Phe Asp Gln Asp Pro Arg
Ile Lys Asp Phe 275 280 285 Glu Ile Ala Cys Ala His Leu Glu Ser Leu
His Ser His Asp Ala Val 290 295 300 Ser Val Ile Ala Lys Gly Val Pro
Gly Gly Phe Arg Ala Asp Phe Ser 305 310 315 320 Asp Phe Lys Ser Leu
Ile Cys 325 451377DNAUnknownObtained from environmental sample
45atgacacaac tggcttttcc atctaacttc atctggggaa cagctacttc cgcttaccaa
60atcgaaggcg cctggaacgc agacggcaag ggcgaatcta tttgggatcg cttttcccat
120acgcagggga agatcattga cggcagcaac ggcgatgtgg cctgcgatca
ctaccaccgc 180tggcgcgagg acgtggccct catgagagac ttgggtatgc
aggcatatcg cttctccatc 240tcctggccac gcatcctgcc caccggtcat
ggacagatca atcaggctgg gctggacttt 300tacaatcgcc tggtggacgg
gttgctggaa gctggcatca agccctttgc caccctctac 360cactgggacc
tgccgctggc gctacaggct gacggcggct ggccggagcg ctccacggcc
420aaggcctttg tcgaatacgc cgacgtggtc agccgcgcgc tgggcgatcg
ggtgaagagc 480tggatcaccc
ataacgaacc gtggtgcatc agcatgctga gccatcaaat tggggagcat
540gcgcccggct ggcgggactg gcaggctgcg ttggcggccg cgcaccacgt
cctcctttcg 600catggttggg ccgtgccgga actgcgtcgc aacagccgcg
atgcagaaat cggcatcacg 660ttgaacttta ccccggcgga gccagcttcg
aacagcgcag ccgatttcaa ggcctatcgc 720cagttcgatg gctacttcaa
ccgctggttc ctggacccgc tctatggccg ccactatccg 780gcagatatgg
tgcacgatta catcgcgcaa ggctacctgc catcacaggg tttgactttc
840gtggaagctg gtgacctgga cgcgatcgcg acgcgcaccg atttcctggg
tgtgaactat 900tacacgcgcg aagtggtccg tagccaggaa atcccagaga
gtgagaacgc gccgcgcaca 960gtcttgcgcg cgccacagga agagtggaca
gagatgggct gggaagtgta tcctgagggc 1020ctctacaggt tgctcaatcg
gttgcacttt gaataccagc cgcgcaagct ctacgtgacc 1080gagagcggtt
gcagctactc cgatggaccc ggccccaacg gtcggatacc ggaccaacgc
1140cgtatcaact acctgcgcga tcacttcgca gcggcgcatc aggcgataca
atgcggcgtc 1200ccgctggccg gctacttcgt ctggtcgttc atggacaact
tcgagtgggc caaagggtac 1260acccaacgtt ttggtatcgt atgggtggat
tatcaatcgc aacgacggat accgaaagac 1320agcgcctact ggtatcgcga
tgtcgtcgcc gccaacgcgg tgcaagttcc tgattag
137746458PRTUnknownObtained from environmental sample 46Met Thr Gln
Leu Ala Phe Pro Ser Asn Phe Ile Trp Gly Thr Ala Thr 1 5 10 15 Ser
Ala Tyr Gln Ile Glu Gly Ala Trp Asn Ala Asp Gly Lys Gly Glu 20 25
30 Ser Ile Trp Asp Arg Phe Ser His Thr Gln Gly Lys Ile Ile Asp Gly
35 40 45 Ser Asn Gly Asp Val Ala Cys Asp His Tyr His Arg Trp Arg
Glu Asp 50 55 60 Val Ala Leu Met Arg Asp Leu Gly Met Gln Ala Tyr
Arg Phe Ser Ile 65 70 75 80 Ser Trp Pro Arg Ile Leu Pro Thr Gly His
Gly Gln Ile Asn Gln Ala 85 90 95 Gly Leu Asp Phe Tyr Asn Arg Leu
Val Asp Gly Leu Leu Glu Ala Gly 100 105 110 Ile Lys Pro Phe Ala Thr
Leu Tyr His Trp Asp Leu Pro Leu Ala Leu 115 120 125 Gln Ala Asp Gly
Gly Trp Pro Glu Arg Ser Thr Ala Lys Ala Phe Val 130 135 140 Glu Tyr
Ala Asp Val Val Ser Arg Ala Leu Gly Asp Arg Val Lys Ser 145 150 155
160 Trp Ile Thr His Asn Glu Pro Trp Cys Ile Ser Met Leu Ser His Gln
165 170 175 Ile Gly Glu His Ala Pro Gly Trp Arg Asp Trp Gln Ala Ala
Leu Ala 180 185 190 Ala Ala His His Val Leu Leu Ser His Gly Trp Ala
Val Pro Glu Leu 195 200 205 Arg Arg Asn Ser Arg Asp Ala Glu Ile Gly
Ile Thr Leu Asn Phe Thr 210 215 220 Pro Ala Glu Pro Ala Ser Asn Ser
Ala Ala Asp Phe Lys Ala Tyr Arg 225 230 235 240 Gln Phe Asp Gly Tyr
Phe Asn Arg Trp Phe Leu Asp Pro Leu Tyr Gly 245 250 255 Arg His Tyr
Pro Ala Asp Met Val His Asp Tyr Ile Ala Gln Gly Tyr 260 265 270 Leu
Pro Ser Gln Gly Leu Thr Phe Val Glu Ala Gly Asp Leu Asp Ala 275 280
285 Ile Ala Thr Arg Thr Asp Phe Leu Gly Val Asn Tyr Tyr Thr Arg Glu
290 295 300 Val Val Arg Ser Gln Glu Ile Pro Glu Ser Glu Asn Ala Pro
Arg Thr 305 310 315 320 Val Leu Arg Ala Pro Gln Glu Glu Trp Thr Glu
Met Gly Trp Glu Val 325 330 335 Tyr Pro Glu Gly Leu Tyr Arg Leu Leu
Asn Arg Leu His Phe Glu Tyr 340 345 350 Gln Pro Arg Lys Leu Tyr Val
Thr Glu Ser Gly Cys Ser Tyr Ser Asp 355 360 365 Gly Pro Gly Pro Asn
Gly Arg Ile Pro Asp Gln Arg Arg Ile Asn Tyr 370 375 380 Leu Arg Asp
His Phe Ala Ala Ala His Gln Ala Ile Gln Cys Gly Val 385 390 395 400
Pro Leu Ala Gly Tyr Phe Val Trp Ser Phe Met Asp Asn Phe Glu Trp 405
410 415 Ala Lys Gly Tyr Thr Gln Arg Phe Gly Ile Val Trp Val Asp Tyr
Gln 420 425 430 Ser Gln Arg Arg Ile Pro Lys Asp Ser Ala Tyr Trp Tyr
Arg Asp Val 435 440 445 Val Ala Ala Asn Ala Val Gln Val Pro Asp 450
455 471353DNAUnknownObtained from environmental sample 47atgaaaaaat
acctttttcc tgaaaatttt ttatggggtg ctgccacagc ttcgtatcaa 60atcgaaggtt
ctccctctgc tgatggcaaa ggtgaatcga tatgggaccg tttttctcac
120acaccgggga acatttggaa cgctgaaacc ggggatatcg cctgcgatca
ttaccggcgt 180tacgtggatg atgtaaagct gatttcacaa atcgggctta
acgcgtaccg tttttcaatt 240tcctggccca gggtatttcc agaggggaga
ggaaaagcaa atgaaaaagg actcgatttt 300taccgcaggt tgattgaaca
gctgcagcaa catcgaatca aaacggcagt gacactttac 360cactgggatc
ttccacaagt tctgcaggat cgcggcgggt gggcaaaccg tgatacggcg
420aagtattttt ctgagtatgc cacctttctc tttgaaaaac tcgatctccc
cgttgacatg 480tggattactc ttaacgaacc atgggttatc gctattctgg
ggcatgcttt tggtatccac 540gctccaggga tgagtgactt cagcacagcc
ctccaggtct cgcataacct gcttctgggg 600cacgggttgg cggttaaagc
atttcgggag tctaagaggg gtgatgaacc ggtaggtatt 660acccttaacc
ttgccccggt tgaaccgctg accgaaaagc ccgccgatct aaaggcagct
720ttactttctg acggttttat gaaccgctgg taccttgatc ccctgttcaa
aggtggttac 780cctgaagata tgatggatat ctattcccgg aactttgaac
tgcccaaaat tgaaaagggg 840gatgctcagg ttattgccga accgatcgac
ttcctgggca taaataacta taccagggtt 900ctcgtggaag ccagcggtga
tgaaaatgcc tttatgggca accctgtcaa cccccagggc 960tctgaatata
ctgaaatggg ttgggaggtt tatccgcagg gtctctacga cctgctgacc
1020agggttcacc gggattacgg gccaatgccg ctatatataa ctgaaaacgg
ggcagccttt 1080cccgatgaac ttgacagcaa tgggcagata gatgatccaa
ggcggataaa ttacctggaa 1140acttatcttc atcagtgctg gaaggcagtt
caggacggtg tgcctctaaa aggctatttt 1200gtctggaccc tgatggataa
cttcgagtgg gctttcggtt tcagcaagcg atttgggctc 1260atatacgtag
attaccagga tcagaaacgt tacttgaaaa acagcgccta ctggtatagc
1320aaggttattg ggcgaaacgg cctcgagcta taa
135348450PRTUnknownObtained from environmental sample 48Met Lys Lys
Tyr Leu Phe Pro Glu Asn Phe Leu Trp Gly Ala Ala Thr 1 5 10 15 Ala
Ser Tyr Gln Ile Glu Gly Ser Pro Ser Ala Asp Gly Lys Gly Glu 20 25
30 Ser Ile Trp Asp Arg Phe Ser His Thr Pro Gly Asn Ile Trp Asn Ala
35 40 45 Glu Thr Gly Asp Ile Ala Cys Asp His Tyr Arg Arg Tyr Val
Asp Asp 50 55 60 Val Lys Leu Ile Ser Gln Ile Gly Leu Asn Ala Tyr
Arg Phe Ser Ile 65 70 75 80 Ser Trp Pro Arg Val Phe Pro Glu Gly Arg
Gly Lys Ala Asn Glu Lys 85 90 95 Gly Leu Asp Phe Tyr Arg Arg Leu
Ile Glu Gln Leu Gln Gln His Arg 100 105 110 Ile Lys Thr Ala Val Thr
Leu Tyr His Trp Asp Leu Pro Gln Val Leu 115 120 125 Gln Asp Arg Gly
Gly Trp Ala Asn Arg Asp Thr Ala Lys Tyr Phe Ser 130 135 140 Glu Tyr
Ala Thr Phe Leu Phe Glu Lys Leu Asp Leu Pro Val Asp Met 145 150 155
160 Trp Ile Thr Leu Asn Glu Pro Trp Val Ile Ala Ile Leu Gly His Ala
165 170 175 Phe Gly Ile His Ala Pro Gly Met Ser Asp Phe Ser Thr Ala
Leu Gln 180 185 190 Val Ser His Asn Leu Leu Leu Gly His Gly Leu Ala
Val Lys Ala Phe 195 200 205 Arg Glu Ser Lys Arg Gly Asp Glu Pro Val
Gly Ile Thr Leu Asn Leu 210 215 220 Ala Pro Val Glu Pro Leu Thr Glu
Lys Pro Ala Asp Leu Lys Ala Ala 225 230 235 240 Leu Leu Ser Asp Gly
Phe Met Asn Arg Trp Tyr Leu Asp Pro Leu Phe 245 250 255 Lys Gly Gly
Tyr Pro Glu Asp Met Met Asp Ile Tyr Ser Arg Asn Phe 260 265 270 Glu
Leu Pro Lys Ile Glu Lys Gly Asp Ala Gln Val Ile Ala Glu Pro 275 280
285 Ile Asp Phe Leu Gly Ile Asn Asn Tyr Thr Arg Val Leu Val Glu Ala
290 295 300 Ser Gly Asp Glu Asn Ala Phe Met Gly Asn Pro Val Asn Pro
Gln Gly 305 310 315 320 Ser Glu Tyr Thr Glu Met Gly Trp Glu Val Tyr
Pro Gln Gly Leu Tyr 325 330 335 Asp Leu Leu Thr Arg Val His Arg Asp
Tyr Gly Pro Met Pro Leu Tyr 340 345 350 Ile Thr Glu Asn Gly Ala Ala
Phe Pro Asp Glu Leu Asp Ser Asn Gly 355 360 365 Gln Ile Asp Asp Pro
Arg Arg Ile Asn Tyr Leu Glu Thr Tyr Leu His 370 375 380 Gln Cys Trp
Lys Ala Val Gln Asp Gly Val Pro Leu Lys Gly Tyr Phe 385 390 395 400
Val Trp Thr Leu Met Asp Asn Phe Glu Trp Ala Phe Gly Phe Ser Lys 405
410 415 Arg Phe Gly Leu Ile Tyr Val Asp Tyr Gln Asp Gln Lys Arg Tyr
Leu 420 425 430 Lys Asn Ser Ala Tyr Trp Tyr Ser Lys Val Ile Gly Arg
Asn Gly Leu 435 440 445 Glu Leu 450 49591DNAUnknownObtained from
environmental sample 49atggactttg agcgggcagt tgacaggaat atcattagat
tacgctcttc gttaaaggaa 60gaaatgaagg atctagttgc agttgaagct ccggtaacaa
tatttttaaa tggcagcgag 120ctggtaaccc tgctctgcac cccggagaaa
attgatcgtt tggccctcgg tttccttcat 180tcagaagggc tgcttaactc
acttgatgat cttagtatga tcaggaccag ggagagcgaa 240ggcctggttg
aaattgaact taaagaggcc tcgccggcac ttgataaatt atacgggaag
300aggacaatta cttccggttg cggtaaggga acaatttttt ttaatgttct
cgattctctg 360cgcagtaaac cactcgacgg aaagcttgtg attacaaccg
aagagattca taaattaatg 420gatgacctgc aggggcgggc ggaactgttc
aaggctaccg ggggtgttca cagcgctgcg 480cttgccgaca gaaaggaaat
actctttttc agtgaagata tcggccgcca taatgctatc 540gataaaattg
tgggagagtg tttgctggag ggggtatctc ctgaagataa g
59150197PRTUnknownObtained from environmental sample 50Met Asp Phe
Glu Arg Ala Val Asp Arg Asn Ile Ile Arg Leu Arg Ser 1 5 10 15 Ser
Leu Lys Glu Glu Met Lys Asp Leu Val Ala Val Glu Ala Pro Val 20 25
30 Thr Ile Phe Leu Asn Gly Ser Glu Leu Val Thr Leu Leu Cys Thr Pro
35 40 45 Glu Lys Ile Asp Arg Leu Ala Leu Gly Phe Leu His Ser Glu
Gly Leu 50 55 60 Leu Asn Ser Leu Asp Asp Leu Ser Met Ile Arg Thr
Arg Glu Ser Glu 65 70 75 80 Gly Leu Val Glu Ile Glu Leu Lys Glu Ala
Ser Pro Ala Leu Asp Lys 85 90 95 Leu Tyr Gly Lys Arg Thr Ile Thr
Ser Gly Cys Gly Lys Gly Thr Ile 100 105 110 Phe Phe Asn Val Leu Asp
Ser Leu Arg Ser Lys Pro Leu Asp Gly Lys 115 120 125 Leu Val Ile Thr
Thr Glu Glu Ile His Lys Leu Met Asp Asp Leu Gln 130 135 140 Gly Arg
Ala Glu Leu Phe Lys Ala Thr Gly Gly Val His Ser Ala Ala 145 150 155
160 Leu Ala Asp Arg Lys Glu Ile Leu Phe Phe Ser Glu Asp Ile Gly Arg
165 170 175 His Asn Ala Ile Asp Lys Ile Val Gly Glu Cys Leu Leu Glu
Gly Val 180 185 190 Ser Pro Glu Asp Lys 195
511014DNAUnknownObtained from environmental sample 51atgtccaggg
gcatcctgat cctcgtcatg ctgtctgttc tgagcggcgc ggcgctggcc 60caaccggccg
ggctgccgcc gcgttcgccg gtgcagcgct gcatcaacct gggcaatatg
120ctggaagcgc cggaggaggg ctggtggggg ctgcgcgtcg agcgcgacta
cctgacgacg 180atcgccgggg ccgggttcga tgcggtgcgc atcccgataa
gctggtcaac ccatgctgcc 240agcgagccgc cctacaccat cgatccggct
ttcttcgccc gcgttgatga agtcgtcggc 300tgggcgctgg cggacgggct
gaaggccatc atcaacgtgc accactacga ggagatgatg 360agcgatccgg
cggggcattt cccccggctg cgcgcgctgt gggcgcagat cgcggagcac
420tacgccgact acccgcccgc gctgatgttc gagctgctca acgaaccgtt
cgaggcgctg 480acgccgctgc ggtggaacga gtacgccgcc gatctgatcg
cgctgatccg ccagaccaac 540ccggggcgca ccctgatcgt cggcgggggc
tggtggaaca gtgtggaagg gctgatgcag 600ctccgcctgc cggatgatcc
cgatctgctg gcgacgttcc attactacca cccgttcgag 660ttcacgcatc
agggggcgga gtggtcaccg gaagtgactg acctgagcgg gatcgcctgg
720gggacgggcg aggaacggct cgatctggag tccaatatcc gtattgcggc
ggcctgggcg 780gtgtacaacc ggcgcccgct gctgttgggc gaattcggcg
tctatggccg ggtggccgat 840ctcgattcgc gcctgcgctg gacgacggcg
gtgcgcgccg aggccgaggc gcagggcatc 900ggctggtgct actgggaatt
cgccgccggc ttcggcattt acgacccgga aagccggacg 960ttcaacccgc
tgtaccgcgc gctgatcccg caggccgggc cggcgcgccc ctga
101452337PRTUnknownObtained from environmental sample 52Met Ser Arg
Gly Ile Leu Ile Leu Val Met Leu Ser Val Leu Ser Gly 1 5 10 15 Ala
Ala Leu Ala Gln Pro Ala Gly Leu Pro Pro Arg Ser Pro Val Gln 20 25
30 Arg Cys Ile Asn Leu Gly Asn Met Leu Glu Ala Pro Glu Glu Gly Trp
35 40 45 Trp Gly Leu Arg Val Glu Arg Asp Tyr Leu Thr Thr Ile Ala
Gly Ala 50 55 60 Gly Phe Asp Ala Val Arg Ile Pro Ile Ser Trp Ser
Thr His Ala Ala 65 70 75 80 Ser Glu Pro Pro Tyr Thr Ile Asp Pro Ala
Phe Phe Ala Arg Val Asp 85 90 95 Glu Val Val Gly Trp Ala Leu Ala
Asp Gly Leu Lys Ala Ile Ile Asn 100 105 110 Val His His Tyr Glu Glu
Met Met Ser Asp Pro Ala Gly His Phe Pro 115 120 125 Arg Leu Arg Ala
Leu Trp Ala Gln Ile Ala Glu His Tyr Ala Asp Tyr 130 135 140 Pro Pro
Ala Leu Met Phe Glu Leu Leu Asn Glu Pro Phe Glu Ala Leu 145 150 155
160 Thr Pro Leu Arg Trp Asn Glu Tyr Ala Ala Asp Leu Ile Ala Leu Ile
165 170 175 Arg Gln Thr Asn Pro Gly Arg Thr Leu Ile Val Gly Gly Gly
Trp Trp 180 185 190 Asn Ser Val Glu Gly Leu Met Gln Leu Arg Leu Pro
Asp Asp Pro Asp 195 200 205 Leu Leu Ala Thr Phe His Tyr Tyr His Pro
Phe Glu Phe Thr His Gln 210 215 220 Gly Ala Glu Trp Ser Pro Glu Val
Thr Asp Leu Ser Gly Ile Ala Trp 225 230 235 240 Gly Thr Gly Glu Glu
Arg Leu Asp Leu Glu Ser Asn Ile Arg Ile Ala 245 250 255 Ala Ala Trp
Ala Val Tyr Asn Arg Arg Pro Leu Leu Leu Gly Glu Phe 260 265 270 Gly
Val Tyr Gly Arg Val Ala Asp Leu Asp Ser Arg Leu Arg Trp Thr 275 280
285 Thr Ala Val Arg Ala Glu Ala Glu Ala Gln Gly Ile Gly Trp Cys Tyr
290 295 300 Trp Glu Phe Ala Ala Gly Phe Gly Ile Tyr Asp Pro Glu Ser
Arg Thr 305 310 315 320 Phe Asn Pro Leu Tyr Arg Ala Leu Ile Pro Gln
Ala Gly Pro Ala Arg 325 330 335 Pro 531377DNAUnknownObtained from
environmental sample 53atgtggatgg ttcaagcgac atctttaatt caaaaataca
atgtgcctgg cccacgctac 60accagttatc caacggttcc ttattgggaa agtgagaatt
tttcactaaa gcagtggcaa 120caaacgctca aaaaatcctt tgatgagtcg
aatcaaagtg aaggcatcag tctgtatatc 180catttgccat tttgcgaaag
tttatgcacc ttctgtggtt gccataaacg tgtgactaaa 240aagcatgaga
tggaaaagcc ttatatccaa gcggtattaa aagaatggga tttatattgc
300caacttttgg tggataaacc tgtcattaaa gaaattcatt tgggtggggg
aactccgaca 360ttttttagtc ctgaacattt aacgcagctg attaagggga
tattggctaa agccgaagtt 420gcagatgagc atgagtttag ttttgaagga
catcccaaca atacgacacg tgaacatttg 480caagcgctct atgatgttgg
atttcgacgt gtcagttatg gcgtgcagga ctataacgaa 540actgtgcaaa
aagccattca ccgcattcag ccctatgaaa atgttaaaaa tgtcaccgag
600tgggcgcgtg agattggcta tacctctatt tcgcatgatt tggtctttgg
cctgccgttt 660caaagtttag acgatgtctt aaatacgatt gatcaaacca
ataccttaat gccggatcgt 720ttggctttgt atagctatgc ccatgtgcca
tggattaaag gcaatggtca acgcggtttt 780aaagatgctg atgtcccgaa
agacgagatt aaacgtcaat gttatgagga aggcaaaaaa 840aaattattag
aacatggcta tcatgaaatt ggtatggatc attttgctct agaacaagac
900agtatgtatc agtcttttaa agcagggagc ttgcatcgta atttcatggg
ttataccgca 960tcgaaaacgc aagtgatgat tgggcttggg atttcatcaa
ttagtgacag ttggtacagc 1020tttgcgcaaa acgtgaaaac attagatgaa
tattatacct tgctagaaaa aaatcagatt 1080cccgtgttta aagggcatgt
cttgaatcag gaagatttga tcatccgtaa acatatttta 1140aatttgatgt
gtggcttcca aacctcatgg gcaaatcccg atatgcaatt tcctgaaatt
1200cagtctgttt tggcacaatt agcagaaatg cagcaagatg gtttgattca
aattgaagac 1260gcatcggtca cagttttaga agcgggcaag ccttttgttc
gaaatatttg tatggccttt 1320gatttaagac tcaagcgcaa caagcctgag
aatcggattt tttcgatgac gatttaa
137754458PRTUnknownObtained from environmental sample 54Met Trp Met
Val Gln Ala Thr Ser Leu Ile Gln Lys Tyr Asn Val Pro 1 5 10 15 Gly
Pro Arg Tyr Thr Ser Tyr Pro Thr Val Pro Tyr Trp Glu Ser Glu 20 25
30 Asn Phe Ser Leu Lys Gln Trp Gln Gln Thr Leu Lys Lys Ser Phe Asp
35 40 45 Glu Ser Asn Gln Ser Glu Gly Ile Ser Leu Tyr Ile His Leu
Pro Phe 50 55 60 Cys Glu Ser Leu Cys Thr Phe Cys Gly Cys His Lys
Arg Val Thr Lys 65 70 75 80 Lys His Glu Met Glu Lys Pro Tyr Ile Gln
Ala Val Leu Lys Glu Trp 85 90 95 Asp Leu Tyr Cys Gln Leu Leu Val
Asp Lys Pro Val Ile Lys Glu Ile 100 105 110 His Leu Gly Gly Gly Thr
Pro Thr Phe Phe Ser Pro Glu His Leu Thr 115 120 125 Gln Leu Ile Lys
Gly Ile Leu Ala Lys Ala Glu Val Ala Asp Glu His 130 135 140 Glu Phe
Ser Phe Glu Gly His Pro Asn Asn Thr Thr Arg Glu His Leu 145 150 155
160 Gln Ala Leu Tyr Asp Val Gly Phe Arg Arg Val Ser Tyr Gly Val Gln
165 170 175 Asp Tyr Asn Glu Thr Val Gln Lys Ala Ile His Arg Ile Gln
Pro Tyr 180 185 190 Glu Asn Val Lys Asn Val Thr Glu Trp Ala Arg Glu
Ile Gly Tyr Thr 195 200 205 Ser Ile Ser His Asp Leu Val Phe Gly Leu
Pro Phe Gln Ser Leu Asp 210 215 220 Asp Val Leu Asn Thr Ile Asp Gln
Thr Asn Thr Leu Met Pro Asp Arg 225 230 235 240 Leu Ala Leu Tyr Ser
Tyr Ala His Val Pro Trp Ile Lys Gly Asn Gly 245 250 255 Gln Arg Gly
Phe Lys Asp Ala Asp Val Pro Lys Asp Glu Ile Lys Arg 260 265 270 Gln
Cys Tyr Glu Glu Gly Lys Lys Lys Leu Leu Glu His Gly Tyr His 275 280
285 Glu Ile Gly Met Asp His Phe Ala Leu Glu Gln Asp Ser Met Tyr Gln
290 295 300 Ser Phe Lys Ala Gly Ser Leu His Arg Asn Phe Met Gly Tyr
Thr Ala 305 310 315 320 Ser Lys Thr Gln Val Met Ile Gly Leu Gly Ile
Ser Ser Ile Ser Asp 325 330 335 Ser Trp Tyr Ser Phe Ala Gln Asn Val
Lys Thr Leu Asp Glu Tyr Tyr 340 345 350 Thr Leu Leu Glu Lys Asn Gln
Ile Pro Val Phe Lys Gly His Val Leu 355 360 365 Asn Gln Glu Asp Leu
Ile Ile Arg Lys His Ile Leu Asn Leu Met Cys 370 375 380 Gly Phe Gln
Thr Ser Trp Ala Asn Pro Asp Met Gln Phe Pro Glu Ile 385 390 395 400
Gln Ser Val Leu Ala Gln Leu Ala Glu Met Gln Gln Asp Gly Leu Ile 405
410 415 Gln Ile Glu Asp Ala Ser Val Thr Val Leu Glu Ala Gly Lys Pro
Phe 420 425 430 Val Arg Asn Ile Cys Met Ala Phe Asp Leu Arg Leu Lys
Arg Asn Lys 435 440 445 Pro Glu Asn Arg Ile Phe Ser Met Thr Ile 450
455 551389DNAUnknownObtained from environmental sample 55atgagcgctt
cgagtccctc ccgccccctg tccttcccag agcagttcgt ctggggtgct 60gccgcggcct
cctaccaagt cgagggcgcc gtccacgagg acgggaaggg cccctccgtc
120tgggacatgt tctgcgagaa gcccggagcg gtcttccagg ggcacgacgg
ggcggtggct 180tgcgaccact atcaccgcta ccgagaggac gtggcgttga
tgcgacaggt gggcctgcac 240gcctaccgcc tgagcgtgtg ctggccccga
gtgctcccgg agggcgtcgg gcagcccaac 300gagaagggcc tcgacttcta
ctcgcggttg gtggacgcgc tgctcgaggc agggattacg 360ccctgggtaa
cgctttttca ttgggactac cccttggctc tctatcaccg ggggggctgg
420ctcaaccggg atagcgcgga ttggtttgcc gagtacgcgg gcctaatcgc
cgatcgcctc 480tccgaccggg tgcagcattt cttcactcag aacgagcccc
aggtctatat cggcttcgga 540cacctcgagg gtaagcatgc tccaggagac
accttgccca tgtcccaggt gctgcttgcg 600gggcatcata gcctactggc
gcacggcaag gccgtgcagg cgctccgcgc ccaggcgaag 660cagcagctgc
gcgtcggcta cgctcccgtc ggcatgcccc tccatccctt cacggactcg
720gccgaggacg tggccgctgc gcggaaggcg accttttggg ttcgggagaa
gaactcctgg 780aacaacgcct ggtggatgga cccggtgttc ttgggtgagt
acccggctca gggcctcgcc 840ttcttcggcc gggacgtgcc gcaggtgcgc
gagggagaca tgcagctcat cgcgcagccc 900ttggacttct ttggggtcaa
catctaccag agcacccccg tgcgcgcgtc tagcgccgaa 960agcggcttcg
aggtcgtccc ccatccaacg ggctatccta tcactgcctt caactggccg
1020atcacgcccc aggccctcta ctggggtccg cgcttcttct acgagcgcta
ccagaagccg 1080atcgtcatca cggagaacgg actgtcctgt cgggacgtcg
tcgctgtgga cgggaaggtt 1140cacgatccgg ctcgcatcga tttcaccacc
cgctatctgc gcgagctcca ccgagccgtc 1200gcggacggcg tcgcggtcga
gggctacttc cactggtcca tcatggacaa cttcgaatgg 1260gctgccggct
accgcgagcg gttcgggctc attcacgtcg actacgagac cctggcgcgg
1320acgcccaagg cgtccgctgc gtggtatcgc aaggtaatcg agagcaacgg
agcgaccctt 1380ttcggatga 138956462PRTUnknownObtained from
environmental sample 56Met Ser Ala Ser Ser Pro Ser Arg Pro Leu Ser
Phe Pro Glu Gln Phe 1 5 10 15 Val Trp Gly Ala Ala Ala Ala Ser Tyr
Gln Val Glu Gly Ala Val His 20 25 30 Glu Asp Gly Lys Gly Pro Ser
Val Trp Asp Met Phe Cys Glu Lys Pro 35 40 45 Gly Ala Val Phe Gln
Gly His Asp Gly Ala Val Ala Cys Asp His Tyr 50 55 60 His Arg Tyr
Arg Glu Asp Val Ala Leu Met Arg Gln Val Gly Leu His 65 70 75 80 Ala
Tyr Arg Leu Ser Val Cys Trp Pro Arg Val Leu Pro Glu Gly Val 85 90
95 Gly Gln Pro Asn Glu Lys Gly Leu Asp Phe Tyr Ser Arg Leu Val Asp
100 105 110 Ala Leu Leu Glu Ala Gly Ile Thr Pro Trp Val Thr Leu Phe
His Trp 115 120 125 Asp Tyr Pro Leu Ala Leu Tyr His Arg Gly Gly Trp
Leu Asn Arg Asp 130 135 140 Ser Ala Asp Trp Phe Ala Glu Tyr Ala Gly
Leu Ile Ala Asp Arg Leu 145 150 155 160 Ser Asp Arg Val Gln His Phe
Phe Thr Gln Asn Glu Pro Gln Val Tyr 165 170 175 Ile Gly Phe Gly His
Leu Glu Gly Lys His Ala Pro Gly Asp Thr Leu 180 185 190 Pro Met Ser
Gln Val Leu Leu Ala Gly His His Ser Leu Leu Ala His 195 200 205 Gly
Lys Ala Val Gln Ala Leu Arg Ala Gln Ala Lys Gln Gln Leu Arg 210 215
220 Val Gly Tyr Ala Pro Val Gly Met Pro Leu His Pro Phe Thr Asp Ser
225 230 235 240 Ala Glu Asp Val Ala Ala Ala Arg Lys Ala Thr Phe Trp
Val Arg Glu 245 250 255 Lys Asn Ser Trp Asn Asn Ala Trp Trp Met Asp
Pro Val Phe Leu Gly 260 265 270 Glu Tyr Pro Ala Gln Gly Leu Ala Phe
Phe Gly Arg Asp Val Pro Gln 275 280 285 Val Arg Glu Gly Asp Met Gln
Leu Ile Ala Gln Pro Leu Asp Phe Phe 290 295 300 Gly Val Asn Ile Tyr
Gln Ser Thr Pro Val Arg Ala Ser Ser Ala Glu 305 310 315 320 Ser Gly
Phe Glu Val Val Pro His Pro Thr Gly Tyr Pro Ile Thr Ala 325 330 335
Phe Asn Trp Pro Ile Thr Pro Gln Ala Leu Tyr Trp Gly Pro Arg Phe 340
345 350 Phe Tyr Glu Arg Tyr Gln Lys Pro Ile Val Ile Thr Glu Asn Gly
Leu 355 360 365 Ser Cys Arg Asp Val Val Ala Val Asp Gly Lys Val His
Asp Pro Ala 370 375 380 Arg Ile Asp Phe Thr Thr Arg Tyr Leu Arg Glu
Leu His Arg Ala Val 385 390 395 400 Ala Asp Gly Val Ala Val Glu Gly
Tyr Phe His Trp Ser Ile Met Asp 405 410 415 Asn Phe Glu Trp Ala Ala
Gly Tyr Arg Glu Arg Phe Gly Leu Ile His 420 425 430 Val Asp Tyr Glu
Thr Leu Ala Arg Thr Pro Lys Ala Ser Ala Ala Trp 435 440 445 Tyr Arg
Lys Val Ile Glu Ser Asn Gly Ala Thr Leu Phe Gly 450 455 460
57414DNAUnknownObtained from environmental sample 57atgattgctt
catctatgtt ctatggaacg gttcgtggaa tacaagagct aactcaaaac 60gttattgcat
tggataccgc aatggtttcg cttaccagag ttgctgacgg aagtgatttt
120gagtttgata gagttattga acgctcgatt gaaaacgtaa ccgaactatc
aggtaagcta 180actgattaca tggatttagt aacggagttt gctagaactg
gtaaaacaat agatgaatct 240tttaatttag ctaatacaac acaaatgtta
atgaatattt ctgaattaac agcagatgaa 300tcagtaaata gtttaactgc
cgcaatgatt gcttttaata ttaacgcaga tgatagtatt 360agaattgctg
ataagttgaa tgaggttaac aatatcagcc tccttttgtg gtaa
41458137PRTUnknownObtained from environmental sample 58Met Ile Ala
Ser Ser Met Phe Tyr Gly Thr Val Arg Gly Ile Gln Glu 1 5 10 15 Leu
Thr Gln Asn Val Ile Ala Leu Asp Thr Ala Met Val Ser Leu Thr 20 25
30 Arg Val Ala Asp Gly Ser Asp Phe Glu Phe Asp Arg Val Ile Glu Arg
35 40 45 Ser Ile Glu Asn Val Thr Glu Leu Ser Gly Lys Leu Thr Asp
Tyr Met 50 55 60 Asp Leu Val Thr Glu Phe Ala Arg Thr Gly Lys Thr
Ile Asp Glu Ser 65 70 75 80 Phe Asn Leu Ala Asn Thr Thr Gln Met Leu
Met Asn Ile Ser Glu Leu 85 90 95 Thr Ala Asp Glu Ser Val Asn Ser
Leu Thr Ala Ala Met Ile Ala Phe 100 105 110 Asn Ile Asn Ala Asp Asp
Ser Ile Arg Ile Ala Asp Lys Leu Asn Glu 115 120 125 Val Asn Asn Ile
Ser Leu Leu Leu Trp 130 135 591044DNAUnknownObtained from
environmental sample 59atgagaattt ttgaaggatt tcagcgaggt gtaaaccttg
gcggctggat ctcccagttc 60gacaagtacg accatgagca tttccgcagc tttattacgg
aaaatgacat cgccgccatt 120gcagctcttg gttttgacca tgtccgcgtg
ccggtggatt ataacgtgct ggaggatgag 180gagggcaacc gcatcgacag
cggatttgtc tacctgagaa gctgctacga gtggtgccgc 240aaacacgacc
tgaacatgct ggtggatctt cacgagtgct acggctactc cttcgatccg
300ctgaaaaaag atatggaccg caaacgcttc ttctatgccg aagctctgca
ggagcgtttt 360ctgaagctct gggagcagat ctgtgaaacc tttaaagacg
atcctgtgca cgtggcattc 420gagccgctga atgagatcgt tttaggagag
gtcgcagacg cctggaacgt gatgatccgc 480aaatatatca agaccgtccg
cgccatctgc ccggagcact atctggtcct tggaagcgtg 540cactacagcc
acgttaccac catccctctt cttgaggcac cggcagatga caagatcgtc
600ttcaacttcc actgctacga gccgctggtc ttcacccacc agggcgcata
ctggctggag 660gatatgattc cggatttccg catgacctat cctgccacca
tggaagagtt ctacgaagca 720acaaagaaga tcctgccaaa catgagtccg
gatggattta aggatttcga tcaggagatg 780ggtccgggct tctttgagaa
gatcttcaca ccggccctga aacgtgccga gcaggacaat 840gtagccctct
actgcggcga gtacggcgtc attgatctgg cagataacca tgccaagatc
900cgctggctca aagacatcca caccaccttc tccaaatacg gcatcggaag
tgccctctgg 960aactacaagg gcaaggattt cggctatgta gatgatcgct
tcgccgagtg cagagaagca 1020tttatcgagt gcctgaaggc ctga
104460347PRTUnknownObtained from environmental sample 60Met Arg Ile
Phe Glu Gly Phe Gln Arg Gly Val Asn Leu Gly Gly Trp 1 5 10 15 Ile
Ser Gln Phe Asp Lys Tyr Asp His Glu His Phe Arg Ser Phe Ile 20 25
30 Thr Glu Asn Asp Ile Ala Ala Ile Ala Ala Leu Gly Phe Asp His Val
35 40 45 Arg Val Pro Val Asp Tyr Asn Val Leu Glu Asp Glu Glu Gly
Asn Arg 50 55 60 Ile Asp Ser Gly Phe Val Tyr Leu Arg Ser Cys Tyr
Glu Trp Cys Arg 65 70 75 80 Lys His Asp Leu Asn Met Leu Val Asp Leu
His Glu Cys Tyr Gly Tyr 85 90 95 Ser Phe Asp Pro Leu Lys Lys Asp
Met Asp Arg Lys Arg Phe Phe Tyr 100 105 110 Ala Glu Ala Leu Gln Glu
Arg Phe Leu Lys Leu Trp Glu Gln Ile Cys 115 120 125 Glu Thr Phe Lys
Asp Asp Pro Val His Val Ala Phe Glu Pro Leu Asn 130 135 140 Glu Ile
Val Leu Gly Glu Val Ala Asp Ala Trp Asn Val Met Ile Arg 145 150 155
160 Lys Tyr Ile Lys Thr Val Arg Ala Ile Cys Pro Glu His Tyr Leu Val
165 170 175 Leu Gly Ser Val His Tyr Ser His Val Thr Thr Ile Pro Leu
Leu Glu 180 185 190 Ala Pro Ala Asp Asp Lys Ile Val Phe Asn Phe His
Cys Tyr Glu Pro 195 200 205 Leu Val Phe Thr His Gln Gly Ala Tyr Trp
Leu Glu Asp Met Ile Pro 210 215 220 Asp Phe Arg Met Thr Tyr Pro Ala
Thr Met Glu Glu Phe Tyr Glu Ala 225 230 235 240 Thr Lys Lys Ile Leu
Pro Asn Met Ser Pro Asp Gly Phe Lys Asp Phe 245 250 255 Asp Gln Glu
Met Gly Pro Gly Phe Phe Glu Lys Ile Phe Thr Pro Ala 260 265 270 Leu
Lys Arg Ala Glu Gln Asp Asn Val Ala Leu Tyr Cys Gly Glu Tyr 275 280
285 Gly Val Ile Asp Leu Ala Asp Asn His Ala Lys Ile Arg Trp Leu Lys
290 295 300 Asp Ile His Thr Thr Phe Ser Lys Tyr Gly Ile Gly Ser Ala
Leu Trp 305 310 315 320 Asn Tyr Lys Gly Lys Asp Phe Gly Tyr Val Asp
Asp Arg Phe Ala Glu 325 330 335 Cys Arg Glu Ala Phe Ile Glu Cys Leu
Lys Ala 340 345 611230DNAUnknownObtained from environmental sample
61ttggtatgga caccagctcg atcaacgctt gctggatctt ctgaaatccc actaatgaca
60atgaatatat tccccaatag aaaagactca cgaatgtccc tctggatcaa gcttggcata
120ctttgtatga tggctggaac ggtgatggtt cacggagcgc agactggtca
aggagaagca 180acaatgaatc aagcaaatgg cttcaaggta agcaacggga
ccaatatcag ccattggttg 240tcccagtgtt ttgaaacaat gccaccccgg
cgcggatttt tctccgaact ggatgttatc 300ttcatccgct cgctggggat
ggatcatttc cgtcttccgg tggacgagaa ggaactttgg 360acggaggatc
ttgagaagat tcccgaagcg tgggattacc tcaggaatgc tctaagctgg
420gctagaaagc atgagcttcg tgtgattgtg gatcttcacg tcgtgcggtc
ccatcacttt 480aatgcggcaa atgaaggggg aaccaacact ctgtgggatg
atccggaggc gcaggaaagt 540ttcctcaacc tttggaggca gctttcggca
gagctcgcct acaccgatgt ggactgggtg 600gcctatgaga tcatgaatga
ggccgtcgcg gatgatccgg aggactggaa tcgtctcatc 660gccaaagccc
actccttgat ccgcgagcgt gagccaaggc gcacactcgt catcggatcc
720aaccggtggc aaattccgtc aacgttcccg gatctgaaga ttccggacgg
agatccgaac 780atcctcctga gtttccattt ctacgcgcct ctgcttttca
cccactatcg ggcaacctgg 840gttgcctttt acgattatga tgggccggtt
tcctatcctg gcaggatcgt tgatgatgca 900gctcttgaga aaaatgatta
tactcctgca ttcaaagaca agattcgtgc gttgaatggt 960gtgtatgaca
tcgacgctct cgaaaaagaa atgcagccgg ctatcgaata cgcaaaacag
1020aaagggttac cactgtattg cggagagtgg ggatgttttc atgctgtgga
aagaaaacaa 1080cgcttgcaat ggtacaaaga tatatccact attttgaaac
gcaatgggat cgcccatgcc 1140acatgggatt acaagggcga gttcggcatt
gtggacactt ggacactagg tgttgattgg 1200aatttggtag gagcaatcct
gtcagagtag 123062409PRTUnknownObtained from environmental sample
62Met Val Trp Thr Pro Ala Arg Ser Thr Leu Ala Gly Ser Ser Glu Ile 1
5 10 15 Pro Leu Met Thr Met Asn Ile Phe Pro Asn Arg Lys Asp Ser Arg
Met 20 25 30 Ser Leu Trp Ile Lys Leu Gly Ile Leu Cys Met Met Ala
Gly Thr Val 35 40 45 Met Val His Gly Ala Gln Thr Gly Gln Gly Glu
Ala Thr Met Asn Gln 50 55 60 Ala Asn Gly Phe Lys Val Ser Asn Gly
Thr Asn Ile Ser His Trp Leu 65 70 75 80 Ser Gln Cys Phe Glu Thr Met
Pro Pro Arg Arg Gly Phe Phe Ser Glu 85 90 95 Leu Asp Val Ile Phe
Ile Arg Ser Leu Gly Met Asp His Phe Arg Leu 100 105 110 Pro Val Asp
Glu Lys Glu Leu Trp Thr Glu Asp Leu Glu Lys Ile Pro 115 120 125 Glu
Ala Trp Asp Tyr Leu Arg Asn Ala Leu Ser Trp Ala Arg Lys His 130 135
140 Glu Leu Arg Val Ile Val Asp Leu His Val Val Arg Ser His His Phe
145 150 155 160 Asn Ala Ala Asn Glu Gly Gly Thr Asn Thr Leu Trp Asp
Asp Pro Glu 165 170 175 Ala Gln Glu Ser Phe Leu Asn Leu Trp Arg Gln
Leu Ser Ala Glu Leu 180 185 190
Ala Tyr Thr Asp Val Asp Trp Val Ala Tyr Glu Ile Met Asn Glu Ala 195
200 205 Val Ala Asp Asp Pro Glu Asp Trp Asn Arg Leu Ile Ala Lys Ala
His 210 215 220 Ser Leu Ile Arg Glu Arg Glu Pro Arg Arg Thr Leu Val
Ile Gly Ser 225 230 235 240 Asn Arg Trp Gln Ile Pro Ser Thr Phe Pro
Asp Leu Lys Ile Pro Asp 245 250 255 Gly Asp Pro Asn Ile Leu Leu Ser
Phe His Phe Tyr Ala Pro Leu Leu 260 265 270 Phe Thr His Tyr Arg Ala
Thr Trp Val Ala Phe Tyr Asp Tyr Asp Gly 275 280 285 Pro Val Ser Tyr
Pro Gly Arg Ile Val Asp Asp Ala Ala Leu Glu Lys 290 295 300 Asn Asp
Tyr Thr Pro Ala Phe Lys Asp Lys Ile Arg Ala Leu Asn Gly 305 310 315
320 Val Tyr Asp Ile Asp Ala Leu Glu Lys Glu Met Gln Pro Ala Ile Glu
325 330 335 Tyr Ala Lys Gln Lys Gly Leu Pro Leu Tyr Cys Gly Glu Trp
Gly Cys 340 345 350 Phe His Ala Val Glu Arg Lys Gln Arg Leu Gln Trp
Tyr Lys Asp Ile 355 360 365 Ser Thr Ile Leu Lys Arg Asn Gly Ile Ala
His Ala Thr Trp Asp Tyr 370 375 380 Lys Gly Glu Phe Gly Ile Val Asp
Thr Trp Thr Leu Gly Val Asp Trp 385 390 395 400 Asn Leu Val Gly Ala
Ile Leu Ser Glu 405 631152DNAUnknownObtained from environmental
sample 63atgaaacgga gggaattcat gttggggggt gcgggtgttg ctgcgttggc
atcgactctt 60ggagtctccg ccggttccac ttccgggcag ggagtgaacg agaatgtgag
ggtataccgg 120aatgcgattc cccgttggag ggggttcaac ctcatgccct
ttttctcggc aatgagcacc 180aacccggaat acaatggtct gacggtgccg
gaggatgacc taaactggat ccgcgactgg 240ggttttgact atgtccggct
tccgattgat tactggattc tggttgattc cgattggcga 300gatgcaaagc
gcatgcgggt agaggatgtt cgcaaggccg accagaaggg atattcacgg
360ctggacgctg tgattgaagc ctgtatcgcg aagggtttgc acctcaacct
gaatatgcat 420cggtgtcccg ggtattgcat caatggctgg gaactggagc
cctataacct cttcaaggat 480gagcaggcgg aggatgattt tgtctaccat
tgggagttgc tcgcgagacg ctataaggga 540atcgatcctt cgctgctgag
tttcaatctg ctgaatgagg ctcccaatcc tggagacaag 600atgtcgtcgg
aggattatcg tcgggtgatg cttcgatccg ctgctgttat tcgggggata
660agcccggacc gcatgattat tgtggacggg ctggaaatcg gtaaatcagt
tgttccaggg 720ctgatgcatg agccatttgc ccaagctgtt catgcctacg
agccccacga gttgagccat 780tataatgcgc cttggacgtc ggtgtttatg
ggtattcctg agccatcctg gccgacagtt 840cgtttggatg gttctctgtt
cgaccgcaag cgactggagt tgtatttcgc gccgtggggg 900gagttggtcc
gccagggggt aggggtccac tgtggggaga ccggttgcta cattcatacg
960ccccatcggg tgtttctgtc ctggttcgaa gatgttttgg atatcctgac
cggatacgac 1020atagggtggg ctctatggaa tttccgggga gatttcggaa
tacttgattc caaacgcaag 1080gatgtgcaat atgtcgattg gtatggacac
cagctcgatc aacgcttgct ggatcttctg 1140aaatcccact aa
115264383PRTUnknownObtained from environmental sample 64Met Lys Arg
Arg Glu Phe Met Leu Gly Gly Ala Gly Val Ala Ala Leu 1 5 10 15 Ala
Ser Thr Leu Gly Val Ser Ala Gly Ser Thr Ser Gly Gln Gly Val 20 25
30 Asn Glu Asn Val Arg Val Tyr Arg Asn Ala Ile Pro Arg Trp Arg Gly
35 40 45 Phe Asn Leu Met Pro Phe Phe Ser Ala Met Ser Thr Asn Pro
Glu Tyr 50 55 60 Asn Gly Leu Thr Val Pro Glu Asp Asp Leu Asn Trp
Ile Arg Asp Trp 65 70 75 80 Gly Phe Asp Tyr Val Arg Leu Pro Ile Asp
Tyr Trp Ile Leu Val Asp 85 90 95 Ser Asp Trp Arg Asp Ala Lys Arg
Met Arg Val Glu Asp Val Arg Lys 100 105 110 Ala Asp Gln Lys Gly Tyr
Ser Arg Leu Asp Ala Val Ile Glu Ala Cys 115 120 125 Ile Ala Lys Gly
Leu His Leu Asn Leu Asn Met His Arg Cys Pro Gly 130 135 140 Tyr Cys
Ile Asn Gly Trp Glu Leu Glu Pro Tyr Asn Leu Phe Lys Asp 145 150 155
160 Glu Gln Ala Glu Asp Asp Phe Val Tyr His Trp Glu Leu Leu Ala Arg
165 170 175 Arg Tyr Lys Gly Ile Asp Pro Ser Leu Leu Ser Phe Asn Leu
Leu Asn 180 185 190 Glu Ala Pro Asn Pro Gly Asp Lys Met Ser Ser Glu
Asp Tyr Arg Arg 195 200 205 Val Met Leu Arg Ser Ala Ala Val Ile Arg
Gly Ile Ser Pro Asp Arg 210 215 220 Met Ile Ile Val Asp Gly Leu Glu
Ile Gly Lys Ser Val Val Pro Gly 225 230 235 240 Leu Met His Glu Pro
Phe Ala Gln Ala Val His Ala Tyr Glu Pro His 245 250 255 Glu Leu Ser
His Tyr Asn Ala Pro Trp Thr Ser Val Phe Met Gly Ile 260 265 270 Pro
Glu Pro Ser Trp Pro Thr Val Arg Leu Asp Gly Ser Leu Phe Asp 275 280
285 Arg Lys Arg Leu Glu Leu Tyr Phe Ala Pro Trp Gly Glu Leu Val Arg
290 295 300 Gln Gly Val Gly Val His Cys Gly Glu Thr Gly Cys Tyr Ile
His Thr 305 310 315 320 Pro His Arg Val Phe Leu Ser Trp Phe Glu Asp
Val Leu Asp Ile Leu 325 330 335 Thr Gly Tyr Asp Ile Gly Trp Ala Leu
Trp Asn Phe Arg Gly Asp Phe 340 345 350 Gly Ile Leu Asp Ser Lys Arg
Lys Asp Val Gln Tyr Val Asp Trp Tyr 355 360 365 Gly His Gln Leu Asp
Gln Arg Leu Leu Asp Leu Leu Lys Ser His 370 375 380
651131DNAUnknownObtained from environmental sample 65atgaacacac
tcctaccacg gcggcgactg tggtcctcca cggcgatcct gcgcacgctg 60gcggccgggg
cgctggcggc cggtatggtc ctggcacccg tcagtgccgc caacgcggcc
120accaccctcg gtgcctcggc ggcggagaag ggccggtact tcggtgcggc
cgtcgggacg 180tacaagttca acgacagcac ctacatgtcg gtgctgaacc
gcgagttcaa cagcctggtc 240gccgagaacg agatgaagtg ggacgcgacc
gagccccagc gcggcgtgtt caactacagc 300gccggggacc gcatcgtcaa
ccacgcccga tcccagggca tgaaggtacg cggacacgcc 360ctgttgtggc
acgcccagca gccacgctgg acggagggcc tgtccggcgg cgacctgcgc
420aacgccgcga tcaaccatgt cacccaggtg gccagccact tccgggggca
gatctactcc 480tgggacgtgg tgaacgaggc tttcgccgac ggtggcagcg
gtgcccggcg ggactcgaac 540ctccagcgca ccggcaacga ctggatcgag
gcggcgttcc gtgccgcccg ggcagccgat 600cccaacgcca agctctgcta
caacgactac aacaccgacg ggatcaacgc gaagtccacc 660ggcgtctaca
acatggtgcg tgacttcaag tcccgtgggg tgccgatcga ctgcgtgggc
720ttccagtcac acctgggcac caccctcccc ggtgactacc aggccaacct
tcagcgcttc 780gccgacctgg gcgtcgacgt ggagatcacc gagctggaca
tcacccaggg cggaaaccag 840gccaacatgt acggcgccgt cacccgcgcc
tgcctggcga tctcgcgctg caccggcatc 900accgtgtggg gggtacggga
ctgcgactcc tggcgtggtg gggacaacgc cctgctgttc 960gactgcgccg
gcaacaagaa gcccgcgtac acggccgtcc tcgacgccct caacagcggc
1020tcgaacccga accccaaccc caccggcaac cggctgcgca acgaggcctc
cggtcgatgc 1080ctggacgtca acggcgcaag ctccgccaac gggtcacaaa
tgatccaaag a 113166377PRTUnknownObtained from environmental sample
66Met Asn Thr Leu Leu Pro Arg Arg Arg Leu Trp Ser Ser Thr Ala Ile 1
5 10 15 Leu Arg Thr Leu Ala Ala Gly Ala Leu Ala Ala Gly Met Val Leu
Ala 20 25 30 Pro Val Ser Ala Ala Asn Ala Ala Thr Thr Leu Gly Ala
Ser Ala Ala 35 40 45 Glu Lys Gly Arg Tyr Phe Gly Ala Ala Val Gly
Thr Tyr Lys Phe Asn 50 55 60 Asp Ser Thr Tyr Met Ser Val Leu Asn
Arg Glu Phe Asn Ser Leu Val 65 70 75 80 Ala Glu Asn Glu Met Lys Trp
Asp Ala Thr Glu Pro Gln Arg Gly Val 85 90 95 Phe Asn Tyr Ser Ala
Gly Asp Arg Ile Val Asn His Ala Arg Ser Gln 100 105 110 Gly Met Lys
Val Arg Gly His Ala Leu Leu Trp His Ala Gln Gln Pro 115 120 125 Arg
Trp Thr Glu Gly Leu Ser Gly Gly Asp Leu Arg Asn Ala Ala Ile 130 135
140 Asn His Val Thr Gln Val Ala Ser His Phe Arg Gly Gln Ile Tyr Ser
145 150 155 160 Trp Asp Val Val Asn Glu Ala Phe Ala Asp Gly Gly Ser
Gly Ala Arg 165 170 175 Arg Asp Ser Asn Leu Gln Arg Thr Gly Asn Asp
Trp Ile Glu Ala Ala 180 185 190 Phe Arg Ala Ala Arg Ala Ala Asp Pro
Asn Ala Lys Leu Cys Tyr Asn 195 200 205 Asp Tyr Asn Thr Asp Gly Ile
Asn Ala Lys Ser Thr Gly Val Tyr Asn 210 215 220 Met Val Arg Asp Phe
Lys Ser Arg Gly Val Pro Ile Asp Cys Val Gly 225 230 235 240 Phe Gln
Ser His Leu Gly Thr Thr Leu Pro Gly Asp Tyr Gln Ala Asn 245 250 255
Leu Gln Arg Phe Ala Asp Leu Gly Val Asp Val Glu Ile Thr Glu Leu 260
265 270 Asp Ile Thr Gln Gly Gly Asn Gln Ala Asn Met Tyr Gly Ala Val
Thr 275 280 285 Arg Ala Cys Leu Ala Ile Ser Arg Cys Thr Gly Ile Thr
Val Trp Gly 290 295 300 Val Arg Asp Cys Asp Ser Trp Arg Gly Gly Asp
Asn Ala Leu Leu Phe 305 310 315 320 Asp Cys Ala Gly Asn Lys Lys Pro
Ala Tyr Thr Ala Val Leu Asp Ala 325 330 335 Leu Asn Ser Gly Ser Asn
Pro Asn Pro Asn Pro Thr Gly Asn Arg Leu 340 345 350 Arg Asn Glu Ala
Ser Gly Arg Cys Leu Asp Val Asn Gly Ala Ser Ser 355 360 365 Ala Asn
Gly Ser Gln Met Ile Gln Arg 370 375 671023DNAUnknownObtained from
environmental sample 67atgaaatata tattttcgta tataataatg atgattttaa
tcggttttat accggtctat 60ggattcggcg attcacctga ccaaacatac tctctcccct
tcctcagcgt agaaggaaat 120tcattcgtcg atgaaaacgg tgaggaggtt
attttgcggg gtgtatcgtt tcccgatccc 180aatcgattgg atgatgctac
tcaatggaac aaacggtatt tccaggcagc aaaagattgg 240aactgtaatg
tcgtcagaat accggttcat ccgcaaagat ggcgggaaag gggaaaagaa
300aattatctga aacttttaga taagggtatc gagtgggccg gtgaactcgg
tatgtacgtg 360atcattgact ggcacactat cggcaatccg attaccgaag
tgttcttcgg cgagctctat 420aatacgaccc agaccgaaac gttccggttc
tggagaacaa tagcggagcg atatgcaggt 480aatcccgttg ttgcatttta
tgaattgttt aatgaaccga ccgattataa cggtcggctc 540gggaggatga
cctgggatca atataaagaa ttcatcgaag agatcattta tataatttat
600gcacacgacg aaaccgtgat accgcttgta ggcggtttcg attggggata
tgatctcagg 660aatgttagag ataatccgat aaatgccccg ggtatcgcgt
atgttactca cccgtatccg 720caaaagcggg accaaccgtg ggaagaaaaa
tgggaaaggg atttcggttt cgtagccgac 780acctaccctg tgtttgctac
cgagttcgga tttatgagtg aggatggttt gggtgcacat 840attcccgtta
tcggtgatga aacatacggt gaagcgatca tcagttactt caatgagaaa
900ggtatatcgt ggacggcctg ggtgttcgat ccgctctggt cgccgcagct
tattaaagac 960tggtatttta ccccgacccg gcagggacag ttttttaaag
agaagctaat ggagttgaat 1020taa 102368340PRTUnknownObtained from
environmental sample 68Met Lys Tyr Ile Phe Ser Tyr Ile Ile Met Met
Ile Leu Ile Gly Phe 1 5 10 15 Ile Pro Val Tyr Gly Phe Gly Asp Ser
Pro Asp Gln Thr Tyr Ser Leu 20 25 30 Pro Phe Leu Ser Val Glu Gly
Asn Ser Phe Val Asp Glu Asn Gly Glu 35 40 45 Glu Val Ile Leu Arg
Gly Val Ser Phe Pro Asp Pro Asn Arg Leu Asp 50 55 60 Asp Ala Thr
Gln Trp Asn Lys Arg Tyr Phe Gln Ala Ala Lys Asp Trp 65 70 75 80 Asn
Cys Asn Val Val Arg Ile Pro Val His Pro Gln Arg Trp Arg Glu 85 90
95 Arg Gly Lys Glu Asn Tyr Leu Lys Leu Leu Asp Lys Gly Ile Glu Trp
100 105 110 Ala Gly Glu Leu Gly Met Tyr Val Ile Ile Asp Trp His Thr
Ile Gly 115 120 125 Asn Pro Ile Thr Glu Val Phe Phe Gly Glu Leu Tyr
Asn Thr Thr Gln 130 135 140 Thr Glu Thr Phe Arg Phe Trp Arg Thr Ile
Ala Glu Arg Tyr Ala Gly 145 150 155 160 Asn Pro Val Val Ala Phe Tyr
Glu Leu Phe Asn Glu Pro Thr Asp Tyr 165 170 175 Asn Gly Arg Leu Gly
Arg Met Thr Trp Asp Gln Tyr Lys Glu Phe Ile 180 185 190 Glu Glu Ile
Ile Tyr Ile Ile Tyr Ala His Asp Glu Thr Val Ile Pro 195 200 205 Leu
Val Gly Gly Phe Asp Trp Gly Tyr Asp Leu Arg Asn Val Arg Asp 210 215
220 Asn Pro Ile Asn Ala Pro Gly Ile Ala Tyr Val Thr His Pro Tyr Pro
225 230 235 240 Gln Lys Arg Asp Gln Pro Trp Glu Glu Lys Trp Glu Arg
Asp Phe Gly 245 250 255 Phe Val Ala Asp Thr Tyr Pro Val Phe Ala Thr
Glu Phe Gly Phe Met 260 265 270 Ser Glu Asp Gly Leu Gly Ala His Ile
Pro Val Ile Gly Asp Glu Thr 275 280 285 Tyr Gly Glu Ala Ile Ile Ser
Tyr Phe Asn Glu Lys Gly Ile Ser Trp 290 295 300 Thr Ala Trp Val Phe
Asp Pro Leu Trp Ser Pro Gln Leu Ile Lys Asp 305 310 315 320 Trp Tyr
Phe Thr Pro Thr Arg Gln Gly Gln Phe Phe Lys Glu Lys Leu 325 330 335
Met Glu Leu Asn 340 691182DNAUnknownObtained from environmental
sample 69atgagtttta aaaaccacat acttttgtcg ctcctcatag tattgctttt
cttttcagcg 60tgcgatatcg aagaaccgat cgccggagat tatcatacac ttgtggatca
aaacgctata 120tcgcacaccc gcgcattatt caccaacctc gaacgtatcc
gtcacgatca tatcctcttc 180ggtcatcagg atgcgcttgc atacggtgtt
cactggcgca acgatgagcc gggtcgatcg 240gatgtattcg aagtaaccgg
ttcgtatcct gcggtgtatg gctgggagat tggcgatatt 300gaacttggtg
caccggaaaa tctggataac gtaaacttcg atcaaatgca gggctggatt
360cgcgaagggt acgaacgcgg cggtataatt acgattagct ggcatatgaa
caatccggca 420tcgggtggtg attcgtggga tgtgaatgga ggtcataaag
cggtaactaa gatacttccc 480ggcggagaac ttcacgatac gtttaaagaa
tggctggata cgtttgcaaa attcgcgaag 540agccagattg cttttcccga
aacaaataat gaacacctta tcccggtcat attccggccg 600tatcatgaaa
acaccggaag ctggttctgg tggggcgccg accactgtac acctgaagaa
660tataaaaagt tatggcgatt taccgtcgaa tacctgcgcg atgtaaaagg
tgttcacaat 720ctcctctggg cgtattcacc tgccggcaat gctgcggatt
cagaggaagc atattttgct 780cggtatcccg gcgacgacta tgttgatatt
attggattcg acgattacgg cagtgtgcgg 840aaaccgtatc aaatcgaacg
ttttactaac cggattcgaa cgattgtaaa cttcgccgaa 900gcacgaaata
aaatcccggc aataacggaa accggctatg aaactatccc cgatccgcaa
960tggtggacgg gtacattgct tagtgcactt gatcacgatt tgacaacccg
gagaatagca 1020tacgtacttg tgtggcgaaa ttcaaacaat gctaccgacc
ggcagaatca ttattacgct 1080ccgtatcccg gacatccaag tgctgacgat
tttatcgcgt tcaggaatca cccgttgata 1140gttttcgaag atgatctgcc
gggtatgtat acactaccgt aa 118270393PRTUnknownObtained from
environmental sample 70Met Ser Phe Lys Asn His Ile Leu Leu Ser Leu
Leu Ile Val Leu Leu 1 5 10 15 Phe Phe Ser Ala Cys Asp Ile Glu Glu
Pro Ile Ala Gly Asp Tyr His 20 25 30 Thr Leu Val Asp Gln Asn Ala
Ile Ser His Thr Arg Ala Leu Phe Thr 35 40 45 Asn Leu Glu Arg Ile
Arg His Asp His Ile Leu Phe Gly His Gln Asp 50 55 60 Ala Leu Ala
Tyr Gly Val His Trp Arg Asn Asp Glu Pro Gly Arg Ser 65 70 75 80 Asp
Val Phe Glu Val Thr Gly Ser Tyr Pro Ala Val Tyr Gly Trp Glu 85 90
95 Ile Gly Asp Ile Glu Leu Gly Ala Pro Glu Asn Leu Asp Asn Val Asn
100 105 110 Phe Asp Gln Met Gln Gly Trp Ile Arg Glu Gly Tyr Glu Arg
Gly Gly 115 120 125 Ile Ile Thr Ile Ser Trp His Met Asn Asn Pro Ala
Ser Gly Gly Asp 130 135 140 Ser Trp Asp Val Asn Gly Gly His Lys Ala
Val Thr Lys Ile Leu Pro 145 150 155 160 Gly Gly Glu Leu His Asp Thr
Phe Lys Glu Trp Leu Asp Thr Phe Ala 165 170 175 Lys Phe Ala Lys Ser
Gln Ile Ala Phe Pro Glu Thr Asn Asn Glu His 180 185 190 Leu Ile Pro
Val Ile Phe Arg Pro Tyr His Glu Asn Thr Gly Ser Trp 195 200 205 Phe
Trp Trp Gly Ala Asp His Cys Thr Pro Glu Glu
Tyr Lys Lys Leu 210 215 220 Trp Arg Phe Thr Val Glu Tyr Leu Arg Asp
Val Lys Gly Val His Asn 225 230 235 240 Leu Leu Trp Ala Tyr Ser Pro
Ala Gly Asn Ala Ala Asp Ser Glu Glu 245 250 255 Ala Tyr Phe Ala Arg
Tyr Pro Gly Asp Asp Tyr Val Asp Ile Ile Gly 260 265 270 Phe Asp Asp
Tyr Gly Ser Val Arg Lys Pro Tyr Gln Ile Glu Arg Phe 275 280 285 Thr
Asn Arg Ile Arg Thr Ile Val Asn Phe Ala Glu Ala Arg Asn Lys 290 295
300 Ile Pro Ala Ile Thr Glu Thr Gly Tyr Glu Thr Ile Pro Asp Pro Gln
305 310 315 320 Trp Trp Thr Gly Thr Leu Leu Ser Ala Leu Asp His Asp
Leu Thr Thr 325 330 335 Arg Arg Ile Ala Tyr Val Leu Val Trp Arg Asn
Ser Asn Asn Ala Thr 340 345 350 Asp Arg Gln Asn His Tyr Tyr Ala Pro
Tyr Pro Gly His Pro Ser Ala 355 360 365 Asp Asp Phe Ile Ala Phe Arg
Asn His Pro Leu Ile Val Phe Glu Asp 370 375 380 Asp Leu Pro Gly Met
Tyr Thr Leu Pro 385 390 711089DNAUnknownObtained from environmental
sample 71atgaaacttt taaaactttt aatctttctc cttattacgg taattttttc
tgatgtttcg 60gctcaaactt ttcaaataca aaaaggcaag aacattagcc attggctgtc
ccaaagtaaa 120agaaggggag aagagcgaaa agagttcttt actaagaatg
acgtagaatt tattgcaggc 180atcggcttcg atcatattcg tattcctatt
gacgaggagc aaatgtggga tgaaaaaggc 240aacaaagagc ctgaagcgtt
tcagttgctg cacaacgcga tagaatggag caggcaatcg 300aacttaaaag
ttattgtgga cctgcatatt ttgaggtcgc attatttcaa cgcggaagaa
360aaaccgcttt ttacggaccc taaagctcag gaacgttttt accaatgttg
ggcggatctg 420tctggtgaat tgaaaaaata tccgaataca ctggtggctt
atgaattaat gaacgaacct 480gtagccgatg atccggaaga ctggaataga
attgtaagag aatcagtaaa aaggctaagg 540gtgcttgagc ccaatagggt
tattgtaatc gggtctaacc gatggcagca ttatgacact 600ctgaaggatt
tatacgtgcc ggaaaacgac aaaaacatca ttttaagctt tcatttttat
660aaccctatgt tgcttacgca ttacagggcc agctgggtaa atttcggcga
ttaccagggt 720cccgttaact acccgggaca gttggtagac tcaaagcatt
tgtcgggact gagcgaagat 780ttaagaaaga aagtcgagca aaacaatggc
gtttataata aggctcggat tgagaaaatg 840atagccgaag ccgttgctgt
agcaaaaaag cacaacctcc ctttgtattg tggtgaatgg 900ggtgcctacg
aaaaagcgcc aagggagccc aggctacaat ggtacagaga catggtggat
960gtgttgaaca aaaacaatat tgcctggact acctgggact ataaaggagg
cttcggcata 1020gttgacgcca aaggaaacaa agacgaacag ttgatcaatg
tattaacagg aaaagagaaa 1080aaaatgtaa 108972362PRTUnknownObtained
from environmental sample 72Met Lys Leu Leu Lys Leu Leu Ile Phe Leu
Leu Ile Thr Val Ile Phe 1 5 10 15 Ser Asp Val Ser Ala Gln Thr Phe
Gln Ile Gln Lys Gly Lys Asn Ile 20 25 30 Ser His Trp Leu Ser Gln
Ser Lys Arg Arg Gly Glu Glu Arg Lys Glu 35 40 45 Phe Phe Thr Lys
Asn Asp Val Glu Phe Ile Ala Gly Ile Gly Phe Asp 50 55 60 His Ile
Arg Ile Pro Ile Asp Glu Glu Gln Met Trp Asp Glu Lys Gly 65 70 75 80
Asn Lys Glu Pro Glu Ala Phe Gln Leu Leu His Asn Ala Ile Glu Trp 85
90 95 Ser Arg Gln Ser Asn Leu Lys Val Ile Val Asp Leu His Ile Leu
Arg 100 105 110 Ser His Tyr Phe Asn Ala Glu Glu Lys Pro Leu Phe Thr
Asp Pro Lys 115 120 125 Ala Gln Glu Arg Phe Tyr Gln Cys Trp Ala Asp
Leu Ser Gly Glu Leu 130 135 140 Lys Lys Tyr Pro Asn Thr Leu Val Ala
Tyr Glu Leu Met Asn Glu Pro 145 150 155 160 Val Ala Asp Asp Pro Glu
Asp Trp Asn Arg Ile Val Arg Glu Ser Val 165 170 175 Lys Arg Leu Arg
Val Leu Glu Pro Asn Arg Val Ile Val Ile Gly Ser 180 185 190 Asn Arg
Trp Gln His Tyr Asp Thr Leu Lys Asp Leu Tyr Val Pro Glu 195 200 205
Asn Asp Lys Asn Ile Ile Leu Ser Phe His Phe Tyr Asn Pro Met Leu 210
215 220 Leu Thr His Tyr Arg Ala Ser Trp Val Asn Phe Gly Asp Tyr Gln
Gly 225 230 235 240 Pro Val Asn Tyr Pro Gly Gln Leu Val Asp Ser Lys
His Leu Ser Gly 245 250 255 Leu Ser Glu Asp Leu Arg Lys Lys Val Glu
Gln Asn Asn Gly Val Tyr 260 265 270 Asn Lys Ala Arg Ile Glu Lys Met
Ile Ala Glu Ala Val Ala Val Ala 275 280 285 Lys Lys His Asn Leu Pro
Leu Tyr Cys Gly Glu Trp Gly Ala Tyr Glu 290 295 300 Lys Ala Pro Arg
Glu Pro Arg Leu Gln Trp Tyr Arg Asp Met Val Asp 305 310 315 320 Val
Leu Asn Lys Asn Asn Ile Ala Trp Thr Thr Trp Asp Tyr Lys Gly 325 330
335 Gly Phe Gly Ile Val Asp Ala Lys Gly Asn Lys Asp Glu Gln Leu Ile
340 345 350 Asn Val Leu Thr Gly Lys Glu Lys Lys Met 355 360
731146DNAUnknownObtained from environmental sample 73gtggatatta
ccggacatcc cgaccacatc gccttcgcgc gggaagttgc cgagcaaagc 60atggtcttgc
tgcaaaaccg tgcgaacctc gccccccttt cggtatctga ctattccacc
120attgccgtga tcggcccgaa tgccaatgac actttgctgg gttcttacag
cggcgttccg 180aaaacctact acacggtact cgacgggata cggtcctatg
tcggtgaccg ggcgaatgtg 240gtttacgctc aggggccgaa gataaccaaa
cccggccatc gggaggacaa tgaagtattt 300ccaccggatc ctgaaaacga
ccggagacga ctggccgaag cgatagctgt cgccgagaac 360gccgacctga
tcatcctcgc gatcggcggc aatgaactga cgggacgaga ggcatgggcg
420gcgcatcatc ccggtgatcg accggatctg tcgttgctcg gtttgcagga
ggatcttgtt 480gacgcagttg gagcgatggg ggttccatct gtcgcattgg
ttttcggtgc acggccgctg 540gacctcggca atgtcgccga aaaaattgat
gtggtcttcc aaaactggta cctgggccag 600gaaaccggca atgccgtcgc
caatgtgctg tttggcgagg tgtcaccgtc cgccaaactc 660cccatcagct
tcccgcggac tgccgggcac attcctgcct actacaatta caaaccatcg
720gctcgacggg tctacctttt tgacgatgtc actccgcgtt accatttcgg
gtacggcctc 780agctatacga cgtttgaata cggggaaccg cagctatcgg
atacactact gtctggcgat 840ggtgaaataa ccctctacgt tgaagttacc
aacaccggag agcgaggcgg ttcggaagtc 900gtgcaactgt acatcaacca
cgaatacaga tccgtcaccc ggccggtaaa ggagctcaag 960ggattcgaaa
aggtgtatct cgagccgaat gaaactgccg gtgtatcgtt caccatcact
1020tcagatcagt tgaggttctg gaatatcgac atggagttta ccgctgaatc
cggtaaagtg 1080aacctgatgg tcggctcatc cagccgtgac gaagacctgc
agacgacggc aatttttctt 1140gaataa 114674381PRTUnknownObtained from
environmental sample 74Met Asp Ile Thr Gly His Pro Asp His Ile Ala
Phe Ala Arg Glu Val 1 5 10 15 Ala Glu Gln Ser Met Val Leu Leu Gln
Asn Arg Ala Asn Leu Ala Pro 20 25 30 Leu Ser Val Ser Asp Tyr Ser
Thr Ile Ala Val Ile Gly Pro Asn Ala 35 40 45 Asn Asp Thr Leu Leu
Gly Ser Tyr Ser Gly Val Pro Lys Thr Tyr Tyr 50 55 60 Thr Val Leu
Asp Gly Ile Arg Ser Tyr Val Gly Asp Arg Ala Asn Val 65 70 75 80 Val
Tyr Ala Gln Gly Pro Lys Ile Thr Lys Pro Gly His Arg Glu Asp 85 90
95 Asn Glu Val Phe Pro Pro Asp Pro Glu Asn Asp Arg Arg Arg Leu Ala
100 105 110 Glu Ala Ile Ala Val Ala Glu Asn Ala Asp Leu Ile Ile Leu
Ala Ile 115 120 125 Gly Gly Asn Glu Leu Thr Gly Arg Glu Ala Trp Ala
Ala His His Pro 130 135 140 Gly Asp Arg Pro Asp Leu Ser Leu Leu Gly
Leu Gln Glu Asp Leu Val 145 150 155 160 Asp Ala Val Gly Ala Met Gly
Val Pro Ser Val Ala Leu Val Phe Gly 165 170 175 Ala Arg Pro Leu Asp
Leu Gly Asn Val Ala Glu Lys Ile Asp Val Val 180 185 190 Phe Gln Asn
Trp Tyr Leu Gly Gln Glu Thr Gly Asn Ala Val Ala Asn 195 200 205 Val
Leu Phe Gly Glu Val Ser Pro Ser Ala Lys Leu Pro Ile Ser Phe 210 215
220 Pro Arg Thr Ala Gly His Ile Pro Ala Tyr Tyr Asn Tyr Lys Pro Ser
225 230 235 240 Ala Arg Arg Val Tyr Leu Phe Asp Asp Val Thr Pro Arg
Tyr His Phe 245 250 255 Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Tyr
Gly Glu Pro Gln Leu 260 265 270 Ser Asp Thr Leu Leu Ser Gly Asp Gly
Glu Ile Thr Leu Tyr Val Glu 275 280 285 Val Thr Asn Thr Gly Glu Arg
Gly Gly Ser Glu Val Val Gln Leu Tyr 290 295 300 Ile Asn His Glu Tyr
Arg Ser Val Thr Arg Pro Val Lys Glu Leu Lys 305 310 315 320 Gly Phe
Glu Lys Val Tyr Leu Glu Pro Asn Glu Thr Ala Gly Val Ser 325 330 335
Phe Thr Ile Thr Ser Asp Gln Leu Arg Phe Trp Asn Ile Asp Met Glu 340
345 350 Phe Thr Ala Glu Ser Gly Lys Val Asn Leu Met Val Gly Ser Ser
Ser 355 360 365 Arg Asp Glu Asp Leu Gln Thr Thr Ala Ile Phe Leu Glu
370 375 380 751014DNAUnknownObtained from environmental sample
75atgctgcgca agttgatcgt ctcggtcttc ggcttcgtca tgctgactag tgcggcagcg
60gcgcagactc ctcccgcctt agcggaatcc gcgcctgctc tccggcgcgg aatgaacgtt
120ctgggctacg acccaatctg gcacgacccg aagaaaggtc ggttcgaaga
gcggcacttc 180gccgagattc gcaagggcgg cttcgacttc gttcgggtga
acctccacgg gttcaaacat 240atgaacgccg cggacaaact cagtccggag
ttcctgagcc gcgtggactg gatcgtgaag 300cacgccagtg cggcgggcct
gtcggtcatc ctagacgagc atgaatatga ggaatgctcg 360gacgacgtcg
caatgtgccg gcggcgtttg gcggcattct ggacgcaggt cgcgccgcgc
420tacaagggcg cgcccgatac ggttctgttc gagcttctca atgagccgca
cgacaagttg 480gatgccgaca cctggaacgc cttgtttccc gacatcctgg
ccatcgtgcg gcagtcgaac 540ccgaagcgcc gcgtggtgat cggcccgact
cagtggaaca acttcagcca gctggacacg 600ctcaagctgc cggcagacga
ccggaacatc gtcgtcacct tccattatta cgatccgttc 660ccgtttaccc
accagggcgc gccgtgggtt ccggacatgc tcaaggtgaa aggcatcgag
720tggaagcccg agcagagggc gaagatcgcc gaggacttcg gcaaggtcgc
ggaatggtcg 780cagaaaaccg gccgcgaaat cttgctcggc gagttcgggg
cctacgatgt gagcggtacg 840ccaaccgcca tgcgttcagc ttatacggaa
gcggtggcgc gcgaggcgga acgccacggc 900ttcgcttggg cctactggca
gttcgacagc aatttcctgg cttgggacat gaagacaaac 960ggctgggtcg
agccgatcca caaggcactc atccccgagg cgaagcagcc ttag
101476337PRTUnknownObtained from environmental sample 76Met Leu Arg
Lys Leu Ile Val Ser Val Phe Gly Phe Val Met Leu Thr 1 5 10 15 Ser
Ala Ala Ala Ala Gln Thr Pro Pro Ala Leu Ala Glu Ser Ala Pro 20 25
30 Ala Leu Arg Arg Gly Met Asn Val Leu Gly Tyr Asp Pro Ile Trp His
35 40 45 Asp Pro Lys Lys Gly Arg Phe Glu Glu Arg His Phe Ala Glu
Ile Arg 50 55 60 Lys Gly Gly Phe Asp Phe Val Arg Val Asn Leu His
Gly Phe Lys His 65 70 75 80 Met Asn Ala Ala Asp Lys Leu Ser Pro Glu
Phe Leu Ser Arg Val Asp 85 90 95 Trp Ile Val Lys His Ala Ser Ala
Ala Gly Leu Ser Val Ile Leu Asp 100 105 110 Glu His Glu Tyr Glu Glu
Cys Ser Asp Asp Val Ala Met Cys Arg Arg 115 120 125 Arg Leu Ala Ala
Phe Trp Thr Gln Val Ala Pro Arg Tyr Lys Gly Ala 130 135 140 Pro Asp
Thr Val Leu Phe Glu Leu Leu Asn Glu Pro His Asp Lys Leu 145 150 155
160 Asp Ala Asp Thr Trp Asn Ala Leu Phe Pro Asp Ile Leu Ala Ile Val
165 170 175 Arg Gln Ser Asn Pro Lys Arg Arg Val Val Ile Gly Pro Thr
Gln Trp 180 185 190 Asn Asn Phe Ser Gln Leu Asp Thr Leu Lys Leu Pro
Ala Asp Asp Arg 195 200 205 Asn Ile Val Val Thr Phe His Tyr Tyr Asp
Pro Phe Pro Phe Thr His 210 215 220 Gln Gly Ala Pro Trp Val Pro Asp
Met Leu Lys Val Lys Gly Ile Glu 225 230 235 240 Trp Lys Pro Glu Gln
Arg Ala Lys Ile Ala Glu Asp Phe Gly Lys Val 245 250 255 Ala Glu Trp
Ser Gln Lys Thr Gly Arg Glu Ile Leu Leu Gly Glu Phe 260 265 270 Gly
Ala Tyr Asp Val Ser Gly Thr Pro Thr Ala Met Arg Ser Ala Tyr 275 280
285 Thr Glu Ala Val Ala Arg Glu Ala Glu Arg His Gly Phe Ala Trp Ala
290 295 300 Tyr Trp Gln Phe Asp Ser Asn Phe Leu Ala Trp Asp Met Lys
Thr Asn 305 310 315 320 Gly Trp Val Glu Pro Ile His Lys Ala Leu Ile
Pro Glu Ala Lys Gln 325 330 335 Pro 771125DNAUnknownObtained from
environmental sample 77atgaaaagga aacgggtttt tattcattct ctaatcgtat
tttttttaat gattggttct 60tttacttctt gtggatcagt cgccgatgat gccgaagaag
ggtttgatat ttttagagga 120accaatatcg ctcattggtt atcacaaagt
aatgcaaggg gcgaagagcg aaaaaatttc 180tttaccgaaa atgatataaa
atttattgct gatgctggtt ttgatcatat tcgtttgcca 240attgacgagg
ttcatttctg ggatgagaat atgaaccggc accaagatgc atttgatctt
300atgcatgact gtattaagtg gtcagagaaa catggtctta gggttgtagt
ggatttgcat 360attattcgtt cacattattt tgttggagat gataatacac
tatgggatga aagacatgaa 420caggaaaagt ttgttgatat ttggatggag
ttatcatctg aactatctca atattcaaac 480tcattagtag cttatgagtt
aatgaatgaa cctgtagccc cttctcatga tgattggaat 540agtttggttg
cggaaactat agaggcaatt cgtaaagttg aacctgagag atatattgta
600gttggatcaa atatgtggca aggtattgat acatttgagt atttggaagt
tcccgaaaat 660gatgatagaa taattcttag ttttcatttt tatgatccct
ttattttgac tcattatact 720gcatcttggg ggtatttaag agattactca
gggcctgtta actatccggg atatcttgtt 780acaaatgacc agctgttgga
tatgtcaaac gaaatgcaaa agttaattag ggagtttcag 840acaaattttg
atatttatac cattgaagaa ctgatatcta ttccatatag tattgcaaag
900gaaaaagggt tgaaattata ttgtggagag tttggtgcaa ttgatcaggc
tccaagagat 960gcgagattgg catggtacag agatgttgtt caggtttttg
agcgatatgg tatagctcat 1020gccaactgga attacaaaga ttatggtacg
tttgggataa agaactatag cgaggagata 1080gatcaggaac tgtttgaaat
cttaattgga acaaaacata aatag 112578374PRTUnknownObtained from
environmental sample 78Met Lys Arg Lys Arg Val Phe Ile His Ser Leu
Ile Val Phe Phe Leu 1 5 10 15 Met Ile Gly Ser Phe Thr Ser Cys Gly
Ser Val Ala Asp Asp Ala Glu 20 25 30 Glu Gly Phe Asp Ile Phe Arg
Gly Thr Asn Ile Ala His Trp Leu Ser 35 40 45 Gln Ser Asn Ala Arg
Gly Glu Glu Arg Lys Asn Phe Phe Thr Glu Asn 50 55 60 Asp Ile Lys
Phe Ile Ala Asp Ala Gly Phe Asp His Ile Arg Leu Pro 65 70 75 80 Ile
Asp Glu Val His Phe Trp Asp Glu Asn Met Asn Arg His Gln Asp 85 90
95 Ala Phe Asp Leu Met His Asp Cys Ile Lys Trp Ser Glu Lys His Gly
100 105 110 Leu Arg Val Val Val Asp Leu His Ile Ile Arg Ser His Tyr
Phe Val 115 120 125 Gly Asp Asp Asn Thr Leu Trp Asp Glu Arg His Glu
Gln Glu Lys Phe 130 135 140 Val Asp Ile Trp Met Glu Leu Ser Ser Glu
Leu Ser Gln Tyr Ser Asn 145 150 155 160 Ser Leu Val Ala Tyr Glu Leu
Met Asn Glu Pro Val Ala Pro Ser His 165 170 175 Asp Asp Trp Asn Ser
Leu Val Ala Glu Thr Ile Glu Ala Ile Arg Lys 180 185 190 Val Glu Pro
Glu Arg Tyr Ile Val Val Gly Ser Asn Met Trp Gln Gly 195 200 205 Ile
Asp Thr Phe Glu Tyr Leu Glu Val Pro Glu Asn Asp Asp Arg Ile 210 215
220 Ile Leu Ser Phe His Phe Tyr Asp Pro Phe Ile Leu Thr His Tyr Thr
225 230 235 240 Ala Ser Trp Gly Tyr Leu Arg Asp Tyr Ser Gly Pro Val
Asn Tyr Pro 245 250 255 Gly Tyr Leu Val Thr Asn Asp Gln Leu Leu Asp
Met Ser Asn Glu Met 260 265 270 Gln Lys Leu Ile Arg Glu Phe Gln Thr
Asn Phe Asp Ile Tyr Thr Ile 275 280 285 Glu Glu Leu Ile Ser Ile Pro
Tyr Ser Ile Ala Lys Glu Lys Gly Leu 290
295 300 Lys Leu Tyr Cys Gly Glu Phe Gly Ala Ile Asp Gln Ala Pro Arg
Asp 305 310 315 320 Ala Arg Leu Ala Trp Tyr Arg Asp Val Val Gln Val
Phe Glu Arg Tyr 325 330 335 Gly Ile Ala His Ala Asn Trp Asn Tyr Lys
Asp Tyr Gly Thr Phe Gly 340 345 350 Ile Lys Asn Tyr Ser Glu Glu Ile
Asp Gln Glu Leu Phe Glu Ile Leu 355 360 365 Ile Gly Thr Lys His Lys
370 791017DNAUnknownObtained from environmental sample 79atgaaatata
aagctatttt tatatacctt attgttttga ttctatttta ctcaattaat 60atttatgcta
atgcagaaaa caaccccctc cccttcctca gtgtcgaagg aaacaggttc
120gtcgatgaag atggaaatac ggtaatcctg cgaggtgtat cgttccccga
tcccgaccgg 180ctggctgagg caactcaatg gaacaagcga tacttccagg
cggcaaaaga ctggaactgt 240aatgtcgtcc ggattcctgt ccatccacag
aaatggcggg aaagaggcga ggaaaattat 300ctgaaacttt tagataaggg
aattcaatgg gcgggtgaac tcgggatgta tgtaatcatc 360gactggcata
ccatcggtaa tccgataacc gaagtatttt tccgcgaact atacaatacg
420tcacgtgcgg agaccttcca gttctggaga acaatcgctg agcgctatgc
cggtaacccg 480gttgttgctt tctatgaact gttcaatgaa ccgaccgact
acaacggccg tctcggaaga 540atgaactggg atcagtataa agagtttatc
gaggagataa ttcacatcat ctattctcac 600gacgatacag ttatccctct
cgttgccggt ttcgactggg cgtatgaact ccgccatata 660aaagataaac
ctatagattt tcccggcatc gcttatgtga ctcaccccta tccccagaaa
720cgcgatccgc catgggaaga aaaatgggaa gaggatttcg ggtttgccgc
cgatatgtat 780ccggtgtttg caaccgagtt cggtttcatg ggggaggatg
aattaggtgc acacataccc 840gtcatcggcg atgaaacata cggcgaagcc
attatcgatt acttttataa aaaggggata 900tcgtggactg catgggtatt
cgatccgctt tggtcgccgc agcttattag agactggtat 960tttaccccgt
cccgacaggg gcagtttttt aaagagaagt tgatggagtt gaattag
101780338PRTUnknownObtained from environmental sample 80Met Lys Tyr
Lys Ala Ile Phe Ile Tyr Leu Ile Val Leu Ile Leu Phe 1 5 10 15 Tyr
Ser Ile Asn Ile Tyr Ala Asn Ala Glu Asn Asn Pro Leu Pro Phe 20 25
30 Leu Ser Val Glu Gly Asn Arg Phe Val Asp Glu Asp Gly Asn Thr Val
35 40 45 Ile Leu Arg Gly Val Ser Phe Pro Asp Pro Asp Arg Leu Ala
Glu Ala 50 55 60 Thr Gln Trp Asn Lys Arg Tyr Phe Gln Ala Ala Lys
Asp Trp Asn Cys 65 70 75 80 Asn Val Val Arg Ile Pro Val His Pro Gln
Lys Trp Arg Glu Arg Gly 85 90 95 Glu Glu Asn Tyr Leu Lys Leu Leu
Asp Lys Gly Ile Gln Trp Ala Gly 100 105 110 Glu Leu Gly Met Tyr Val
Ile Ile Asp Trp His Thr Ile Gly Asn Pro 115 120 125 Ile Thr Glu Val
Phe Phe Arg Glu Leu Tyr Asn Thr Ser Arg Ala Glu 130 135 140 Thr Phe
Gln Phe Trp Arg Thr Ile Ala Glu Arg Tyr Ala Gly Asn Pro 145 150 155
160 Val Val Ala Phe Tyr Glu Leu Phe Asn Glu Pro Thr Asp Tyr Asn Gly
165 170 175 Arg Leu Gly Arg Met Asn Trp Asp Gln Tyr Lys Glu Phe Ile
Glu Glu 180 185 190 Ile Ile His Ile Ile Tyr Ser His Asp Asp Thr Val
Ile Pro Leu Val 195 200 205 Ala Gly Phe Asp Trp Ala Tyr Glu Leu Arg
His Ile Lys Asp Lys Pro 210 215 220 Ile Asp Phe Pro Gly Ile Ala Tyr
Val Thr His Pro Tyr Pro Gln Lys 225 230 235 240 Arg Asp Pro Pro Trp
Glu Glu Lys Trp Glu Glu Asp Phe Gly Phe Ala 245 250 255 Ala Asp Met
Tyr Pro Val Phe Ala Thr Glu Phe Gly Phe Met Gly Glu 260 265 270 Asp
Glu Leu Gly Ala His Ile Pro Val Ile Gly Asp Glu Thr Tyr Gly 275 280
285 Glu Ala Ile Ile Asp Tyr Phe Tyr Lys Lys Gly Ile Ser Trp Thr Ala
290 295 300 Trp Val Phe Asp Pro Leu Trp Ser Pro Gln Leu Ile Arg Asp
Trp Tyr 305 310 315 320 Phe Thr Pro Ser Arg Gln Gly Gln Phe Phe Lys
Glu Lys Leu Met Glu 325 330 335 Leu Asn 811119DNAUnknownObtained
from environmental sample 81atgaatttac ttgctcaata cttttccgga
ctatttctga tttttttgat ctcaattttt 60ttcgttagtt ctgcagcgaa tcatcattat
gaaaaaaata cagtcaacga attttctgat 120gatgtaaatc aaacaacatt
agtccttcaa cccgggatat ccgaagccca gaatactcaa 180aacctgccgc
ggatttcggt tgaaggaaac caatttgtgg atgaatcggg aaacacagtc
240acatttcagg gtgtcagtgt tgccgatccg cacaggctta ataatgccgg
ccaatggaaa 300cgggaactgt ttgaagaaat cgcaaactgg ggagcaaacg
tcgttcgtct gcccatacac 360ccgctctggt ggcgggaacg gggagaggag
caatacctcg aatggattga tgaagccgtg 420gagtgggcca aagagctgga
gatgtacctc atcatcgact ggcacagtat cgggaacctg 480cggacagaac
tctttttcag ggatatctac aacaccaccc gccgtgaaac ttatgaattc
540tggaggctga tttcggatcg ctatgctgat gaaaccacaa ttgcctttta
cgaaatcttt 600aatgaaccca cacggcagca gggcaggctg ggaaccatga
cctggaagca atggaaggaa 660attctaaccg acattatcac aatcatttat
gcccacaatc ctgatgcgat tccgctggta 720gcaggtttta actgggcgta
tgaccttact ccggtccgcc actcacccct cgattttgaa 780ggtattgcct
atgttaccca cccatatccg caaaaaagaa gcaggccctg ggttccaaaa
840tgggaagaag atttcggttt tgtggctgac aaatatcctg tatttgccac
tgaattcggc 900tatatgaggg agtatgagcg gggcgctcat gtgcccgtaa
tcggggacga agaatatggg 960gaaatcctca tcaattattt ccgcgaaaaa
gggatttcgt ggacagcctg ggtattcgat 1020ccaagctggt cgccacagct
cattcaggat tgggattata cacccacacg ctcaggtgag 1080tttttcagaa
atgcgatgag aacgaaaaac aatgaataa 111982372PRTUnknownObtained from
environmental sample 82Met Asn Leu Leu Ala Gln Tyr Phe Ser Gly Leu
Phe Leu Ile Phe Leu 1 5 10 15 Ile Ser Ile Phe Phe Val Ser Ser Ala
Ala Asn His His Tyr Glu Lys 20 25 30 Asn Thr Val Asn Glu Phe Ser
Asp Asp Val Asn Gln Thr Thr Leu Val 35 40 45 Leu Gln Pro Gly Ile
Ser Glu Ala Gln Asn Thr Gln Asn Leu Pro Arg 50 55 60 Ile Ser Val
Glu Gly Asn Gln Phe Val Asp Glu Ser Gly Asn Thr Val 65 70 75 80 Thr
Phe Gln Gly Val Ser Val Ala Asp Pro His Arg Leu Asn Asn Ala 85 90
95 Gly Gln Trp Lys Arg Glu Leu Phe Glu Glu Ile Ala Asn Trp Gly Ala
100 105 110 Asn Val Val Arg Leu Pro Ile His Pro Leu Trp Trp Arg Glu
Arg Gly 115 120 125 Glu Glu Gln Tyr Leu Glu Trp Ile Asp Glu Ala Val
Glu Trp Ala Lys 130 135 140 Glu Leu Glu Met Tyr Leu Ile Ile Asp Trp
His Ser Ile Gly Asn Leu 145 150 155 160 Arg Thr Glu Leu Phe Phe Arg
Asp Ile Tyr Asn Thr Thr Arg Arg Glu 165 170 175 Thr Tyr Glu Phe Trp
Arg Leu Ile Ser Asp Arg Tyr Ala Asp Glu Thr 180 185 190 Thr Ile Ala
Phe Tyr Glu Ile Phe Asn Glu Pro Thr Arg Gln Gln Gly 195 200 205 Arg
Leu Gly Thr Met Thr Trp Lys Gln Trp Lys Glu Ile Leu Thr Asp 210 215
220 Ile Ile Thr Ile Ile Tyr Ala His Asn Pro Asp Ala Ile Pro Leu Val
225 230 235 240 Ala Gly Phe Asn Trp Ala Tyr Asp Leu Thr Pro Val Arg
His Ser Pro 245 250 255 Leu Asp Phe Glu Gly Ile Ala Tyr Val Thr His
Pro Tyr Pro Gln Lys 260 265 270 Arg Ser Arg Pro Trp Val Pro Lys Trp
Glu Glu Asp Phe Gly Phe Val 275 280 285 Ala Asp Lys Tyr Pro Val Phe
Ala Thr Glu Phe Gly Tyr Met Arg Glu 290 295 300 Tyr Glu Arg Gly Ala
His Val Pro Val Ile Gly Asp Glu Glu Tyr Gly 305 310 315 320 Glu Ile
Leu Ile Asn Tyr Phe Arg Glu Lys Gly Ile Ser Trp Thr Ala 325 330 335
Trp Val Phe Asp Pro Ser Trp Ser Pro Gln Leu Ile Gln Asp Trp Asp 340
345 350 Tyr Thr Pro Thr Arg Ser Gly Glu Phe Phe Arg Asn Ala Met Arg
Thr 355 360 365 Lys Asn Asn Glu 370 831089DNAUnknownObtained from
environmental sample 83atgagccttg gcctgactgc aatcgagttg atcaatcgcg
cccgcgccga tctgcgactg 60ggcgtgccga tcgttctgcg cgagggcgac gtgcaggcgc
tggtgctggc ggtcgagcca 120gtaaccgagg cgcggctggg tgggctgcgc
gggctggggc cagggctggt gcttgcaatc 180acgcagcgcc gcgccacgac
actgaaggcg cgcgcctatg atgaggatct tgcgcgagtg 240gtggtgcccg
agggggtagg ctgcgactgg ctgcgggcgg tggcggaccc ctccgacgat
300ctgcgctttc cgatgaaggg cccgctgatg accgctcgcg agggcacggc
cgcgctgcat 360cgcgctgcac ttcaactggt gaaatccgcg cagcttcttc
cggccgcact tgttcagccg 420cttgcggatc ccgaggcgct gcccgtcacg
gggctgacag tgctcgatat cgccgatgtc 480agccgtgaat tggcgcgcga
gacagtgttg tatccagtgg tgcatgcgcg cttgccgatg 540ctggcggcgc
aagcgggccg cgtgcatatc ttccgacccc gcgacggcgg cgttgagcat
600tacgccatcg agatcggcca gcccgaccgt gccgcgcccg tgctcacgcg
gctgcattcg 660gcctgtttca caggcgatgt gctgggctcg ctcaaatgcg
attgcggccc gcaactgcag 720gcagcactcg cgcagatggg cgaggaaggc
gcgggggtgc tgctctatct caatcaggag 780ggtcgcggca tcgggcttgc
caacaagatg cgcgcctatt cgctgcagga tcagggcttt 840gacacggtcg
aggccaatca ccgtctgggg ttcgaggatg acgagcggga tttccgcatc
900ggggccgcgc ttctgcggcg gatggggttc tctcgggcgc ggctgctgac
caacaaccct 960cggaaggtga acatgctgaa tgcgcatcgg gtcgaagtgg
tggaacgggt gccgcttcgg 1020gtgggcgaga cggtcgagaa ccgcgcctat
cttgccacca aggccgccaa atccgggcat 1080ctgttgtga
108984362PRTUnknownObtained from environmental sample 84Met Ser Leu
Gly Leu Thr Ala Ile Glu Leu Ile Asn Arg Ala Arg Ala 1 5 10 15 Asp
Leu Arg Leu Gly Val Pro Ile Val Leu Arg Glu Gly Asp Val Gln 20 25
30 Ala Leu Val Leu Ala Val Glu Pro Val Thr Glu Ala Arg Leu Gly Gly
35 40 45 Leu Arg Gly Leu Gly Pro Gly Leu Val Leu Ala Ile Thr Gln
Arg Arg 50 55 60 Ala Thr Thr Leu Lys Ala Arg Ala Tyr Asp Glu Asp
Leu Ala Arg Val 65 70 75 80 Val Val Pro Glu Gly Val Gly Cys Asp Trp
Leu Arg Ala Val Ala Asp 85 90 95 Pro Ser Asp Asp Leu Arg Phe Pro
Met Lys Gly Pro Leu Met Thr Ala 100 105 110 Arg Glu Gly Thr Ala Ala
Leu His Arg Ala Ala Leu Gln Leu Val Lys 115 120 125 Ser Ala Gln Leu
Leu Pro Ala Ala Leu Val Gln Pro Leu Ala Asp Pro 130 135 140 Glu Ala
Leu Pro Val Thr Gly Leu Thr Val Leu Asp Ile Ala Asp Val 145 150 155
160 Ser Arg Glu Leu Ala Arg Glu Thr Val Leu Tyr Pro Val Val His Ala
165 170 175 Arg Leu Pro Met Leu Ala Ala Gln Ala Gly Arg Val His Ile
Phe Arg 180 185 190 Pro Arg Asp Gly Gly Val Glu His Tyr Ala Ile Glu
Ile Gly Gln Pro 195 200 205 Asp Arg Ala Ala Pro Val Leu Thr Arg Leu
His Ser Ala Cys Phe Thr 210 215 220 Gly Asp Val Leu Gly Ser Leu Lys
Cys Asp Cys Gly Pro Gln Leu Gln 225 230 235 240 Ala Ala Leu Ala Gln
Met Gly Glu Glu Gly Ala Gly Val Leu Leu Tyr 245 250 255 Leu Asn Gln
Glu Gly Arg Gly Ile Gly Leu Ala Asn Lys Met Arg Ala 260 265 270 Tyr
Ser Leu Gln Asp Gln Gly Phe Asp Thr Val Glu Ala Asn His Arg 275 280
285 Leu Gly Phe Glu Asp Asp Glu Arg Asp Phe Arg Ile Gly Ala Ala Leu
290 295 300 Leu Arg Arg Met Gly Phe Ser Arg Ala Arg Leu Leu Thr Asn
Asn Pro 305 310 315 320 Arg Lys Val Asn Met Leu Asn Ala His Arg Val
Glu Val Val Glu Arg 325 330 335 Val Pro Leu Arg Val Gly Glu Thr Val
Glu Asn Arg Ala Tyr Leu Ala 340 345 350 Thr Lys Ala Ala Lys Ser Gly
His Leu Leu 355 360 851284DNAUnknownObtained from environmental
sample 85gtgaacaccg cgcatcgcat cgaattccct cggcaattta tcttcggttc
cgccactgct 60gctcaccaag tggagggcaa caacgttcac aatgattggt gggcccacga
gcatgccacc 120gacacgaatg ccgtggagcc gtcgggcctc gcctgcgacc
actttcggcg ctttgccgac 180gacttccgcc tcttacgcca actcggacag
ccagcgcacc gcctgtcgct ggaatggagc 240cgcatcgaac cggcacccgg
tgaaatcgat cgttcggcat tgtcccacta ccgccgagtc 300ctgggtactt
tgcgagacct cggaatcgag ccatgggtca ccatccacca cttcacttgc
360cctcgctggt tcgtggaaca gggagggttt acacgcatgg attcagcgcg
ctctctcgtt 420cgccataccg aacgcgtggc gagggagttc tccgacctag
tcacaaactg gtgcaccata 480aatgagccaa acgtcgtggc agaactcggt
tatcgcttcg gatactttcc gccgcggttg 540caggacgatg agctggcagc
ggaagtgctc accaacttct ttcgcttaca cgctgaaatg 600gcagaagttt
tgcgcgctca cgcgcagaga tcggcgcaaa tcggtatcac ccttgcgatg
660caagcacacg agccgctgcg catcgaaagc gaagcggacc gcgcactggc
ggcgcggcgc 720gacgccgaga ccaacggcgt catgctcaac gccttgcgaa
ccggtgtatt cgcctacccg 780ggacgggagc cggtggaaat ccctggactg
aaaacgtcat cgaccttcgt gggggtccag 840tactattcgc gggtccgcta
cgacgccgag tcgcaaggtc cagcaatgcc cgacttcgag 900cgcaccctca
gccaaatggg atgggaggtg tatcctgagg ggttcggccc cttgctcgag
960cgcgcagcag aaactggact cgaagtgatc gtcacagaga acgggatggc
gcacgacgat 1020gaccgtgtgc gcgtgcgttt tatcgccgac cacttgcggg
tcgttcaccg ccttctggaa 1080cgcggtgtgc gcatcggagg gtacttttac
tggtcgacca tggacaactt cgaatggaac 1140ttcgggtacg gaccgaagtt
cggcctgatc gaagtggacc gttctaccct ggaacgcagg 1200ccgcggcgaa
gcgcgtattt cttccgtgac atgatccagc agcgagtgct cgacgacgac
1260ctggtcgagc actggactcg ctga 128486427PRTUnknownObtained from
environmental sample 86Met Asn Thr Ala His Arg Ile Glu Phe Pro Arg
Gln Phe Ile Phe Gly 1 5 10 15 Ser Ala Thr Ala Ala His Gln Val Glu
Gly Asn Asn Val His Asn Asp 20 25 30 Trp Trp Ala His Glu His Ala
Thr Asp Thr Asn Ala Val Glu Pro Ser 35 40 45 Gly Leu Ala Cys Asp
His Phe Arg Arg Phe Ala Asp Asp Phe Arg Leu 50 55 60 Leu Arg Gln
Leu Gly Gln Pro Ala His Arg Leu Ser Leu Glu Trp Ser 65 70 75 80 Arg
Ile Glu Pro Ala Pro Gly Glu Ile Asp Arg Ser Ala Leu Ser His 85 90
95 Tyr Arg Arg Val Leu Gly Thr Leu Arg Asp Leu Gly Ile Glu Pro Trp
100 105 110 Val Thr Ile His His Phe Thr Cys Pro Arg Trp Phe Val Glu
Gln Gly 115 120 125 Gly Phe Thr Arg Met Asp Ser Ala Arg Ser Leu Val
Arg His Thr Glu 130 135 140 Arg Val Ala Arg Glu Phe Ser Asp Leu Val
Thr Asn Trp Cys Thr Ile 145 150 155 160 Asn Glu Pro Asn Val Val Ala
Glu Leu Gly Tyr Arg Phe Gly Tyr Phe 165 170 175 Pro Pro Arg Leu Gln
Asp Asp Glu Leu Ala Ala Glu Val Leu Thr Asn 180 185 190 Phe Phe Arg
Leu His Ala Glu Met Ala Glu Val Leu Arg Ala His Ala 195 200 205 Gln
Arg Ser Ala Gln Ile Gly Ile Thr Leu Ala Met Gln Ala His Glu 210 215
220 Pro Leu Arg Ile Glu Ser Glu Ala Asp Arg Ala Leu Ala Ala Arg Arg
225 230 235 240 Asp Ala Glu Thr Asn Gly Val Met Leu Asn Ala Leu Arg
Thr Gly Val 245 250 255 Phe Ala Tyr Pro Gly Arg Glu Pro Val Glu Ile
Pro Gly Leu Lys Thr 260 265 270 Ser Ser Thr Phe Val Gly Val Gln Tyr
Tyr Ser Arg Val Arg Tyr Asp 275 280 285 Ala Glu Ser Gln Gly Pro Ala
Met Pro Asp Phe Glu Arg Thr Leu Ser 290 295 300 Gln Met Gly Trp Glu
Val Tyr Pro Glu Gly Phe Gly Pro Leu Leu Glu 305 310 315 320 Arg Ala
Ala Glu Thr Gly Leu Glu Val Ile Val Thr Glu Asn Gly Met 325 330 335
Ala His Asp Asp Asp Arg Val Arg Val Arg Phe Ile Ala Asp His Leu 340
345 350 Arg Val Val His Arg Leu Leu Glu Arg Gly Val Arg Ile Gly Gly
Tyr 355 360 365 Phe Tyr Trp Ser Thr Met Asp Asn Phe Glu Trp Asn Phe
Gly Tyr Gly 370 375 380 Pro Lys Phe Gly Leu Ile Glu Val
Asp Arg Ser Thr Leu Glu Arg Arg 385 390 395 400 Pro Arg Arg Ser Ala
Tyr Phe Phe Arg Asp Met Ile Gln Gln Arg Val 405 410 415 Leu Asp Asp
Asp Leu Val Glu His Trp Thr Arg 420 425 871167DNAUnknownObtained
from environmental sample 87atgagaaaga gtgtgttcac cctcgccgtg
tttttgtcgg cactgtttgc attcacgtct 60tgtcagaaca agagccagaa cgaggctcaa
gaccaggcag gacaagtcaa taacttccgc 120atcaagcgcg gcacgaacat
cagccactgg ctgtcgcagt cggagcagcg cggtgaggct 180cgcagactgc
atatccagga ggacgacttc gcccgtctgg aagagctggg cttcgacttc
240gtgcgcatcc ccatcgacga ggtgcagttc tgggacgagc agggcaacaa
gctgcccgag 300gcgtgggatc tgctgaacaa cgccctcgac tggagcaaga
agcacaacct gcgtgccatc 360gtcgacctgc acatcatccg tgcgcactat
ttcaatgccg tgaatgaggc agaccaggcc 420gccaataccc tcttcacctc
tgaggaggca caggaaggac tccttaacct gtggcgccag 480ctctccgagt
tcctgaagga ccgcagcaac gactgggtgg cctacgagtt catgaacgag
540ccggtagccc ctgagcacga gatgtggaac cagctggtag ccaaggtaca
caaggccctg 600cgcgaactgg aaccccagcg tacactcgtc gtcggctcga
acatgtggca gggacacgag 660acgatgaagt atctgaaagt gcccgagggc
gataagaaca tcatcctctc gttccactac 720tacaacccga tgctgctgac
gcactacggt gcctggtggt cgccgctgtg tgctgcctac 780aagggtaagg
tgaactatcc cggtgtgctc gtgtcgaagg aagactacga tgccgctcct
840gctgccatca aggatcagct gaagcccttt accgaggaag tatggaacat
cgacaagatc 900cgtgagcagt tcaaggatgc catcgaggcc gccaagaaat
atgacctgca actgttctgc 960ggcgagtggg gtgtctatga gcccgtggac
cgtgagctgg cctacaaatg gtatcgtgac 1020gtgctgacgg tgttcgacga
gttcaacatc gcctggacga cctggtgcta cgatgctgac 1080ttcggtttct
gggatcagca gcgccactgc tacaaagact atccgctggt ggagctcctg
1140atgtcaggaa agaaactggg agaatag 116788388PRTUnknownObtained from
environmental sample 88Met Arg Lys Ser Val Phe Thr Leu Ala Val Phe
Leu Ser Ala Leu Phe 1 5 10 15 Ala Phe Thr Ser Cys Gln Asn Lys Ser
Gln Asn Glu Ala Gln Asp Gln 20 25 30 Ala Gly Gln Val Asn Asn Phe
Arg Ile Lys Arg Gly Thr Asn Ile Ser 35 40 45 His Trp Leu Ser Gln
Ser Glu Gln Arg Gly Glu Ala Arg Arg Leu His 50 55 60 Ile Gln Glu
Asp Asp Phe Ala Arg Leu Glu Glu Leu Gly Phe Asp Phe 65 70 75 80 Val
Arg Ile Pro Ile Asp Glu Val Gln Phe Trp Asp Glu Gln Gly Asn 85 90
95 Lys Leu Pro Glu Ala Trp Asp Leu Leu Asn Asn Ala Leu Asp Trp Ser
100 105 110 Lys Lys His Asn Leu Arg Ala Ile Val Asp Leu His Ile Ile
Arg Ala 115 120 125 His Tyr Phe Asn Ala Val Asn Glu Ala Asp Gln Ala
Ala Asn Thr Leu 130 135 140 Phe Thr Ser Glu Glu Ala Gln Glu Gly Leu
Leu Asn Leu Trp Arg Gln 145 150 155 160 Leu Ser Glu Phe Leu Lys Asp
Arg Ser Asn Asp Trp Val Ala Tyr Glu 165 170 175 Phe Met Asn Glu Pro
Val Ala Pro Glu His Glu Met Trp Asn Gln Leu 180 185 190 Val Ala Lys
Val His Lys Ala Leu Arg Glu Leu Glu Pro Gln Arg Thr 195 200 205 Leu
Val Val Gly Ser Asn Met Trp Gln Gly His Glu Thr Met Lys Tyr 210 215
220 Leu Lys Val Pro Glu Gly Asp Lys Asn Ile Ile Leu Ser Phe His Tyr
225 230 235 240 Tyr Asn Pro Met Leu Leu Thr His Tyr Gly Ala Trp Trp
Ser Pro Leu 245 250 255 Cys Ala Ala Tyr Lys Gly Lys Val Asn Tyr Pro
Gly Val Leu Val Ser 260 265 270 Lys Glu Asp Tyr Asp Ala Ala Pro Ala
Ala Ile Lys Asp Gln Leu Lys 275 280 285 Pro Phe Thr Glu Glu Val Trp
Asn Ile Asp Lys Ile Arg Glu Gln Phe 290 295 300 Lys Asp Ala Ile Glu
Ala Ala Lys Lys Tyr Asp Leu Gln Leu Phe Cys 305 310 315 320 Gly Glu
Trp Gly Val Tyr Glu Pro Val Asp Arg Glu Leu Ala Tyr Lys 325 330 335
Trp Tyr Arg Asp Val Leu Thr Val Phe Asp Glu Phe Asn Ile Ala Trp 340
345 350 Thr Thr Trp Cys Tyr Asp Ala Asp Phe Gly Phe Trp Asp Gln Gln
Arg 355 360 365 His Cys Tyr Lys Asp Tyr Pro Leu Val Glu Leu Leu Met
Ser Gly Lys 370 375 380 Lys Leu Gly Glu 385
891500DNAUnknownObtained from environmental sample 89atgaaacgtt
cagtctctat ctttatcgca tgtttattaa tgacagtatt aacaattagc 60ggtgtcgcgg
caccagaagc atctgcagca ggggcgaaaa cgcctgtagc ccttaatggc
120cagcttagca ttaaaggtac tcagctagtc aatcaaaacg gaaaaccggt
gcagctgaag 180gggatcagct cacacggttt gcagtggttc ggcgattatg
tcaataaaga cactttaaaa 240tggctaagag acgattgggg aattaccgtc
ttccgggcgg caatgtacac ggctgacggc 300ggttatatcg agaatccgtc
tgtgaaaaat aaagtcaaag aagctgttga agcggcaaaa 360gagctcggga
tatatgtcat cattgactgg catattttaa atgacggcaa tccaaatcaa
420aataaagaga aggcgaagga attctttaag gaaatgtcaa gcctttacgg
aagctcacca 480aacgttatat atgaaattgc taatgaaccg aacggtgatg
taaattggaa gcgcgatatc 540aaaccgtatg cggaagaagt gatttctgtt
atccgtaaaa atgacccgga taacatcatt 600attaccggaa caggcacttg
gagccaggat gtcaacgatg ctgcggatga tcagcttaag 660gatgcaaacg
tcatgtacgc gcttcatttt tatgccggta cacacggcca gtttttaagg
720gataaagcgg actatgcgct cagcaaagga gctccgattt ttgtaacgga
atgggggacg 780agtgacgctt ccggaaatgg aggggtatac cttgaccagt
cgagggaatg gctgaattat 840ctcgacagca agaaaatcag ctgggtaaac
tggaaccttt ctgataagca ggaatcatcc 900tcagctttaa agccgggggc
atctaaaaca ggcggctggc cgttatcaga tttatccgct 960tcagggacat
ttgtaagaga aaacattcgc ggctcccaaa attcgagtga agacagatct
1020gagacaccaa agcaagagaa acccgcacag gaaaacagca tctctgtgca
atacagaaca 1080ggggatggaa gtgtgaacag caaccaaatc cgtcctcaga
tcaatgtgaa aaacaacagc 1140aagaccaccg ttaacttaaa aaatgtaact
gtccgctact ggtataacac gaaaaacaaa 1200ggccaaaact tcgactgtga
ttacgcgaag atcggatgca gcaatgtgac gcacaagttt 1260gtgacattac
ataaacctgt aaaaggtgca gatgcctatc tggaacttgg gtttagaaac
1320gggacgctgt caccgggagc aagcaccgga gaaattcaaa ttcgtcttca
caatgaggac 1380tggagcaatt attcacaagc cggggattat tcttttttcc
agtcgaatac gtttaaagat 1440acaaaaaaaa tcacattata taataacgga
aaactgattt ggggaacaga acccaaatag 150090499PRTUnknownObtained from
environmental sample 90Met Lys Arg Ser Val Ser Ile Phe Ile Ala Cys
Leu Leu Met Thr Val 1 5 10 15 Leu Thr Ile Ser Gly Val Ala Ala Pro
Glu Ala Ser Ala Ala Gly Ala 20 25 30 Lys Thr Pro Val Ala Leu Asn
Gly Gln Leu Ser Ile Lys Gly Thr Gln 35 40 45 Leu Val Asn Gln Asn
Gly Lys Pro Val Gln Leu Lys Gly Ile Ser Ser 50 55 60 His Gly Leu
Gln Trp Phe Gly Asp Tyr Val Asn Lys Asp Thr Leu Lys 65 70 75 80 Trp
Leu Arg Asp Asp Trp Gly Ile Thr Val Phe Arg Ala Ala Met Tyr 85 90
95 Thr Ala Asp Gly Gly Tyr Ile Glu Asn Pro Ser Val Lys Asn Lys Val
100 105 110 Lys Glu Ala Val Glu Ala Ala Lys Glu Leu Gly Ile Tyr Val
Ile Ile 115 120 125 Asp Trp His Ile Leu Asn Asp Gly Asn Pro Asn Gln
Asn Lys Glu Lys 130 135 140 Ala Lys Glu Phe Phe Lys Glu Met Ser Ser
Leu Tyr Gly Ser Ser Pro 145 150 155 160 Asn Val Ile Tyr Glu Ile Ala
Asn Glu Pro Asn Gly Asp Val Asn Trp 165 170 175 Lys Arg Asp Ile Lys
Pro Tyr Ala Glu Glu Val Ile Ser Val Ile Arg 180 185 190 Lys Asn Asp
Pro Asp Asn Ile Ile Ile Thr Gly Thr Gly Thr Trp Ser 195 200 205 Gln
Asp Val Asn Asp Ala Ala Asp Asp Gln Leu Lys Asp Ala Asn Val 210 215
220 Met Tyr Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Phe Leu Arg
225 230 235 240 Asp Lys Ala Asp Tyr Ala Leu Ser Lys Gly Ala Pro Ile
Phe Val Thr 245 250 255 Glu Trp Gly Thr Ser Asp Ala Ser Gly Asn Gly
Gly Val Tyr Leu Asp 260 265 270 Gln Ser Arg Glu Trp Leu Asn Tyr Leu
Asp Ser Lys Lys Ile Ser Trp 275 280 285 Val Asn Trp Asn Leu Ser Asp
Lys Gln Glu Ser Ser Ser Ala Leu Lys 290 295 300 Pro Gly Ala Ser Lys
Thr Gly Gly Trp Pro Leu Ser Asp Leu Ser Ala 305 310 315 320 Ser Gly
Thr Phe Val Arg Glu Asn Ile Arg Gly Ser Gln Asn Ser Ser 325 330 335
Glu Asp Arg Ser Glu Thr Pro Lys Gln Glu Lys Pro Ala Gln Glu Asn 340
345 350 Ser Ile Ser Val Gln Tyr Arg Thr Gly Asp Gly Ser Val Asn Ser
Asn 355 360 365 Gln Ile Arg Pro Gln Ile Asn Val Lys Asn Asn Ser Lys
Thr Thr Val 370 375 380 Asn Leu Lys Asn Val Thr Val Arg Tyr Trp Tyr
Asn Thr Lys Asn Lys 385 390 395 400 Gly Gln Asn Phe Asp Cys Asp Tyr
Ala Lys Ile Gly Cys Ser Asn Val 405 410 415 Thr His Lys Phe Val Thr
Leu His Lys Pro Val Lys Gly Ala Asp Ala 420 425 430 Tyr Leu Glu Leu
Gly Phe Arg Asn Gly Thr Leu Ser Pro Gly Ala Ser 435 440 445 Thr Gly
Glu Ile Gln Ile Arg Leu His Asn Glu Asp Trp Ser Asn Tyr 450 455 460
Ser Gln Ala Gly Asp Tyr Ser Phe Phe Gln Ser Asn Thr Phe Lys Asp 465
470 475 480 Thr Lys Lys Ile Thr Leu Tyr Asn Asn Gly Lys Leu Ile Trp
Gly Thr 485 490 495 Glu Pro Lys 911725DNAUnknownObtained from
environmental sample 91atgctgaaat taagtgataa cctaactttc ttgaaaagca
aaccattttt tcttaatgaa 60aaagaaatga agtgggtgga gaaaacactt caatccatgt
ccttacatga aaaagtaggg 120caattatttt gtcccattgg cggttcagat
aataaacaag aattagaagc ctttattaag 180gaatatcatc ctggcggcat
catgtaccgt cctaatacag gagcaaaaat acaggaaaca 240catcggttgt
tacaagagct atccccggta cctttattaa tttctgctaa cttagaggcc
300ggtggtaatg ggattgctac ggatggtact tacttcggaa agcaaatgca
ggtggctgca 360acagataatg aagaaatggc ctataaatta ggattagttg
ctggccgtga aggccgtgtg 420gccggttgta actgggcttt tgcaccaatt
gttgatattg atatgaacta tcgaaaccca 480attacaaacg taagaacgta
tgggtctgac ccaattagag ttgcccaaat gtctaaagct 540tttatgaagg
gaattcatga aagcggactc gcagcagctg ttaagcattt cccaggggat
600ggagtggatg atagagatca gcatctttta tcatctgtaa acaccttatc
taccgaagaa 660tgggatcaaa cctttgggat ggtttatcaa gaaatgatag
acagtggggc aaaatcgatt 720atggcgggcc atatcatgct ccctgaatat
tcaagagaac tattgccggg tattgaagac 780gaacaaatca tgcccgccac
actagcacca gagttactta atggtttatt aagggaaaag 840ttaggtttta
atggtttaat cgtgactgat gcatccccta tgttagggtt cactacttcg
900gaaagaagag aaattgctgt tcctaaggcg attgcttcgg gctgtgatat
gtttctcttc 960aaccgtaaca taaaagaaga ttatgagttc atgctgaatg
gaattgaaac tggaattcta 1020accttggaaa gagtagatga agctgttact
agagtacttg ctcttaaagc atctctaggt 1080ctgaatgtac aaaaggaatt
gggaatatta gtacctgaag aagcggaatt gtcggtatta 1140caatctgaag
aacatttgga ttgggcaaga gaatgtgcag accaatcggt tacattagta
1200aaggatacac aaaaactgct gcctattagt gctgatcagt ataaacgggt
tcgactttat 1260gtattgggtg atcaagaagg agggctaaag gaaggcggct
ccgtcactca accgtttatc 1320gattctctta aaaatgctgg ctttgaagta
gatttatata atgacaagca agttaatttc 1380caagaactgt ttatgagtgt
aaacgagttt aaaaagaact atgatctgat catttatgtc 1440gccaaccttg
aaaccgctag taaccaaacg acagtcagaa ttaattggca gcagccgcta
1500aatgccaacg ctccatggtt tgttaaagat ataccgacat tatttatttc
ggttgctaac 1560ccataccatc tacaggacgt accaatggtt aagacctata
taaatgctta ttcatctaat 1620gaatatgtgg tagaagcaat tgtagataaa
atcttaggaa aatcagagtt taaagggaag 1680aatcccgtcg atccgttttg
tgggaaatgg gataccagac tttaa 172592574PRTUnknownObtained from
environmental sample 92Met Leu Lys Leu Ser Asp Asn Leu Thr Phe Leu
Lys Ser Lys Pro Phe 1 5 10 15 Phe Leu Asn Glu Lys Glu Met Lys Trp
Val Glu Lys Thr Leu Gln Ser 20 25 30 Met Ser Leu His Glu Lys Val
Gly Gln Leu Phe Cys Pro Ile Gly Gly 35 40 45 Ser Asp Asn Lys Gln
Glu Leu Glu Ala Phe Ile Lys Glu Tyr His Pro 50 55 60 Gly Gly Ile
Met Tyr Arg Pro Asn Thr Gly Ala Lys Ile Gln Glu Thr 65 70 75 80 His
Arg Leu Leu Gln Glu Leu Ser Pro Val Pro Leu Leu Ile Ser Ala 85 90
95 Asn Leu Glu Ala Gly Gly Asn Gly Ile Ala Thr Asp Gly Thr Tyr Phe
100 105 110 Gly Lys Gln Met Gln Val Ala Ala Thr Asp Asn Glu Glu Met
Ala Tyr 115 120 125 Lys Leu Gly Leu Val Ala Gly Arg Glu Gly Arg Val
Ala Gly Cys Asn 130 135 140 Trp Ala Phe Ala Pro Ile Val Asp Ile Asp
Met Asn Tyr Arg Asn Pro 145 150 155 160 Ile Thr Asn Val Arg Thr Tyr
Gly Ser Asp Pro Ile Arg Val Ala Gln 165 170 175 Met Ser Lys Ala Phe
Met Lys Gly Ile His Glu Ser Gly Leu Ala Ala 180 185 190 Ala Val Lys
His Phe Pro Gly Asp Gly Val Asp Asp Arg Asp Gln His 195 200 205 Leu
Leu Ser Ser Val Asn Thr Leu Ser Thr Glu Glu Trp Asp Gln Thr 210 215
220 Phe Gly Met Val Tyr Gln Glu Met Ile Asp Ser Gly Ala Lys Ser Ile
225 230 235 240 Met Ala Gly His Ile Met Leu Pro Glu Tyr Ser Arg Glu
Leu Leu Pro 245 250 255 Gly Ile Glu Asp Glu Gln Ile Met Pro Ala Thr
Leu Ala Pro Glu Leu 260 265 270 Leu Asn Gly Leu Leu Arg Glu Lys Leu
Gly Phe Asn Gly Leu Ile Val 275 280 285 Thr Asp Ala Ser Pro Met Leu
Gly Phe Thr Thr Ser Glu Arg Arg Glu 290 295 300 Ile Ala Val Pro Lys
Ala Ile Ala Ser Gly Cys Asp Met Phe Leu Phe 305 310 315 320 Asn Arg
Asn Ile Lys Glu Asp Tyr Glu Phe Met Leu Asn Gly Ile Glu 325 330 335
Thr Gly Ile Leu Thr Leu Glu Arg Val Asp Glu Ala Val Thr Arg Val 340
345 350 Leu Ala Leu Lys Ala Ser Leu Gly Leu Asn Val Gln Lys Glu Leu
Gly 355 360 365 Ile Leu Val Pro Glu Glu Ala Glu Leu Ser Val Leu Gln
Ser Glu Glu 370 375 380 His Leu Asp Trp Ala Arg Glu Cys Ala Asp Gln
Ser Val Thr Leu Val 385 390 395 400 Lys Asp Thr Gln Lys Leu Leu Pro
Ile Ser Ala Asp Gln Tyr Lys Arg 405 410 415 Val Arg Leu Tyr Val Leu
Gly Asp Gln Glu Gly Gly Leu Lys Glu Gly 420 425 430 Gly Ser Val Thr
Gln Pro Phe Ile Asp Ser Leu Lys Asn Ala Gly Phe 435 440 445 Glu Val
Asp Leu Tyr Asn Asp Lys Gln Val Asn Phe Gln Glu Leu Phe 450 455 460
Met Ser Val Asn Glu Phe Lys Lys Asn Tyr Asp Leu Ile Ile Tyr Val 465
470 475 480 Ala Asn Leu Glu Thr Ala Ser Asn Gln Thr Thr Val Arg Ile
Asn Trp 485 490 495 Gln Gln Pro Leu Asn Ala Asn Ala Pro Trp Phe Val
Lys Asp Ile Pro 500 505 510 Thr Leu Phe Ile Ser Val Ala Asn Pro Tyr
His Leu Gln Asp Val Pro 515 520 525 Met Val Lys Thr Tyr Ile Asn Ala
Tyr Ser Ser Asn Glu Tyr Val Val 530 535 540 Glu Ala Ile Val Asp Lys
Ile Leu Gly Lys Ser Glu Phe Lys Gly Lys 545 550 555 560 Asn Pro Val
Asp Pro Phe Cys Gly Lys Trp Asp Thr Arg Leu 565 570
93546DNAUnknownObtained from environmental sample 93atgagaataa
aaaatttaaa aacgaaccgt atcacaaacc cgctgggatt tgatatagga 60aaaccacgta
tatcttttgt cacttatgac actacggcta aaaagcaaac agcagcgcaa
120atacaggttg cgctagatca agagtttacg aacctaacat ttgacagtgg
gaaaagcacg 180gagatagata gtctagcata cgaactgcca tttcaattag
agtcttacac tcgctactac 240tggcgtgtga ccgtttgggc ggataatggg
gatgtggcca caagtgaaat tgcttggttt 300gaaacagcca aactaggcga
ttcttgggag gccaagtgga ttacccccga ttttgataag 360gaaatccatc
ccgtactatc aagggaattt gatttgtcaa aagaagtcgt ttctgcccgt
420gcctatgttt gcggtttggg attatatgaa atggagatta atggtctaaa
ggctggggat
480gaatatctga cccctaattt caacgcctat gataaatggc tgcagtacca
gacctatgat 540attaca 54694182PRTUnknownObtained from environmental
sample 94Met Arg Ile Lys Asn Leu Lys Thr Asn Arg Ile Thr Asn Pro
Leu Gly 1 5 10 15 Phe Asp Ile Gly Lys Pro Arg Ile Ser Phe Val Thr
Tyr Asp Thr Thr 20 25 30 Ala Lys Lys Gln Thr Ala Ala Gln Ile Gln
Val Ala Leu Asp Gln Glu 35 40 45 Phe Thr Asn Leu Thr Phe Asp Ser
Gly Lys Ser Thr Glu Ile Asp Ser 50 55 60 Leu Ala Tyr Glu Leu Pro
Phe Gln Leu Glu Ser Tyr Thr Arg Tyr Tyr 65 70 75 80 Trp Arg Val Thr
Val Trp Ala Asp Asn Gly Asp Val Ala Thr Ser Glu 85 90 95 Ile Ala
Trp Phe Glu Thr Ala Lys Leu Gly Asp Ser Trp Glu Ala Lys 100 105 110
Trp Ile Thr Pro Asp Phe Asp Lys Glu Ile His Pro Val Leu Ser Arg 115
120 125 Glu Phe Asp Leu Ser Lys Glu Val Val Ser Ala Arg Ala Tyr Val
Cys 130 135 140 Gly Leu Gly Leu Tyr Glu Met Glu Ile Asn Gly Leu Lys
Ala Gly Asp 145 150 155 160 Glu Tyr Leu Thr Pro Asn Phe Asn Ala Tyr
Asp Lys Trp Leu Gln Tyr 165 170 175 Gln Thr Tyr Asp Ile Thr 180
952298DNAUnknownObtained from environmental sample 95atgatcaatc
aagatataaa acaattaatc tcacaaatga ccttggaaga aaaagctggt 60ctttgttctg
gattagattt ttggaattta aaaggtatcg aaagactggg aataccctcg
120ataatggtaa ccgatggtcc gcatggactc cgtaaacaaa aaatgggagc
agatcattta 180gggctgtttg acagtattcc tgcgacatgt ttcccatctg
cagccggttt agctagtact 240tggaataaag agttaatata tgaagttggg
gttgcattag gaaaggaatg ccaggcagag 300gatgtggcaa tacttcttgg
ccctggagca aacattaagc gctcacccct ttgtggcaga 360aactttgaat
atttttcgga agatccattc ctttcatcag aaatggctgc gtcccatatc
420aagggtgttc aaagtgaggg ggttgggaca tcacttaagc acttcgctgc
aaataatcaa 480gaacaccgaa gaatgtcgac agatgctatt gtggatgaaa
ggacgttgcg agaaatatat 540ttggccagct ttgaaaacgc tgtaaagaaa
gcgcagccat ggactgtgat gtgcgcctac 600aacaaggtca atggagactt
tgcatcagaa aataaaacat tgttaactga catcctgcga 660gatgagtggg
gctttgaagg aattgttgtt tctgactggg gggcggttaa tgaacctgtt
720gacggattaa atgccgggtt agacctggaa atgccttcaa gtagtgggat
tggtgaaaag 780aaaatcatca atgctgtaag aaatggtcag cttttagaag
ataaactaga tcaggcagtt 840gaaagaattc tacgtattat cttaatggca
gtagaaaaca agaaagaaac cgctgactat 900gataaagaac aacatcataa
gcttgcaaga aaagcagcaa gtgaaagtat ggttttatta 960aagaatgaag
ataatatcct gccgttaaag aaagaaggaa ccatttcgat tattggttca
1020tttgccaaaa aaccaaggta tcaaggcggt ggaagctcac acattaaccc
gacaaagctt 1080gaaaatatct atgaagaaat agagaaaaca gcgggccaaa
atgtgaacgt tttatacgcg 1140gaaggatatc atcttgaaaa ggatttaatc
gatgatcaat taattgaaga ggcaaaaaaa 1200acggcagcaa aatccgatgt
aaccgtattg tttgtaggtc ttcctgaccg atatgaatct 1260gaaggatatg
atagagagca cctgaatata ccggagaatc accgtctttt agtcgaagcg
1320gttgcggaag tacaaaagaa tatagttgtt gtactaagta atggggcacc
gcttgttatg 1380ccatggcttg ataaggtgaa ggggctgctg gaaagttacc
tgggaggtca ggcactagga 1440ggtgcgattg cagacatcct attcggagaa
gttaatccaa gtggaaagct tgccgaaact 1500tttcccgtaa aattaggtga
caatccttct tatctcaact ttccaggaga gagggataaa 1560gttgagtata
aagaaggcat ctttgttggt tatcgttatt acgatacaaa acagattgag
1620ccgctgtttc catttggata tggtttaagc tatacaaact ttgaatataa
aaaccttgta 1680attgataaaa aagaaataaa agatacagaa attgtcacag
ttaccgtgaa tgtgaaaaat 1740acaggaaaag tgcctgggaa agaaatcatc
cagttatatg taaaagatat aaaaagcagt 1800gtagttcgtc ctgaaaaaga
gttaaaaggc tttggaaagg tttccttaca gcctggggaa 1860gacaaaacta
tttcctttaa attggataaa cgcgcatttg catattacaa cacggaattg
1920aaggattggt atgtagaatc aggagaattt gaaattttgg tggggaaatc
gtccagagaa 1980attgaactaa cagaaaaaat tatggttcac tctacttccc
cagttttctt ggaggttcac 2040cgaaattcca cggtcggaga tcttttaact
gatccaattc taggtgaaaa agctaatgct 2100ctaattagag agctaacaaa
aggaagtcca ttatttgatg ctgggtcaga tcacggagag 2160ggtgcagaaa
tgatggaagc gatgttaaaa tacatgcctt tgcgtgctct tatgaatttt
2220agtggtggag acattaccga agagaaacta actgaattta ttaaggaact
taattcaact 2280aattttgtaa gcctttaa 229896765PRTUnknownObtained from
environmental sample 96Met Ile Asn Gln Asp Ile Lys Gln Leu Ile Ser
Gln Met Thr Leu Glu 1 5 10 15 Glu Lys Ala Gly Leu Cys Ser Gly Leu
Asp Phe Trp Asn Leu Lys Gly 20 25 30 Ile Glu Arg Leu Gly Ile Pro
Ser Ile Met Val Thr Asp Gly Pro His 35 40 45 Gly Leu Arg Lys Gln
Lys Met Gly Ala Asp His Leu Gly Leu Phe Asp 50 55 60 Ser Ile Pro
Ala Thr Cys Phe Pro Ser Ala Ala Gly Leu Ala Ser Thr 65 70 75 80 Trp
Asn Lys Glu Leu Ile Tyr Glu Val Gly Val Ala Leu Gly Lys Glu 85 90
95 Cys Gln Ala Glu Asp Val Ala Ile Leu Leu Gly Pro Gly Ala Asn Ile
100 105 110 Lys Arg Ser Pro Leu Cys Gly Arg Asn Phe Glu Tyr Phe Ser
Glu Asp 115 120 125 Pro Phe Leu Ser Ser Glu Met Ala Ala Ser His Ile
Lys Gly Val Gln 130 135 140 Ser Glu Gly Val Gly Thr Ser Leu Lys His
Phe Ala Ala Asn Asn Gln 145 150 155 160 Glu His Arg Arg Met Ser Thr
Asp Ala Ile Val Asp Glu Arg Thr Leu 165 170 175 Arg Glu Ile Tyr Leu
Ala Ser Phe Glu Asn Ala Val Lys Lys Ala Gln 180 185 190 Pro Trp Thr
Val Met Cys Ala Tyr Asn Lys Val Asn Gly Asp Phe Ala 195 200 205 Ser
Glu Asn Lys Thr Leu Leu Thr Asp Ile Leu Arg Asp Glu Trp Gly 210 215
220 Phe Glu Gly Ile Val Val Ser Asp Trp Gly Ala Val Asn Glu Pro Val
225 230 235 240 Asp Gly Leu Asn Ala Gly Leu Asp Leu Glu Met Pro Ser
Ser Ser Gly 245 250 255 Ile Gly Glu Lys Lys Ile Ile Asn Ala Val Arg
Asn Gly Gln Leu Leu 260 265 270 Glu Asp Lys Leu Asp Gln Ala Val Glu
Arg Ile Leu Arg Ile Ile Leu 275 280 285 Met Ala Val Glu Asn Lys Lys
Glu Thr Ala Asp Tyr Asp Lys Glu Gln 290 295 300 His His Lys Leu Ala
Arg Lys Ala Ala Ser Glu Ser Met Val Leu Leu 305 310 315 320 Lys Asn
Glu Asp Asn Ile Leu Pro Leu Lys Lys Glu Gly Thr Ile Ser 325 330 335
Ile Ile Gly Ser Phe Ala Lys Lys Pro Arg Tyr Gln Gly Gly Gly Ser 340
345 350 Ser His Ile Asn Pro Thr Lys Leu Glu Asn Ile Tyr Glu Glu Ile
Glu 355 360 365 Lys Thr Ala Gly Gln Asn Val Asn Val Leu Tyr Ala Glu
Gly Tyr His 370 375 380 Leu Glu Lys Asp Leu Ile Asp Asp Gln Leu Ile
Glu Glu Ala Lys Lys 385 390 395 400 Thr Ala Ala Lys Ser Asp Val Thr
Val Leu Phe Val Gly Leu Pro Asp 405 410 415 Arg Tyr Glu Ser Glu Gly
Tyr Asp Arg Glu His Leu Asn Ile Pro Glu 420 425 430 Asn His Arg Leu
Leu Val Glu Ala Val Ala Glu Val Gln Lys Asn Ile 435 440 445 Val Val
Val Leu Ser Asn Gly Ala Pro Leu Val Met Pro Trp Leu Asp 450 455 460
Lys Val Lys Gly Leu Leu Glu Ser Tyr Leu Gly Gly Gln Ala Leu Gly 465
470 475 480 Gly Ala Ile Ala Asp Ile Leu Phe Gly Glu Val Asn Pro Ser
Gly Lys 485 490 495 Leu Ala Glu Thr Phe Pro Val Lys Leu Gly Asp Asn
Pro Ser Tyr Leu 500 505 510 Asn Phe Pro Gly Glu Arg Asp Lys Val Glu
Tyr Lys Glu Gly Ile Phe 515 520 525 Val Gly Tyr Arg Tyr Tyr Asp Thr
Lys Gln Ile Glu Pro Leu Phe Pro 530 535 540 Phe Gly Tyr Gly Leu Ser
Tyr Thr Asn Phe Glu Tyr Lys Asn Leu Val 545 550 555 560 Ile Asp Lys
Lys Glu Ile Lys Asp Thr Glu Ile Val Thr Val Thr Val 565 570 575 Asn
Val Lys Asn Thr Gly Lys Val Pro Gly Lys Glu Ile Ile Gln Leu 580 585
590 Tyr Val Lys Asp Ile Lys Ser Ser Val Val Arg Pro Glu Lys Glu Leu
595 600 605 Lys Gly Phe Gly Lys Val Ser Leu Gln Pro Gly Glu Asp Lys
Thr Ile 610 615 620 Ser Phe Lys Leu Asp Lys Arg Ala Phe Ala Tyr Tyr
Asn Thr Glu Leu 625 630 635 640 Lys Asp Trp Tyr Val Glu Ser Gly Glu
Phe Glu Ile Leu Val Gly Lys 645 650 655 Ser Ser Arg Glu Ile Glu Leu
Thr Glu Lys Ile Met Val His Ser Thr 660 665 670 Ser Pro Val Phe Leu
Glu Val His Arg Asn Ser Thr Val Gly Asp Leu 675 680 685 Leu Thr Asp
Pro Ile Leu Gly Glu Lys Ala Asn Ala Leu Ile Arg Glu 690 695 700 Leu
Thr Lys Gly Ser Pro Leu Phe Asp Ala Gly Ser Asp His Gly Glu 705 710
715 720 Gly Ala Glu Met Met Glu Ala Met Leu Lys Tyr Met Pro Leu Arg
Ala 725 730 735 Leu Met Asn Phe Ser Gly Gly Asp Ile Thr Glu Glu Lys
Leu Thr Glu 740 745 750 Phe Ile Lys Glu Leu Asn Ser Thr Asn Phe Val
Ser Leu 755 760 765 97615DNAUnknownObtained from environmental
sample 97atgttatacc caattataac tgaaactcgc agtatcatcg atttaaatgg
tatctggaaa 60tttaaattag ataatggtga aggactgcag gaaaaatggt atgaaaacgg
attaacagac 120acgatcagta tggctgtacc atcttccttt aatgatattg
gagtaaatgc cagtatacgc 180gatcatgttg gctgggtatg gtatgagcgg
gaattttctg tccccgccat ccttcaatct 240gagcgtgtgg ttttgcgatt
cggttccgca acacatctag ctaaggtttt cgtaaatggt 300gaacttgttg
ttgaacataa gggcggtttt ttaccgtttg aagcagaaat aaataagttt
360ttacaaaaag ggaaaaatcg aataacggtt gctgtcaaca atattcttga
ttactcaact 420ttacccgttg gcacagtaat agaaaaggat attcctggag
ttggcaaagt aatacgcaat 480cagccaaatt ttgacttctt caactacgct
ggcttgcacc gtccagtgaa aatatatact 540acaccgacta cttatgtgaa
ggatgtaacc attgtaacgg aaatagatgg acaggttcac 600tattcaattg attaa
61598204PRTUnknownObtained from environmental sample 98Met Leu Tyr
Pro Ile Ile Thr Glu Thr Arg Ser Ile Ile Asp Leu Asn 1 5 10 15 Gly
Ile Trp Lys Phe Lys Leu Asp Asn Gly Glu Gly Leu Gln Glu Lys 20 25
30 Trp Tyr Glu Asn Gly Leu Thr Asp Thr Ile Ser Met Ala Val Pro Ser
35 40 45 Ser Phe Asn Asp Ile Gly Val Asn Ala Ser Ile Arg Asp His
Val Gly 50 55 60 Trp Val Trp Tyr Glu Arg Glu Phe Ser Val Pro Ala
Ile Leu Gln Ser 65 70 75 80 Glu Arg Val Val Leu Arg Phe Gly Ser Ala
Thr His Leu Ala Lys Val 85 90 95 Phe Val Asn Gly Glu Leu Val Val
Glu His Lys Gly Gly Phe Leu Pro 100 105 110 Phe Glu Ala Glu Ile Asn
Lys Phe Leu Gln Lys Gly Lys Asn Arg Ile 115 120 125 Thr Val Ala Val
Asn Asn Ile Leu Asp Tyr Ser Thr Leu Pro Val Gly 130 135 140 Thr Val
Ile Glu Lys Asp Ile Pro Gly Val Gly Lys Val Ile Arg Asn 145 150 155
160 Gln Pro Asn Phe Asp Phe Phe Asn Tyr Ala Gly Leu His Arg Pro Val
165 170 175 Lys Ile Tyr Thr Thr Pro Thr Thr Tyr Val Lys Asp Val Thr
Ile Val 180 185 190 Thr Glu Ile Asp Gly Gln Val His Tyr Ser Ile Asp
195 200 991404DNAUnknownObtained from environmental sample
99atgaatcatt ccctttcatt tccgccatcc tttgtatggg gcgcggcaac cgcaagctac
60caactggaag gatcaaccca aggcgtggac ggctgcgccg agtccgtctg ggatatgcac
120tgccgaagat ccggcgcgat caaggacggc tcgaacggat tcgtcgcctg
cgatcactac 180catcgctatc gcgaggatgt ggcgctcatg aacgagcttg
gcttgaatgc ctatcgattc 240tcaatcatgt ggccccgcgt catgcccgaa
ggcaccggcg cggtgaacga gaagggcatg 300gatttctacg atcggttggt
tgatgaactg ctcgccgccg gcatcacacc ttgggttact 360ttgttccact
gggactttcc cctagccttg ttccaacgcg gtggctggct gaatgcggat
420tccccgcaat ggtttgagga ttacactcgg gaagtggtta aacgcttgtc
ggatcgtgtg 480catcactggc taacgctcaa cgaaccggcg tgcttcattg
agtttggcca ccgtaccggc 540atgcatgcac ccggcttgca actggcggac
aaggaagcct gccgggtctg gcaccatgcc 600atgctggccc acggtcgcgc
cgttcgcgct attcgccagg aatccgtgca tccatcaccc 660caggtcggct
acgcgccggt cttccgcact accatcccgg acactgaaga tcctgccgac
720atcgaagcgg cccggacctc gatgtttgct catcaggccg gcaacctgtt
cgatacgcgg 780tggaacctcg acccctgctt tcggggcgcg tatccggaga
tcatgatgca gtattggggc 840gatgccgcgc cgcgcatcca ggacggcgac
atggagttga tccgtcagga actcgatttt 900ctcggcctga atatttacca
gtccgagcgc attcgggccg gtgcggatgg cgcacccgag 960gtggtgccat
accctgcgga ttatccgcgc aaccagctcg gttggcccat cacgccggag
1020gccctgcgct gggcgaccct ctttctcttt gaggagtacg ggaaacccct
gatcatcaca 1080gaaaacggaa tcaccctcga cgacaagccc aatgcagacg
gcgaggtgaa tgatgtccag 1140cggatcgctt ttctgaatga ctatcttagc
ggtctccagc gcagcgtgga cgacggcatc 1200cctgtactgg gctatttcca
ctggtcgctg tgcgacaact ttgagtgggc agaaggctat 1260gtccctcgct
tcggcctgat ccatgtggac tatgccagtc aacgcagaac catcaaggcc
1320tcaggacggt tttaccgcga catcattcgg ggccagacag ccacgccctg
catcgcccaa 1380tccagtcagc cggaaacaac ctaa
1404100467PRTUnknownObtained from environmental sample 100Met Asn
His Ser Leu Ser Phe Pro Pro Ser Phe Val Trp Gly Ala Ala 1 5 10 15
Thr Ala Ser Tyr Gln Leu Glu Gly Ser Thr Gln Gly Val Asp Gly Cys 20
25 30 Ala Glu Ser Val Trp Asp Met His Cys Arg Arg Ser Gly Ala Ile
Lys 35 40 45 Asp Gly Ser Asn Gly Phe Val Ala Cys Asp His Tyr His
Arg Tyr Arg 50 55 60 Glu Asp Val Ala Leu Met Asn Glu Leu Gly Leu
Asn Ala Tyr Arg Phe 65 70 75 80 Ser Ile Met Trp Pro Arg Val Met Pro
Glu Gly Thr Gly Ala Val Asn 85 90 95 Glu Lys Gly Met Asp Phe Tyr
Asp Arg Leu Val Asp Glu Leu Leu Ala 100 105 110 Ala Gly Ile Thr Pro
Trp Val Thr Leu Phe His Trp Asp Phe Pro Leu 115 120 125 Ala Leu Phe
Gln Arg Gly Gly Trp Leu Asn Ala Asp Ser Pro Gln Trp 130 135 140 Phe
Glu Asp Tyr Thr Arg Glu Val Val Lys Arg Leu Ser Asp Arg Val 145 150
155 160 His His Trp Leu Thr Leu Asn Glu Pro Ala Cys Phe Ile Glu Phe
Gly 165 170 175 His Arg Thr Gly Met His Ala Pro Gly Leu Gln Leu Ala
Asp Lys Glu 180 185 190 Ala Cys Arg Val Trp His His Ala Met Leu Ala
His Gly Arg Ala Val 195 200 205 Arg Ala Ile Arg Gln Glu Ser Val His
Pro Ser Pro Gln Val Gly Tyr 210 215 220 Ala Pro Val Phe Arg Thr Thr
Ile Pro Asp Thr Glu Asp Pro Ala Asp 225 230 235 240 Ile Glu Ala Ala
Arg Thr Ser Met Phe Ala His Gln Ala Gly Asn Leu 245 250 255 Phe Asp
Thr Arg Trp Asn Leu Asp Pro Cys Phe Arg Gly Ala Tyr Pro 260 265 270
Glu Ile Met Met Gln Tyr Trp Gly Asp Ala Ala Pro Arg Ile Gln Asp 275
280 285 Gly Asp Met Glu Leu Ile Arg Gln Glu Leu Asp Phe Leu Gly Leu
Asn 290 295 300 Ile Tyr Gln Ser Glu Arg Ile Arg Ala Gly Ala Asp Gly
Ala Pro Glu 305 310 315 320 Val Val Pro Tyr Pro Ala Asp Tyr Pro Arg
Asn Gln Leu Gly Trp Pro 325 330 335 Ile Thr Pro Glu Ala Leu Arg Trp
Ala Thr Leu Phe Leu Phe Glu Glu 340 345 350 Tyr Gly Lys Pro Leu Ile
Ile Thr Glu Asn Gly Ile Thr Leu Asp Asp 355 360 365 Lys Pro Asn Ala
Asp Gly Glu Val Asn Asp Val Gln Arg Ile Ala Phe 370 375 380 Leu Asn
Asp Tyr Leu Ser Gly Leu Gln Arg Ser Val Asp Asp Gly Ile 385 390 395
400 Pro Val Leu Gly Tyr Phe His Trp Ser Leu Cys Asp
Asn Phe Glu Trp 405 410 415 Ala Glu Gly Tyr Val Pro Arg Phe Gly Leu
Ile His Val Asp Tyr Ala 420 425 430 Ser Gln Arg Arg Thr Ile Lys Ala
Ser Gly Arg Phe Tyr Arg Asp Ile 435 440 445 Ile Arg Gly Gln Thr Ala
Thr Pro Cys Ile Ala Gln Ser Ser Gln Pro 450 455 460 Glu Thr Thr 465
1011101DNAUnknownObtained from environmental sample 101atgagaaatc
atctgaatgt acccttttac tttatcttct tttttttaat agcgtcaata 60tttacagtct
gttcatcatc aactgcttct gataacaatg agcatccacc gccagtggaa
120gtcgcggatc aggacgcttt tcgtgatgct tttgaagtga atgaattact
tggacgcggt 180attaatctgg gtaatgccct tgaagcgccc aatgaaggcg
aatggggaat ggtaatccag 240gaagagtttc ttgatctgat acttgcagca
ggttttgagt ctgtacgaat tccgattcgc 300tggaatgccc atgccagtga
aagtcaccct ttcaccattc aacgatcgtt ttttgatcgg 360gttgatgaag
tcatccaatg gtcgctggat cgtggccttt ctgtaatgat caatattcat
420cactacaatg aactgatgca aaacccgcag cagcaccggc agcggttttt
gcgactctgg 480aaccagattg ctacacacta taaagattat ccggataatc
tggtttttga aatccttaat 540gaacctcatg ataatctgac tccttctatc
tggaatagtt atttgaggga tgctattggc 600atgattcgcc agacaaaccc
acgcagggtt atcgctatcg gaacagcaaa ctggggtggt 660ttcggagcat
tatcacaact tgaaatcccc tcaaacgatc gccagatcat tgcaactgtt
720cattattatg aacccttcag gttcacccat cagggggctg aatgggcagg
accggaaaca 780aacgattggc tggggacacg atgggatgga tcggatgagg
aaaaatttga tattgaaagt 840ggttttgatg ccgtacagtc ctgggcagtg
acaaataacc ggcctgttca tctcggagaa 900ttcggtgctt acagtactgc
cgataatgaa tcacgcgaac gctggacaac ctttgttcgg 960gaatccgctg
agcaacgcaa tttcagctgg gcatactggg aatttgcagc cggttttggg
1020atctatgacc gtaatcagtg gcaatggagg gattatctgt tgagggcttt
gataccggat 1080agcccggtcc tgttggagta a 1101102366PRTUnknownObtained
from environmental sample 102Met Arg Asn His Leu Asn Val Pro Phe
Tyr Phe Ile Phe Phe Phe Leu 1 5 10 15 Ile Ala Ser Ile Phe Thr Val
Cys Ser Ser Ser Thr Ala Ser Asp Asn 20 25 30 Asn Glu His Pro Pro
Pro Val Glu Val Ala Asp Gln Asp Ala Phe Arg 35 40 45 Asp Ala Phe
Glu Val Asn Glu Leu Leu Gly Arg Gly Ile Asn Leu Gly 50 55 60 Asn
Ala Leu Glu Ala Pro Asn Glu Gly Glu Trp Gly Met Val Ile Gln 65 70
75 80 Glu Glu Phe Leu Asp Leu Ile Leu Ala Ala Gly Phe Glu Ser Val
Arg 85 90 95 Ile Pro Ile Arg Trp Asn Ala His Ala Ser Glu Ser His
Pro Phe Thr 100 105 110 Ile Gln Arg Ser Phe Phe Asp Arg Val Asp Glu
Val Ile Gln Trp Ser 115 120 125 Leu Asp Arg Gly Leu Ser Val Met Ile
Asn Ile His His Tyr Asn Glu 130 135 140 Leu Met Gln Asn Pro Gln Gln
His Arg Gln Arg Phe Leu Arg Leu Trp 145 150 155 160 Asn Gln Ile Ala
Thr His Tyr Lys Asp Tyr Pro Asp Asn Leu Val Phe 165 170 175 Glu Ile
Leu Asn Glu Pro His Asp Asn Leu Thr Pro Ser Ile Trp Asn 180 185 190
Ser Tyr Leu Arg Asp Ala Ile Gly Met Ile Arg Gln Thr Asn Pro Arg 195
200 205 Arg Val Ile Ala Ile Gly Thr Ala Asn Trp Gly Gly Phe Gly Ala
Leu 210 215 220 Ser Gln Leu Glu Ile Pro Ser Asn Asp Arg Gln Ile Ile
Ala Thr Val 225 230 235 240 His Tyr Tyr Glu Pro Phe Arg Phe Thr His
Gln Gly Ala Glu Trp Ala 245 250 255 Gly Pro Glu Thr Asn Asp Trp Leu
Gly Thr Arg Trp Asp Gly Ser Asp 260 265 270 Glu Glu Lys Phe Asp Ile
Glu Ser Gly Phe Asp Ala Val Gln Ser Trp 275 280 285 Ala Val Thr Asn
Asn Arg Pro Val His Leu Gly Glu Phe Gly Ala Tyr 290 295 300 Ser Thr
Ala Asp Asn Glu Ser Arg Glu Arg Trp Thr Thr Phe Val Arg 305 310 315
320 Glu Ser Ala Glu Gln Arg Asn Phe Ser Trp Ala Tyr Trp Glu Phe Ala
325 330 335 Ala Gly Phe Gly Ile Tyr Asp Arg Asn Gln Trp Gln Trp Arg
Asp Tyr 340 345 350 Leu Leu Arg Ala Leu Ile Pro Asp Ser Pro Val Leu
Leu Glu 355 360 365 1031101DNAUnknownObtained from environmental
sample 103atgctgataa ttggaggcct tcttgtttta ctgggatttt cttcttgcgg
gcggcaggca 60gaacctgctg ctgactcttt cagggggttt catgactttg acatcaggcg
tggggtgaac 120atcagccact ggttgtcgca gagtggaagg cgtggtgctg
atcgggaggc gttctttacc 180agggcggatg tggaggccat cgccggcttc
ggttatgatc acattcgttt gcccattgat 240gaggagcaga tgtgggatga
gtcgggcaac aaggaaccac gtgcctttga attgctgcat 300gaagccattg
gctgggcttt ggacaatgag ctcagggtca ttgtcgacct gcacatcatc
360aggtcgcact attttaatgc gcctgagaac ccgctttgga ccgatcgtgc
tgaacagttg 420aaatttgttg agatgtggcg acagttgtct gatgagctgc
agggctatcc gctcgatagg 480gtggcctatg aattgatgaa tgaggccgtg
gctgatgatc cggacgattg gaaccggctt 540gtggctgaga cgatggaggc
gctacggatg ctggaaccgg agcgcaagat tgtcattggc 600tccaaccgct
ggcagtctgt gcatacattt cctgacctgg tgatcccgga taatgacccg
660catatcatat tgagttttca cttctacgaa ccatttctgc tgacgcacca
caaggcctcc 720tggacacaca tccgtgatta caccggtccg gtgaactatc
cgggtttgac tgtagacccg 780acccacctgg aggggttgtc tgaagaactg
gtgacccgga ttggccatca caatggggtg 840tatacaaaag aaacgatgga
ggagatgatc atgatcccac tgcaatatgc caaagaccgg 900gggctccccc
tttattgtgg agagtgggga tgtttcccga ccatgcccca ggagatgcgc
960ctgcaatggt acgccgatgt gcgtgcgatc ctggaaaagc atgagattgc
ctgggcaaac 1020tgggattaca agggtggttt cggtgtggtt gaccgcaacg
gcgaacccca ccatgattta 1080ttggaagtgc tcttaaaata a
1101104366PRTUnknownObtained from environmental sample 104Met Leu
Ile Ile Gly Gly Leu Leu Val Leu Leu Gly Phe Ser Ser Cys 1 5 10 15
Gly Arg Gln Ala Glu Pro Ala Ala Asp Ser Phe Arg Gly Phe His Asp 20
25 30 Phe Asp Ile Arg Arg Gly Val Asn Ile Ser His Trp Leu Ser Gln
Ser 35 40 45 Gly Arg Arg Gly Ala Asp Arg Glu Ala Phe Phe Thr Arg
Ala Asp Val 50 55 60 Glu Ala Ile Ala Gly Phe Gly Tyr Asp His Ile
Arg Leu Pro Ile Asp 65 70 75 80 Glu Glu Gln Met Trp Asp Glu Ser Gly
Asn Lys Glu Pro Arg Ala Phe 85 90 95 Glu Leu Leu His Glu Ala Ile
Gly Trp Ala Leu Asp Asn Glu Leu Arg 100 105 110 Val Ile Val Asp Leu
His Ile Ile Arg Ser His Tyr Phe Asn Ala Pro 115 120 125 Glu Asn Pro
Leu Trp Thr Asp Arg Ala Glu Gln Leu Lys Phe Val Glu 130 135 140 Met
Trp Arg Gln Leu Ser Asp Glu Leu Gln Gly Tyr Pro Leu Asp Arg 145 150
155 160 Val Ala Tyr Glu Leu Met Asn Glu Ala Val Ala Asp Asp Pro Asp
Asp 165 170 175 Trp Asn Arg Leu Val Ala Glu Thr Met Glu Ala Leu Arg
Met Leu Glu 180 185 190 Pro Glu Arg Lys Ile Val Ile Gly Ser Asn Arg
Trp Gln Ser Val His 195 200 205 Thr Phe Pro Asp Leu Val Ile Pro Asp
Asn Asp Pro His Ile Ile Leu 210 215 220 Ser Phe His Phe Tyr Glu Pro
Phe Leu Leu Thr His His Lys Ala Ser 225 230 235 240 Trp Thr His Ile
Arg Asp Tyr Thr Gly Pro Val Asn Tyr Pro Gly Leu 245 250 255 Thr Val
Asp Pro Thr His Leu Glu Gly Leu Ser Glu Glu Leu Val Thr 260 265 270
Arg Ile Gly His His Asn Gly Val Tyr Thr Lys Glu Thr Met Glu Glu 275
280 285 Met Ile Met Ile Pro Leu Gln Tyr Ala Lys Asp Arg Gly Leu Pro
Leu 290 295 300 Tyr Cys Gly Glu Trp Gly Cys Phe Pro Thr Met Pro Gln
Glu Met Arg 305 310 315 320 Leu Gln Trp Tyr Ala Asp Val Arg Ala Ile
Leu Glu Lys His Glu Ile 325 330 335 Ala Trp Ala Asn Trp Asp Tyr Lys
Gly Gly Phe Gly Val Val Asp Arg 340 345 350 Asn Gly Glu Pro His His
Asp Leu Leu Glu Val Leu Leu Lys 355 360 365
1051047DNAUnknownObtained from environmental sample 105atgcaacact
tcatcaacgg cgtcaacctg ggaggctggc tctcccaata ccagaaatac 60gaccatgagc
acttccgcac cttcatcacc cggcgcgata tcgaacaaat cgcatcctgg
120ggcttcgacc acatccgcct gccggtcgat tatccggttc tcgaatcgga
cgacgcgccc 180ggtatctatc atgaagatgg ctttgcctat cttgactctt
gcctggaatg gtgccaggcc 240gctgggctgg cagtcgtctt cgacctgcat
catgcccccg gctacagttt cacgaacacg 300ctcaagcctg aaaccctgca
cctgaacgta ctcttcgagc aggaaatcgc ccaaaatcga 360tttatcgccc
tctgggaaac cattgttcgg cgctaccagg ccgccggctt gcctatcatc
420tttgaactac tgaatgaaat ggtgctgcca gacagcggcc cctggaacgc
cctggcccac 480aaaaccgtcg ccgccctgcg acagatttcg cccgattgca
aaatcatgat tggcggcaat 540aactacaacg ccgcatccga actcaaaaac
ataaccctgc acaacgaccc caacatccta 600tacaccttcc atttctacga
accggccctg ttcacccacc agaaagcccc ctgggtgcag 660attgctgtcg
aatacaacca ggaactcgaa taccctggct cgtacaccaa cctggccgcc
720tttctccggc gcaatcccca ctatcaagaa tcctatggat ggcaggtcaa
ccgccgtatc 780gaccgcgacc tcctgctcga attcacccaa cccgccctgg
actttgtcca gcagaccggg 840cgcgacctgt actgcggtga attcggcgtc
attgaatacg tcgagcctgc cagccgccaa 900aactggcacg ccgacctgct
ggacatcctg cgccagcaga agattggccg cgccgtctgg 960acttataaac
aaatggattt tggcctggtg gacgcggacg gcaaggtggt cgaccccaaa
1020cttctcgaaa tcttgtgtca atcctga 1047106348PRTUnknownObtained from
environmental sample 106Met Gln His Phe Ile Asn Gly Val Asn Leu Gly
Gly Trp Leu Ser Gln 1 5 10 15 Tyr Gln Lys Tyr Asp His Glu His Phe
Arg Thr Phe Ile Thr Arg Arg 20 25 30 Asp Ile Glu Gln Ile Ala Ser
Trp Gly Phe Asp His Ile Arg Leu Pro 35 40 45 Val Asp Tyr Pro Val
Leu Glu Ser Asp Asp Ala Pro Gly Ile Tyr His 50 55 60 Glu Asp Gly
Phe Ala Tyr Leu Asp Ser Cys Leu Glu Trp Cys Gln Ala 65 70 75 80 Ala
Gly Leu Ala Val Val Phe Asp Leu His His Ala Pro Gly Tyr Ser 85 90
95 Phe Thr Asn Thr Leu Lys Pro Glu Thr Leu His Leu Asn Val Leu Phe
100 105 110 Glu Gln Glu Ile Ala Gln Asn Arg Phe Ile Ala Leu Trp Glu
Thr Ile 115 120 125 Val Arg Arg Tyr Gln Ala Ala Gly Leu Pro Ile Ile
Phe Glu Leu Leu 130 135 140 Asn Glu Met Val Leu Pro Asp Ser Gly Pro
Trp Asn Ala Leu Ala His 145 150 155 160 Lys Thr Val Ala Ala Leu Arg
Gln Ile Ser Pro Asp Cys Lys Ile Met 165 170 175 Ile Gly Gly Asn Asn
Tyr Asn Ala Ala Ser Glu Leu Lys Asn Ile Thr 180 185 190 Leu His Asn
Asp Pro Asn Ile Leu Tyr Thr Phe His Phe Tyr Glu Pro 195 200 205 Ala
Leu Phe Thr His Gln Lys Ala Pro Trp Val Gln Ile Ala Val Glu 210 215
220 Tyr Asn Gln Glu Leu Glu Tyr Pro Gly Ser Tyr Thr Asn Leu Ala Ala
225 230 235 240 Phe Leu Arg Arg Asn Pro His Tyr Gln Glu Ser Tyr Gly
Trp Gln Val 245 250 255 Asn Arg Arg Ile Asp Arg Asp Leu Leu Leu Glu
Phe Thr Gln Pro Ala 260 265 270 Leu Asp Phe Val Gln Gln Thr Gly Arg
Asp Leu Tyr Cys Gly Glu Phe 275 280 285 Gly Val Ile Glu Tyr Val Glu
Pro Ala Ser Arg Gln Asn Trp His Ala 290 295 300 Asp Leu Leu Asp Ile
Leu Arg Gln Gln Lys Ile Gly Arg Ala Val Trp 305 310 315 320 Thr Tyr
Lys Gln Met Asp Phe Gly Leu Val Asp Ala Asp Gly Lys Val 325 330 335
Val Asp Pro Lys Leu Leu Glu Ile Leu Cys Gln Ser 340 345
1071137DNAUnknownObtained from environmental sample 107atggaaaagc
aaatctgttc aaatgttttc agtacgatgc tgataattgg aggccttctt 60gttttactgg
gattttcttc ttgcgggcgg caggcagaac ctgctgctga ctctttcagg
120gggtttcacg actttgacat caggcgcggg gtgaacatca gccattggtt
gtcgcagagt 180ggaaggcgtg gtgctgatcg ggaggcgttc tttaccaggg
cggatgtgga ggccatcgcc 240ggcttcggtt atgatcacat tcgtttgccc
atcgatgaag agcagatgtg ggatgagtcg 300ggcaacaagg agccacgtgc
ctttgaattg ctgcatgagg ccattggctg ggctttggac 360aatgagctca
gggtcattgt tgacctgcac atcatcaggt cgcactattt taatgcgcct
420gagaacccgc tttggaccga tcgtgctgaa cagttgaaat ttgttgagat
gtggcgacag 480ttgtctgatg agctgcaggg ctatccgctc gatagggtgg
cctatgaatt gatgaatgag 540gccgtggctg atgatccgga cgattggaac
cggcttgtgg ctgagacgat ggaggcgcta 600cggatgctgg aaccggagcg
caagattgtc attggctcca accgctggca gtctgtgcat 660acatttcctg
acctggtgat cccggataat gacccgcata tcatattgag ttttcacttc
720tacgaaccat ttctgctgac gcaccacaag gcctcctgga cacacatccg
tgattacacc 780ggtccggtga actatccggg tttgactgta gacccgaccc
acctggaggg gttgtctgaa 840gaactggtga cccggattgg ccatcacaat
ggggtgtata caaaagaaac gatggaggag 900atgatcatga tcccactgca
atatgccaaa gaacgggggc tccccctgta ttgcggggag 960tggggatgtt
tcccgaccat gccccaggag atgcgcctgc aatggtacgc cgatgtgcgt
1020gcgatcctgg aaaagcatga gattgcctgg gcaaactggg attacaaggg
tggtttcggt 1080gtggttgacc gcaacggcga accccaccat gatttattgg
aagtcttact aaaataa 1137108378PRTUnknownObtained from environmental
sample 108Met Glu Lys Gln Ile Cys Ser Asn Val Phe Ser Thr Met Leu
Ile Ile 1 5 10 15 Gly Gly Leu Leu Val Leu Leu Gly Phe Ser Ser Cys
Gly Arg Gln Ala 20 25 30 Glu Pro Ala Ala Asp Ser Phe Arg Gly Phe
His Asp Phe Asp Ile Arg 35 40 45 Arg Gly Val Asn Ile Ser His Trp
Leu Ser Gln Ser Gly Arg Arg Gly 50 55 60 Ala Asp Arg Glu Ala Phe
Phe Thr Arg Ala Asp Val Glu Ala Ile Ala 65 70 75 80 Gly Phe Gly Tyr
Asp His Ile Arg Leu Pro Ile Asp Glu Glu Gln Met 85 90 95 Trp Asp
Glu Ser Gly Asn Lys Glu Pro Arg Ala Phe Glu Leu Leu His 100 105 110
Glu Ala Ile Gly Trp Ala Leu Asp Asn Glu Leu Arg Val Ile Val Asp 115
120 125 Leu His Ile Ile Arg Ser His Tyr Phe Asn Ala Pro Glu Asn Pro
Leu 130 135 140 Trp Thr Asp Arg Ala Glu Gln Leu Lys Phe Val Glu Met
Trp Arg Gln 145 150 155 160 Leu Ser Asp Glu Leu Gln Gly Tyr Pro Leu
Asp Arg Val Ala Tyr Glu 165 170 175 Leu Met Asn Glu Ala Val Ala Asp
Asp Pro Asp Asp Trp Asn Arg Leu 180 185 190 Val Ala Glu Thr Met Glu
Ala Leu Arg Met Leu Glu Pro Glu Arg Lys 195 200 205 Ile Val Ile Gly
Ser Asn Arg Trp Gln Ser Val His Thr Phe Pro Asp 210 215 220 Leu Val
Ile Pro Asp Asn Asp Pro His Ile Ile Leu Ser Phe His Phe 225 230 235
240 Tyr Glu Pro Phe Leu Leu Thr His His Lys Ala Ser Trp Thr His Ile
245 250 255 Arg Asp Tyr Thr Gly Pro Val Asn Tyr Pro Gly Leu Thr Val
Asp Pro 260 265 270 Thr His Leu Glu Gly Leu Ser Glu Glu Leu Val Thr
Arg Ile Gly His 275 280 285 His Asn Gly Val Tyr Thr Lys Glu Thr Met
Glu Glu Met Ile Met Ile 290 295 300 Pro Leu Gln Tyr Ala Lys Glu Arg
Gly Leu Pro Leu Tyr Cys Gly Glu 305 310 315 320 Trp Gly Cys Phe Pro
Thr Met Pro Gln Glu Met Arg Leu Gln Trp Tyr 325 330 335 Ala Asp Val
Arg Ala Ile Leu Glu Lys His Glu Ile Ala Trp Ala Asn 340 345 350 Trp
Asp Tyr Lys Gly Gly Phe Gly Val Val Asp Arg Asn Gly Glu Pro 355 360
365 His His Asp Leu Leu Glu Val Leu Leu Lys 370 375
1091248DNAUnknownObtained from environmental sample 109atgaagacac
atagcttcaa cctcagatca cggatcacct tgttgaccgc ggcactgctt 60ttcatcgggg
caacggccgg ggccgccacg acacctatca ccctcaaaga cgcctacaaa
120gaccatttcc ttatgggtgt agccatcaac cgcctgattg caatgggcga
tacgaatgtc 180cgggccgaca
acgccagccg gaccccggaa cagctcaagg gggacattgc cctggtcaag
240gcgcagttca acctgatcgt caatgagaac gatctgaaac cgattctcat
tcacccgagg 300ccaggaccgg acgggtacga cttcgcccca gcggatgcct
tcgtgaagtt cggcatggac 360aacaatatgt atatcgtggg ccacaccctc
ctctggcaca gccaggtgcc caactggttc 420ttccaagggt ctgctccggc
gactccggaa acgccacctg ctgccacgga cgcggcggtc 480gcaccccgcg
gcggacgagg aggtcgcggc gggattaccg gccccctggc gacccgcgag
540gagttgatcg aacgcatgcg cgagcacatt cacaccgtcg tcggccgcta
taagggaaag 600atcaaggtct gggacgtcgt caacgaagcc ctcgccgacg
gcggcaccga gaccctgcga 660agcacgtact ggacccaaat catcgggccg
gaattcatcg ccatggcctt tcgattcgcc 720cacgaagccg atccggatgc
gatccttcgt tacaacgatt atggcctgga gaaccctgcc 780aagcgtgaga
aactcaagaa gctgatcgcg tcgctccagg agcagaacgt tccggttcat
840gccatcggca cgcaaaccca tatcagcgtc tccacgacgt tcgaaagaat
ggatgagacc 900ttgagggacc tggcatccat cgggcttccc gtccacatca
ccgaactgga tgtcaacgcc 960gccgcggggg gccagagggg caccaatgcg
gacattgccg gcactgccga gcgtacggcg 1020ggcggcgtgg tcagtgaagc
cgacaagcgg ctggccgacg cctacgcgaa tctcttccgc 1080gcgatcatga
agcacaagga ctcggtgaag atggtcacgt tctggggcgt caatgacgcg
1140gtttcgtggc tcgcacgcgg caccccgctg ctgttcgacg gcaacaatca
gcccaagccg 1200gctttcgatg cggtcattcg cgtcgccacg gaggcggcac agaactga
1248110415PRTUnknownObtained from environmental sample 110Met Lys
Thr His Ser Phe Asn Leu Arg Ser Arg Ile Thr Leu Leu Thr 1 5 10 15
Ala Ala Leu Leu Phe Ile Gly Ala Thr Ala Gly Ala Ala Thr Thr Pro 20
25 30 Ile Thr Leu Lys Asp Ala Tyr Lys Asp His Phe Leu Met Gly Val
Ala 35 40 45 Ile Asn Arg Leu Ile Ala Met Gly Asp Thr Asn Val Arg
Ala Asp Asn 50 55 60 Ala Ser Arg Thr Pro Glu Gln Leu Lys Gly Asp
Ile Ala Leu Val Lys 65 70 75 80 Ala Gln Phe Asn Leu Ile Val Asn Glu
Asn Asp Leu Lys Pro Ile Leu 85 90 95 Ile His Pro Arg Pro Gly Pro
Asp Gly Tyr Asp Phe Ala Pro Ala Asp 100 105 110 Ala Phe Val Lys Phe
Gly Met Asp Asn Asn Met Tyr Ile Val Gly His 115 120 125 Thr Leu Leu
Trp His Ser Gln Val Pro Asn Trp Phe Phe Gln Gly Ser 130 135 140 Ala
Pro Ala Thr Pro Glu Thr Pro Pro Ala Ala Thr Asp Ala Ala Val 145 150
155 160 Ala Pro Arg Gly Gly Arg Gly Gly Arg Gly Gly Ile Thr Gly Pro
Leu 165 170 175 Ala Thr Arg Glu Glu Leu Ile Glu Arg Met Arg Glu His
Ile His Thr 180 185 190 Val Val Gly Arg Tyr Lys Gly Lys Ile Lys Val
Trp Asp Val Val Asn 195 200 205 Glu Ala Leu Ala Asp Gly Gly Thr Glu
Thr Leu Arg Ser Thr Tyr Trp 210 215 220 Thr Gln Ile Ile Gly Pro Glu
Phe Ile Ala Met Ala Phe Arg Phe Ala 225 230 235 240 His Glu Ala Asp
Pro Asp Ala Ile Leu Arg Tyr Asn Asp Tyr Gly Leu 245 250 255 Glu Asn
Pro Ala Lys Arg Glu Lys Leu Lys Lys Leu Ile Ala Ser Leu 260 265 270
Gln Glu Gln Asn Val Pro Val His Ala Ile Gly Thr Gln Thr His Ile 275
280 285 Ser Val Ser Thr Thr Phe Glu Arg Met Asp Glu Thr Leu Arg Asp
Leu 290 295 300 Ala Ser Ile Gly Leu Pro Val His Ile Thr Glu Leu Asp
Val Asn Ala 305 310 315 320 Ala Ala Gly Gly Gln Arg Gly Thr Asn Ala
Asp Ile Ala Gly Thr Ala 325 330 335 Glu Arg Thr Ala Gly Gly Val Val
Ser Glu Ala Asp Lys Arg Leu Ala 340 345 350 Asp Ala Tyr Ala Asn Leu
Phe Arg Ala Ile Met Lys His Lys Asp Ser 355 360 365 Val Lys Met Val
Thr Phe Trp Gly Val Asn Asp Ala Val Ser Trp Leu 370 375 380 Ala Arg
Gly Thr Pro Leu Leu Phe Asp Gly Asn Asn Gln Pro Lys Pro 385 390 395
400 Ala Phe Asp Ala Val Ile Arg Val Ala Thr Glu Ala Ala Gln Asn 405
410 415 1111131DNAUnknownObtained from environmental sample
111atgcgaagac tgatcaccat catccttgcg acggctgtcg caatcttatc
gaccacatca 60tgctccaaga ccgctgaacg agagggcttc ttgatcaagc gaggaaccaa
cctcagccat 120tggctctccc agagcaagga aaggggagag gctcgcaggc
tccatatcca ggaggatgac 180tttgctcgcc tcgacagcct cggtttcgac
catgtgcgca tccctgtcga cgaggaacaa 240ctctgggacg aggatggcaa
caagctcaca gaagcatggg aactgctcga tttcgccctc 300gacatggcgc
gcaagtacaa cctgcgcgct atcgtggacc ttcacatcat ccgcgcccat
360tacttcaacg ccgtcaacga aggcgcgtcg aatactctct tcaccagcga
ggaggcgcag 420cagggcctga tcaacctttg gtaccagctt tccgacttcc
tcaaggaccg cagcgtcgac 480tgggttgcct acgagttcat gaacgagcca
gtcgctcctg agcatgagca atggaacgcc 540ctcgtcgcaa aggtgcacaa
ggcgcttcgt gagcgtgaac cggagcgtac cctcgtgatc 600ggttctaacc
tgtggcaggg tcaccagacc ttcaagtacc tccgcgtacc tgagaatgac
660ccgaacatca tcctgagctt ccactactac aacccttcga tcctcaccca
caacatggct 720ccgtggactc cggtgggcaa atataccggt tccatcaatt
atccgggcgt catcgtctct 780gctgaggatt acgctgcgca gagccctgag
gtgcaggccg aggtgaagca gtatacggag 840atggtctgga accgcgacac
gatctacagc cagatgaagg atgcgatcga ggtggctgcc 900agctatggac
tgcagctctt ctgcggcgaa tggggcgtgt atgaacctgt cgaccgtgag
960cttgcatacg catggaccaa ggatatgctg tcggtgttcg acgagttcga
catcgcatgg 1020acgacctggt gttacgatgc cgacttcggc ttctgggacc
aggcgaaaca tgatttcaag 1080gacaagcctc ttgtcgatct cctgatgggt
tccaagggtc ttgaacaata g 1131112376PRTUnknownObtained from
environmental sample 112Met Arg Arg Leu Ile Thr Ile Ile Leu Ala Thr
Ala Val Ala Ile Leu 1 5 10 15 Ser Thr Thr Ser Cys Ser Lys Thr Ala
Glu Arg Glu Gly Phe Leu Ile 20 25 30 Lys Arg Gly Thr Asn Leu Ser
His Trp Leu Ser Gln Ser Lys Glu Arg 35 40 45 Gly Glu Ala Arg Arg
Leu His Ile Gln Glu Asp Asp Phe Ala Arg Leu 50 55 60 Asp Ser Leu
Gly Phe Asp His Val Arg Ile Pro Val Asp Glu Glu Gln 65 70 75 80 Leu
Trp Asp Glu Asp Gly Asn Lys Leu Thr Glu Ala Trp Glu Leu Leu 85 90
95 Asp Phe Ala Leu Asp Met Ala Arg Lys Tyr Asn Leu Arg Ala Ile Val
100 105 110 Asp Leu His Ile Ile Arg Ala His Tyr Phe Asn Ala Val Asn
Glu Gly 115 120 125 Ala Ser Asn Thr Leu Phe Thr Ser Glu Glu Ala Gln
Gln Gly Leu Ile 130 135 140 Asn Leu Trp Tyr Gln Leu Ser Asp Phe Leu
Lys Asp Arg Ser Val Asp 145 150 155 160 Trp Val Ala Tyr Glu Phe Met
Asn Glu Pro Val Ala Pro Glu His Glu 165 170 175 Gln Trp Asn Ala Leu
Val Ala Lys Val His Lys Ala Leu Arg Glu Arg 180 185 190 Glu Pro Glu
Arg Thr Leu Val Ile Gly Ser Asn Leu Trp Gln Gly His 195 200 205 Gln
Thr Phe Lys Tyr Leu Arg Val Pro Glu Asn Asp Pro Asn Ile Ile 210 215
220 Leu Ser Phe His Tyr Tyr Asn Pro Ser Ile Leu Thr His Asn Met Ala
225 230 235 240 Pro Trp Thr Pro Val Gly Lys Tyr Thr Gly Ser Ile Asn
Tyr Pro Gly 245 250 255 Val Ile Val Ser Ala Glu Asp Tyr Ala Ala Gln
Ser Pro Glu Val Gln 260 265 270 Ala Glu Val Lys Gln Tyr Thr Glu Met
Val Trp Asn Arg Asp Thr Ile 275 280 285 Tyr Ser Gln Met Lys Asp Ala
Ile Glu Val Ala Ala Ser Tyr Gly Leu 290 295 300 Gln Leu Phe Cys Gly
Glu Trp Gly Val Tyr Glu Pro Val Asp Arg Glu 305 310 315 320 Leu Ala
Tyr Ala Trp Thr Lys Asp Met Leu Ser Val Phe Asp Glu Phe 325 330 335
Asp Ile Ala Trp Thr Thr Trp Cys Tyr Asp Ala Asp Phe Gly Phe Trp 340
345 350 Asp Gln Ala Lys His Asp Phe Lys Asp Lys Pro Leu Val Asp Leu
Leu 355 360 365 Met Gly Ser Lys Gly Leu Glu Gln 370 375
1131095DNAUnknownObtained from environmental sample 113atgaaggtga
cccgaacagc tgtcgcgggc attgtcgccg cagcggtcct catcacgatc 60ggcacgtcga
ccgcgtcggc tgaggatgaa ccaaccagcg agaacacgtc gacggatcag
120ccgttgcgcg tcctggcagc caaagccggg atcgcgttcg gcacggccgt
cgacatgaac 180gcgtacaaca acgacgcgac ctaccgtgag ctcgtcggcc
aggagttctc gagcgtcacg 240gccgagaacg tcatgaagtg gcagctcctc
gagccgcagc gaggggtcta caactggggt 300ccggccgatc agctcgtgcg
cgtagccaac gagaacggcc agaaggtgcg cgggcacacg 360ctcatctggc
acaaccagct gcccacctgg cttaccagcg gagtcgcctc cggtgagatc
420acaccggacg agctccggca gctcctgagg aaccacatct tcacggtgat
gcgccacttc 480aagggcgaga tccaccagtg ggatgtcgcc aacgaggtca
tcgacgacag cggcaacctg 540cgcaacacga tctggctgca gaacctgggt
ccgagctaca tcgcggacgc gttccggtgg 600gctcgcaagg ccgacccgga
cgccgccctc tatctgaacg actacaacgt cgagggcccg 660aacgccaagg
ccgatgcgta ctacgccctg gtcaagcagc tcctcgccga cgacgtgccg
720gtggacggct tcggaataca ggggcacctc ggtgtgcagt tcggcttctg
gcccgcgagt 780gcggtggccg acaacatggg gcgcttcgag gcactcggcc
tgcagacggc ggtcaccgag 840gcggatgtcc ggatgatcat gccgcccgac
gaggacaagc tggccgcaca ggcacgtggc 900tacagcacgt tggtccaggg
ctgcctgatg gccaagcgtt gcaggtcgtt caccgtctgg 960ggcttcaccg
acaagtactc ctgggttccg ggcaccttcc ccggccaggg cgcggcgaac
1020ctcctggccg aggacttcca gcccaagccg gcttactacg ccgtccagga
tgacctcgcg 1080cgcgccggac ggtag 1095114364PRTUnknownObtained from
environmental sample 114Met Lys Val Thr Arg Thr Ala Val Ala Gly Ile
Val Ala Ala Ala Val 1 5 10 15 Leu Ile Thr Ile Gly Thr Ser Thr Ala
Ser Ala Glu Asp Glu Pro Thr 20 25 30 Ser Glu Asn Thr Ser Thr Asp
Gln Pro Leu Arg Val Leu Ala Ala Lys 35 40 45 Ala Gly Ile Ala Phe
Gly Thr Ala Val Asp Met Asn Ala Tyr Asn Asn 50 55 60 Asp Ala Thr
Tyr Arg Glu Leu Val Gly Gln Glu Phe Ser Ser Val Thr 65 70 75 80 Ala
Glu Asn Val Met Lys Trp Gln Leu Leu Glu Pro Gln Arg Gly Val 85 90
95 Tyr Asn Trp Gly Pro Ala Asp Gln Leu Val Arg Val Ala Asn Glu Asn
100 105 110 Gly Gln Lys Val Arg Gly His Thr Leu Ile Trp His Asn Gln
Leu Pro 115 120 125 Thr Trp Leu Thr Ser Gly Val Ala Ser Gly Glu Ile
Thr Pro Asp Glu 130 135 140 Leu Arg Gln Leu Leu Arg Asn His Ile Phe
Thr Val Met Arg His Phe 145 150 155 160 Lys Gly Glu Ile His Gln Trp
Asp Val Ala Asn Glu Val Ile Asp Asp 165 170 175 Ser Gly Asn Leu Arg
Asn Thr Ile Trp Leu Gln Asn Leu Gly Pro Ser 180 185 190 Tyr Ile Ala
Asp Ala Phe Arg Trp Ala Arg Lys Ala Asp Pro Asp Ala 195 200 205 Ala
Leu Tyr Leu Asn Asp Tyr Asn Val Glu Gly Pro Asn Ala Lys Ala 210 215
220 Asp Ala Tyr Tyr Ala Leu Val Lys Gln Leu Leu Ala Asp Asp Val Pro
225 230 235 240 Val Asp Gly Phe Gly Ile Gln Gly His Leu Gly Val Gln
Phe Gly Phe 245 250 255 Trp Pro Ala Ser Ala Val Ala Asp Asn Met Gly
Arg Phe Glu Ala Leu 260 265 270 Gly Leu Gln Thr Ala Val Thr Glu Ala
Asp Val Arg Met Ile Met Pro 275 280 285 Pro Asp Glu Asp Lys Leu Ala
Ala Gln Ala Arg Gly Tyr Ser Thr Leu 290 295 300 Val Gln Gly Cys Leu
Met Ala Lys Arg Cys Arg Ser Phe Thr Val Trp 305 310 315 320 Gly Phe
Thr Asp Lys Tyr Ser Trp Val Pro Gly Thr Phe Pro Gly Gln 325 330 335
Gly Ala Ala Asn Leu Leu Ala Glu Asp Phe Gln Pro Lys Pro Ala Tyr 340
345 350 Tyr Ala Val Gln Asp Asp Leu Ala Arg Ala Gly Arg 355 360
115774DNAUnknownObtained from environmental sample 115atggacttgc
agctaggcgg aaagcgcgtg ctgatcacgg gtgcgtccaa aggcatcggc 60ctggcctgcg
ccgtcgcctt tgcgcgcgag ggtgccgacc cgattctggt ggcgcgcgat
120gatgcggcgt tgcatcacgc cacgtccgcc atccgcgaac aaagcggccg
cgcggcacat 180gccatcacgc tggacctggc cctgcctggc gcggcggaaa
agctggccaa ggaaaccggc 240cccatcgaca tactggtcaa caacgcgggc
gcggtgcccg gcggcgcgct ggaccaggtg 300caagacgaac gctggcgcgc
gggctgggaa ttgaaagtgc acggctacat cagcctggcg 360cgctgctact
acccgcacat gcgcgaagcg ggcgcgggcg tcatcgccaa catcatcggc
420atggcgggcg cggcgccccg cgccgactac atctgcggcg cggcggccaa
tgcctcactg 480attgccttta cccgcgcgct gggtggcgaa gcgccccgcc
acggcgtgcg cgtctttggc 540gtcaacccct cgcgcacgcg gaccgaccgc
gtgctgaccc tggcccggca acgcgcgcag 600gcgcgctggg gcgacgaaac
ccgttggcag gaaacgctgt cggacctgcc cttcaaccgg 660ctgatggaac
ccgccgaagt ggccgacatg attgtgttcg gcgcctcgcc gcgcgcgggt
720tacctgagcg gcacggtcat cgacctggac ggcggcgaac agtacgcgaa atag
774116257PRTUnknownObtained from environmental sample 116Met Asp
Leu Gln Leu Gly Gly Lys Arg Val Leu Ile Thr Gly Ala Ser 1 5 10 15
Lys Gly Ile Gly Leu Ala Cys Ala Val Ala Phe Ala Arg Glu Gly Ala 20
25 30 Asp Pro Ile Leu Val Ala Arg Asp Asp Ala Ala Leu His His Ala
Thr 35 40 45 Ser Ala Ile Arg Glu Gln Ser Gly Arg Ala Ala His Ala
Ile Thr Leu 50 55 60 Asp Leu Ala Leu Pro Gly Ala Ala Glu Lys Leu
Ala Lys Glu Thr Gly 65 70 75 80 Pro Ile Asp Ile Leu Val Asn Asn Ala
Gly Ala Val Pro Gly Gly Ala 85 90 95 Leu Asp Gln Val Gln Asp Glu
Arg Trp Arg Ala Gly Trp Glu Leu Lys 100 105 110 Val His Gly Tyr Ile
Ser Leu Ala Arg Cys Tyr Tyr Pro His Met Arg 115 120 125 Glu Ala Gly
Ala Gly Val Ile Ala Asn Ile Ile Gly Met Ala Gly Ala 130 135 140 Ala
Pro Arg Ala Asp Tyr Ile Cys Gly Ala Ala Ala Asn Ala Ser Leu 145 150
155 160 Ile Ala Phe Thr Arg Ala Leu Gly Gly Glu Ala Pro Arg His Gly
Val 165 170 175 Arg Val Phe Gly Val Asn Pro Ser Arg Thr Arg Thr Asp
Arg Val Leu 180 185 190 Thr Leu Ala Arg Gln Arg Ala Gln Ala Arg Trp
Gly Asp Glu Thr Arg 195 200 205 Trp Gln Glu Thr Leu Ser Asp Leu Pro
Phe Asn Arg Leu Met Glu Pro 210 215 220 Ala Glu Val Ala Asp Met Ile
Val Phe Gly Ala Ser Pro Arg Ala Gly 225 230 235 240 Tyr Leu Ser Gly
Thr Val Ile Asp Leu Asp Gly Gly Glu Gln Tyr Ala 245 250 255 Lys
117747DNAUnknownObtained from environmental sample 117atgcccaaag
tcatgctcgt taccggcggc agccgtggca tcggcgccgc cgtcgccaag 60ctggccgcgc
gccgcggcta cgcggtcggc atcaactacc gcacccattc cgacgccgcc
120gacgccgtcg tggccgagat ccagcaggcg ggcggcaccg cgctggccat
ccaggccgac 180gtgtcgcaag aagatgacgt gctgcacatg ttccgcacgc
tggacgagcg cctgggccgc 240atcgacgcgc tggtcaataa cgccggcatc
ctggaaacgc agatgcgcct ggaccagatg 300gaagcggacc gcctgctgcg
cgtgctgtcc accaacgtca tcggcgcttt cctgtgtgcg 360cgcgaagcgg
tgcgcaggat gtcgacgcgc catggcggcg tgggcggcgc catcgtcaac
420gtgtcttcgg cggcggcgcg cctgggctcg cccaatgaat acgtggatta
cgcggcctcc 480aagggcgcgc tggacacgat gaccatcggc ctgtccaaag
aggtagcgcc cgaaggtatc 540cgcgtgaatg gcgtgcgccc cggcaccatc
tacaccgaca tgcacgcaag cggcggcgag 600ccgggccggg tggatcgcct
gaaaagcgtg atcccgctgc ggcgcggcgg ctcggtggaa 660gaagtggcgg
gcgccgtcat gtggctgttt tccgaagaag ccggctatac cagcggctcg
720ttcatcgacg tgtccggcgg tagttga 747118248PRTUnknownObtained from
environmental sample 118Met Pro Lys Val Met Leu Val Thr Gly Gly Ser
Arg Gly Ile Gly Ala 1 5 10 15 Ala Val Ala Lys Leu Ala Ala Arg Arg
Gly Tyr Ala Val Gly Ile Asn 20 25 30 Tyr Arg Thr His Ser Asp Ala
Ala Asp Ala Val Val Ala Glu Ile Gln 35 40 45 Gln Ala Gly Gly Thr
Ala Leu Ala Ile Gln Ala Asp Val Ser Gln Glu 50 55 60 Asp Asp Val
Leu His Met Phe Arg Thr Leu Asp Glu Arg Leu Gly Arg 65
70 75 80 Ile Asp Ala Leu Val Asn Asn Ala Gly Ile Leu Glu Thr Gln
Met Arg 85 90 95 Leu Asp Gln Met Glu Ala Asp Arg Leu Leu Arg Val
Leu Ser Thr Asn 100 105 110 Val Ile Gly Ala Phe Leu Cys Ala Arg Glu
Ala Val Arg Arg Met Ser 115 120 125 Thr Arg His Gly Gly Val Gly Gly
Ala Ile Val Asn Val Ser Ser Ala 130 135 140 Ala Ala Arg Leu Gly Ser
Pro Asn Glu Tyr Val Asp Tyr Ala Ala Ser 145 150 155 160 Lys Gly Ala
Leu Asp Thr Met Thr Ile Gly Leu Ser Lys Glu Val Ala 165 170 175 Pro
Glu Gly Ile Arg Val Asn Gly Val Arg Pro Gly Thr Ile Tyr Thr 180 185
190 Asp Met His Ala Ser Gly Gly Glu Pro Gly Arg Val Asp Arg Leu Lys
195 200 205 Ser Val Ile Pro Leu Arg Arg Gly Gly Ser Val Glu Glu Val
Ala Gly 210 215 220 Ala Val Met Trp Leu Phe Ser Glu Glu Ala Gly Tyr
Thr Ser Gly Ser 225 230 235 240 Phe Ile Asp Val Ser Gly Gly Ser 245
1191611DNAUnknownObtained from environmental sample 119atgcaaaagc
ggtatgacgt cattgtcgtg ggcagcggga tcgccggcct cagttttgcg 60ctaaaagtcg
ccaaggcggg gcatcgcgta gggattttga ccaaaaaaga ccgtgctgaa
120agcaacacca attatgccca aggcggcatc gcggcagtca cttcgcagac
agatgatttc 180gagctgcatg tgcaggacac attgaccgcg ggagatggac
tctgcgacga ggcagtcgtc 240cgcacgatta tcggcgaggc tcccgcccga
atccaggagc tgatcgattt gggggtggcc 300ttctcacatt tggaagatgg
acgggtttcc ctccatcgcg aagggggtca ctcgaaaagg 360cgcattcttc
acgttcagga tgtcaccggc aaagcgattg aagaagccct cctccatgcc
420atcgaacagt cgccgctgat cgacctgaat gagcacgtct ttgccatcga
cttactgact 480gaacgcaagc tggcgctggc gggctttgag gtggaaggtg
ctaaaaaccg ggtggtcgga 540ctctatgcgc tcgatgaagc cactcaggag
gttcacgtat ttgaggctcc agtcgtcatg 600ctggcaacgg gaggcgtcgg
gcaggtctac ctctacagca ccaacccaag gatcgcgacc 660ggtgatggat
tggccatggc ttaccgggct ggcgccgaaa tccgcaacct cgagtgtatc
720caatttcatc ctacagcgct atacaccacc accaatgacc gctttctgat
cagcgaagcc 780gtccggggtg aaggggccat cctccgcaat caggagggag
aggctttcat ggctcgctac 840gatgaccgca aggacctcgc cccccgggat
attgtggcca gagcaattga cagtgaaatg 900aagcagtccg gctcatccca
tgtctggctc gacatcactc atcgggatga aaccgatctg 960cgggagcgtt
tccccaacat tttcgaggcc tgcctgaagg tcggagtcaa catggcgcaa
1020tcctccatcc cggtggttcc ggcgatgcac tacctctgcg gaggcgtagc
caccgacctc 1080aatgcggcca ccgacatcac tggactgttt gcctgtgggg
aagttgcctg cacgggattg 1140catggtgcca accgtctcgc cagcaacagc
ctgctggagg cagtggtcat ggcgcaccgg 1200gcctccgtcg cagtggatgc
atacctcaac agcaaacctc accgctatgc acaattgccg 1260gaatggacgg
atggcaacgt gcaggacagc gacgagcgtg tcgtgatcag ccacaactgg
1320gatgaactca aacgcacgat gtgggactac gtgggcatcg tccgcaccac
caagcggctt 1380cagcgcgcgc aacgacgcat tcgtcacctc cagcaggaaa
tcgaagagta ttactggaat 1440ttcaaggttg agtcctccct tctggagtta
cggaatctgg ttgtggtggc ggatctggtt 1500atccactgtg ccctccaacg
ccatgagagc cgtggcctgc attgcacccg ggattatccc 1560ggcaagttgc
ccaccccgat caataccgcc gttcgcagaa gaaccggtta a
1611120536PRTUnknownObtained from environmental sample 120Met Gln
Lys Arg Tyr Asp Val Ile Val Val Gly Ser Gly Ile Ala Gly 1 5 10 15
Leu Ser Phe Ala Leu Lys Val Ala Lys Ala Gly His Arg Val Gly Ile 20
25 30 Leu Thr Lys Lys Asp Arg Ala Glu Ser Asn Thr Asn Tyr Ala Gln
Gly 35 40 45 Gly Ile Ala Ala Val Thr Ser Gln Thr Asp Asp Phe Glu
Leu His Val 50 55 60 Gln Asp Thr Leu Thr Ala Gly Asp Gly Leu Cys
Asp Glu Ala Val Val 65 70 75 80 Arg Thr Ile Ile Gly Glu Ala Pro Ala
Arg Ile Gln Glu Leu Ile Asp 85 90 95 Leu Gly Val Ala Phe Ser His
Leu Glu Asp Gly Arg Val Ser Leu His 100 105 110 Arg Glu Gly Gly His
Ser Lys Arg Arg Ile Leu His Val Gln Asp Val 115 120 125 Thr Gly Lys
Ala Ile Glu Glu Ala Leu Leu His Ala Ile Glu Gln Ser 130 135 140 Pro
Leu Ile Asp Leu Asn Glu His Val Phe Ala Ile Asp Leu Leu Thr 145 150
155 160 Glu Arg Lys Leu Ala Leu Ala Gly Phe Glu Val Glu Gly Ala Lys
Asn 165 170 175 Arg Val Val Gly Leu Tyr Ala Leu Asp Glu Ala Thr Gln
Glu Val His 180 185 190 Val Phe Glu Ala Pro Val Val Met Leu Ala Thr
Gly Gly Val Gly Gln 195 200 205 Val Tyr Leu Tyr Ser Thr Asn Pro Arg
Ile Ala Thr Gly Asp Gly Leu 210 215 220 Ala Met Ala Tyr Arg Ala Gly
Ala Glu Ile Arg Asn Leu Glu Cys Ile 225 230 235 240 Gln Phe His Pro
Thr Ala Leu Tyr Thr Thr Thr Asn Asp Arg Phe Leu 245 250 255 Ile Ser
Glu Ala Val Arg Gly Glu Gly Ala Ile Leu Arg Asn Gln Glu 260 265 270
Gly Glu Ala Phe Met Ala Arg Tyr Asp Asp Arg Lys Asp Leu Ala Pro 275
280 285 Arg Asp Ile Val Ala Arg Ala Ile Asp Ser Glu Met Lys Gln Ser
Gly 290 295 300 Ser Ser His Val Trp Leu Asp Ile Thr His Arg Asp Glu
Thr Asp Leu 305 310 315 320 Arg Glu Arg Phe Pro Asn Ile Phe Glu Ala
Cys Leu Lys Val Gly Val 325 330 335 Asn Met Ala Gln Ser Ser Ile Pro
Val Val Pro Ala Met His Tyr Leu 340 345 350 Cys Gly Gly Val Ala Thr
Asp Leu Asn Ala Ala Thr Asp Ile Thr Gly 355 360 365 Leu Phe Ala Cys
Gly Glu Val Ala Cys Thr Gly Leu His Gly Ala Asn 370 375 380 Arg Leu
Ala Ser Asn Ser Leu Leu Glu Ala Val Val Met Ala His Arg 385 390 395
400 Ala Ser Val Ala Val Asp Ala Tyr Leu Asn Ser Lys Pro His Arg Tyr
405 410 415 Ala Gln Leu Pro Glu Trp Thr Asp Gly Asn Val Gln Asp Ser
Asp Glu 420 425 430 Arg Val Val Ile Ser His Asn Trp Asp Glu Leu Lys
Arg Thr Met Trp 435 440 445 Asp Tyr Val Gly Ile Val Arg Thr Thr Lys
Arg Leu Gln Arg Ala Gln 450 455 460 Arg Arg Ile Arg His Leu Gln Gln
Glu Ile Glu Glu Tyr Tyr Trp Asn 465 470 475 480 Phe Lys Val Glu Ser
Ser Leu Leu Glu Leu Arg Asn Leu Val Val Val 485 490 495 Ala Asp Leu
Val Ile His Cys Ala Leu Gln Arg His Glu Ser Arg Gly 500 505 510 Leu
His Cys Thr Arg Asp Tyr Pro Gly Lys Leu Pro Thr Pro Ile Asn 515 520
525 Thr Ala Val Arg Arg Arg Thr Gly 530 535
121990DNAUnknownObtained from environmental sample 121atgccttttg
atgccattgg agaaagcttc cgtgccagcc agcaactccc gctgatcaag 60gtcgacggca
accgtttcgt gattgcggag accggtgagc cgatcgtctt ccggggcgtc
120tccgcctccg acccggctgc gctactggaa cgcggtcaat ggggtcgccg
ttactttgaa 180gagatggcca agtggaatgc caacgttgtg cgcattcctg
ttcacccggc agactggcgt 240aatctcggcg aagacatcta tctcgcccta
ctcgaccagg cgattgaatg gtcggctgaa 300ctcggcatgc acgtcatcat
cgactggcac actatcggca atattctgac cggtatttat 360caccgcgaca
tttatgaaac cacccgtgat gagacttacc gtttttggta caccatcgcc
420attcgttatc agggtaaccc gacagtggcc ttttatgaac tctacaatga
gcccaccaac 480cgaggcggtc gcatgggccc ccttccctgg gaagaatatg
cccagttcat cgaagggctg 540atttccatgc tctacgccat cgacgacacc
gttattccac tggtcgctgg cttcgactgg 600ggatatgatt tgagctatgt
tgcggaacgc ccgatccgtt ttccaggagt cgcctatgtc 660acccaccctt
acccgcagaa gcgccccgag ccttgggaac cgatctggca ggaggaatgg
720ggttttgtcg ccgacaccta tcccatgatc gccactgagt ttggcttcat
gagtgaggac 780ggtcccggag cccacaaccc ggttatcggg gatgaacact
atggcgaatc ggtcatccgc 840tttttcgagg aacgcggcat ttcctggacg
gcctgggtgt ttgatcctct ctggtcaccc 900cagcttttcg aagactggga
aacctatacc cccacccggc aaggccgatt ctttaaacag 960aaaatgatgg
aactgaatcc cccgcgttga 990122329PRTUnknownObtained from
environmental sample 122Met Pro Phe Asp Ala Ile Gly Glu Ser Phe Arg
Ala Ser Gln Gln Leu 1 5 10 15 Pro Leu Ile Lys Val Asp Gly Asn Arg
Phe Val Ile Ala Glu Thr Gly 20 25 30 Glu Pro Ile Val Phe Arg Gly
Val Ser Ala Ser Asp Pro Ala Ala Leu 35 40 45 Leu Glu Arg Gly Gln
Trp Gly Arg Arg Tyr Phe Glu Glu Met Ala Lys 50 55 60 Trp Asn Ala
Asn Val Val Arg Ile Pro Val His Pro Ala Asp Trp Arg 65 70 75 80 Asn
Leu Gly Glu Asp Ile Tyr Leu Ala Leu Leu Asp Gln Ala Ile Glu 85 90
95 Trp Ser Ala Glu Leu Gly Met His Val Ile Ile Asp Trp His Thr Ile
100 105 110 Gly Asn Ile Leu Thr Gly Ile Tyr His Arg Asp Ile Tyr Glu
Thr Thr 115 120 125 Arg Asp Glu Thr Tyr Arg Phe Trp Tyr Thr Ile Ala
Ile Arg Tyr Gln 130 135 140 Gly Asn Pro Thr Val Ala Phe Tyr Glu Leu
Tyr Asn Glu Pro Thr Asn 145 150 155 160 Arg Gly Gly Arg Met Gly Pro
Leu Pro Trp Glu Glu Tyr Ala Gln Phe 165 170 175 Ile Glu Gly Leu Ile
Ser Met Leu Tyr Ala Ile Asp Asp Thr Val Ile 180 185 190 Pro Leu Val
Ala Gly Phe Asp Trp Gly Tyr Asp Leu Ser Tyr Val Ala 195 200 205 Glu
Arg Pro Ile Arg Phe Pro Gly Val Ala Tyr Val Thr His Pro Tyr 210 215
220 Pro Gln Lys Arg Pro Glu Pro Trp Glu Pro Ile Trp Gln Glu Glu Trp
225 230 235 240 Gly Phe Val Ala Asp Thr Tyr Pro Met Ile Ala Thr Glu
Phe Gly Phe 245 250 255 Met Ser Glu Asp Gly Pro Gly Ala His Asn Pro
Val Ile Gly Asp Glu 260 265 270 His Tyr Gly Glu Ser Val Ile Arg Phe
Phe Glu Glu Arg Gly Ile Ser 275 280 285 Trp Thr Ala Trp Val Phe Asp
Pro Leu Trp Ser Pro Gln Leu Phe Glu 290 295 300 Asp Trp Glu Thr Tyr
Thr Pro Thr Arg Gln Gly Arg Phe Phe Lys Gln 305 310 315 320 Lys Met
Met Glu Leu Asn Pro Pro Arg 325 1231398DNAUnknownObtained from
environmental sample 123atgccgatga gcacagaaac gacttttcct tctgatttca
cctggggcgc agcaacagcc 60gcctaccaga tcgaaggggg cgatcgcgct ggcgggcgcg
gccgttccgt gtgggacatg 120ttttgcgaga aacgaggagc tatttgggag
gggcatacgg ggcagcgagc gagtctgcat 180cttcagcgct ggcgtgagga
cgtaatgttg atgcaacagc tcggactgcg gggctatcgt 240tttagcgtca
gctggccgcg cgtcttcccg acaggagtcg gcaaagtcaa ccgtgaaggg
300ttggcctttt acgatcagct cgtagacgcc ttgctcgagg ccggcatcac
cccctttata 360acgctatttc attgggactt cccgctcgat ttgtaccacc
gaggcggctg gttgaatcgc 420gacagcgccg actggtttgc ctcctacgcc
gagtgcctcg gcaaggcact gggcgacagg 480gtcaagcact gggtgaccct
caacgagccg caggttttca taggcctcgg tcattacgaa 540gggcgtcatg
ccccggggtt gaagctctcc atcgcggaaa tgctgcgctg cgggcaccac
600gccttgctcg cgcacgggaa ggccgtgcaa gccctgcgcg cttccgtcga
cggcccctgc 660aagattggat ttgctccggt ggggattccc aagcttccgg
cgagtgagag ctcagaggat 720atcgccgcgg cacgaaaggc ccagttcgcg
gcgggagcgc cgccgtattg gacgctgagc 780tggtgggcgg atccggtgtt
tcaggggaca tatcccgctg atgcctgcca ggctctcgga 840gcggacgcgc
cgcaggtggc cgatcacgac atgagcatca tcagcgagcc gactgatttc
900ctgggcctca acctttatca aggggtggtg gtgcgtgccg atcacacggg
tcaaccagaa 960acggtgccgt ttccgccggg attccccgtg actgcgctca
actgggccgt aaccccagag 1020gcgctgtatt ggggcccgcg ctttgccttc
gaacgctaca aaaagccgat tcacatcacg 1080gaaaacgggc tatcctgtcg
tgactggccg tcgctcgacg ggcacgtcca cgacgccgac 1140cgcatcgact
tcatggcccg gcacttgcgc gcagcgcatc gagccattcg cgatgggata
1200ccgatcgaag gctacttcca ctggtctgcg atcgacaact tcgagtgggc
agaaggctac 1260aaggaacgct tcgggctcat ttacgtcgac tatacgagcg
gcgagcgcat tccgaaggac 1320tcgtaccact ggtaccagaa ggtcattgcc
tccgaggggc gggcagcgct cggcgcgccc 1380agtgctgctc gcccataa
1398124465PRTUnknownObtained from environmental sample 124Met Pro
Met Ser Thr Glu Thr Thr Phe Pro Ser Asp Phe Thr Trp Gly 1 5 10 15
Ala Ala Thr Ala Ala Tyr Gln Ile Glu Gly Gly Asp Arg Ala Gly Gly 20
25 30 Arg Gly Arg Ser Val Trp Asp Met Phe Cys Glu Lys Arg Gly Ala
Ile 35 40 45 Trp Glu Gly His Thr Gly Gln Arg Ala Ser Leu His Leu
Gln Arg Trp 50 55 60 Arg Glu Asp Val Met Leu Met Gln Gln Leu Gly
Leu Arg Gly Tyr Arg 65 70 75 80 Phe Ser Val Ser Trp Pro Arg Val Phe
Pro Thr Gly Val Gly Lys Val 85 90 95 Asn Arg Glu Gly Leu Ala Phe
Tyr Asp Gln Leu Val Asp Ala Leu Leu 100 105 110 Glu Ala Gly Ile Thr
Pro Phe Ile Thr Leu Phe His Trp Asp Phe Pro 115 120 125 Leu Asp Leu
Tyr His Arg Gly Gly Trp Leu Asn Arg Asp Ser Ala Asp 130 135 140 Trp
Phe Ala Ser Tyr Ala Glu Cys Leu Gly Lys Ala Leu Gly Asp Arg 145 150
155 160 Val Lys His Trp Val Thr Leu Asn Glu Pro Gln Val Phe Ile Gly
Leu 165 170 175 Gly His Tyr Glu Gly Arg His Ala Pro Gly Leu Lys Leu
Ser Ile Ala 180 185 190 Glu Met Leu Arg Cys Gly His His Ala Leu Leu
Ala His Gly Lys Ala 195 200 205 Val Gln Ala Leu Arg Ala Ser Val Asp
Gly Pro Cys Lys Ile Gly Phe 210 215 220 Ala Pro Val Gly Ile Pro Lys
Leu Pro Ala Ser Glu Ser Ser Glu Asp 225 230 235 240 Ile Ala Ala Ala
Arg Lys Ala Gln Phe Ala Ala Gly Ala Pro Pro Tyr 245 250 255 Trp Thr
Leu Ser Trp Trp Ala Asp Pro Val Phe Gln Gly Thr Tyr Pro 260 265 270
Ala Asp Ala Cys Gln Ala Leu Gly Ala Asp Ala Pro Gln Val Ala Asp 275
280 285 His Asp Met Ser Ile Ile Ser Glu Pro Thr Asp Phe Leu Gly Leu
Asn 290 295 300 Leu Tyr Gln Gly Val Val Val Arg Ala Asp His Thr Gly
Gln Pro Glu 305 310 315 320 Thr Val Pro Phe Pro Pro Gly Phe Pro Val
Thr Ala Leu Asn Trp Ala 325 330 335 Val Thr Pro Glu Ala Leu Tyr Trp
Gly Pro Arg Phe Ala Phe Glu Arg 340 345 350 Tyr Lys Lys Pro Ile His
Ile Thr Glu Asn Gly Leu Ser Cys Arg Asp 355 360 365 Trp Pro Ser Leu
Asp Gly His Val His Asp Ala Asp Arg Ile Asp Phe 370 375 380 Met Ala
Arg His Leu Arg Ala Ala His Arg Ala Ile Arg Asp Gly Ile 385 390 395
400 Pro Ile Glu Gly Tyr Phe His Trp Ser Ala Ile Asp Asn Phe Glu Trp
405 410 415 Ala Glu Gly Tyr Lys Glu Arg Phe Gly Leu Ile Tyr Val Asp
Tyr Thr 420 425 430 Ser Gly Glu Arg Ile Pro Lys Asp Ser Tyr His Trp
Tyr Gln Lys Val 435 440 445 Ile Ala Ser Glu Gly Arg Ala Ala Leu Gly
Ala Pro Ser Ala Ala Arg 450 455 460 Pro 465
1251350DNAUnknownObtained from environmental sample 125atgtcagatg
ccgccccgac tgatccgaaa tccgcaatgc ccagacgctc ggacttcccc 60gagggttttg
tcttcggcgc ggccaccgcg gcctatcaga tcgagggcca tgccttcggc
120ggcgcgggcc cctgccattg ggacagcttc gccgcaaccg ggcgtaacgt
ggtcggcaat 180gaggatggcg cgcgcgcctg cgagcattac acccgctggc
cgcaggatct ggacctgatc 240cgcgaggccg ggctcgacgc ctaccgcttc
tcgacctcct gggcgcgggt gatgcccgat 300ggcgtgaccc tgaaccccga
ggggctggat ttctacgacc gcctcgtcga tggcatgctc 360gagcgcgggc
taaagcccta tctcaccctc taccattggg aattgccctc ggcgcttgcc
420gacaggggcg gctggaccaa tcgcgacacg gccgagcgct ttgccgattt
cgcagcggtg 480gtgatggagc ggttgggcag ccgcgtcgcc cgcacggcca
ccatcaacga gccatggtgc 540gtgagctggc tctcgcattt cgaaggccat
cacgcgccgg gcctgcgcga catccgtgcc 600accgcacgcg ccatgcatca
tgtgcaactg gcgcacggcc tcgcgctcgg gaagctgcgc 660gcgcaggggc
atggcaatct cggcatcgtg ctgaatttct cggaaatcat tcccgccggg
720cgagagcacg cgaaggcggc tgatctcggc gacgcaatct cgaaccgctg
gttcatcgag 780tcagtcgcgc
gtggcaccta tcccgatgtg gtcctcgagg gtctgggcaa gcacatgccc
840gagggctggc aggatgacat gaaaaccatc gcggccccgc tcgactggct
gggtgtgaac 900tactacaccc gcggcatcgt cgcgcatgac ccggacgcgt
cctggccctc gacccgagcg 960gaggaggggc ccctgcccaa gacgcagatg
ggctgggaga tctaccccga gggcttgcgc 1020aacctgctgg tgcgcatggc
gcgcgactat gtgggcgacc ttcccatggt cgtgaccgaa 1080aacgggatgg
cctgggccga cgaggtcgcg gatggcgccg tcagagatac gatccgcacc
1140gaatatgtcg cagcccatct caacgcgacc cgcgaggcgc tggccggcgg
ggcgaatatc 1200gaaggtttct tctattggtc gctgctcgac aattacgaat
gggccttcgg ctatgccaag 1260cgcttcggcc tcgtccatgt cgatttcgac
acgatggcac gcacgccgaa agcctcctac 1320cacgcgctga gggccgcgct
gcagggttga 1350126449PRTUnknownObtained from environmental sample
126Met Ser Asp Ala Ala Pro Thr Asp Pro Lys Ser Ala Met Pro Arg Arg
1 5 10 15 Ser Asp Phe Pro Glu Gly Phe Val Phe Gly Ala Ala Thr Ala
Ala Tyr 20 25 30 Gln Ile Glu Gly His Ala Phe Gly Gly Ala Gly Pro
Cys His Trp Asp 35 40 45 Ser Phe Ala Ala Thr Gly Arg Asn Val Val
Gly Asn Glu Asp Gly Ala 50 55 60 Arg Ala Cys Glu His Tyr Thr Arg
Trp Pro Gln Asp Leu Asp Leu Ile 65 70 75 80 Arg Glu Ala Gly Leu Asp
Ala Tyr Arg Phe Ser Thr Ser Trp Ala Arg 85 90 95 Val Met Pro Asp
Gly Val Thr Leu Asn Pro Glu Gly Leu Asp Phe Tyr 100 105 110 Asp Arg
Leu Val Asp Gly Met Leu Glu Arg Gly Leu Lys Pro Tyr Leu 115 120 125
Thr Leu Tyr His Trp Glu Leu Pro Ser Ala Leu Ala Asp Arg Gly Gly 130
135 140 Trp Thr Asn Arg Asp Thr Ala Glu Arg Phe Ala Asp Phe Ala Ala
Val 145 150 155 160 Val Met Glu Arg Leu Gly Ser Arg Val Ala Arg Thr
Ala Thr Ile Asn 165 170 175 Glu Pro Trp Cys Val Ser Trp Leu Ser His
Phe Glu Gly His His Ala 180 185 190 Pro Gly Leu Arg Asp Ile Arg Ala
Thr Ala Arg Ala Met His His Val 195 200 205 Gln Leu Ala His Gly Leu
Ala Leu Gly Lys Leu Arg Ala Gln Gly His 210 215 220 Gly Asn Leu Gly
Ile Val Leu Asn Phe Ser Glu Ile Ile Pro Ala Gly 225 230 235 240 Arg
Glu His Ala Lys Ala Ala Asp Leu Gly Asp Ala Ile Ser Asn Arg 245 250
255 Trp Phe Ile Glu Ser Val Ala Arg Gly Thr Tyr Pro Asp Val Val Leu
260 265 270 Glu Gly Leu Gly Lys His Met Pro Glu Gly Trp Gln Asp Asp
Met Lys 275 280 285 Thr Ile Ala Ala Pro Leu Asp Trp Leu Gly Val Asn
Tyr Tyr Thr Arg 290 295 300 Gly Ile Val Ala His Asp Pro Asp Ala Ser
Trp Pro Ser Thr Arg Ala 305 310 315 320 Glu Glu Gly Pro Leu Pro Lys
Thr Gln Met Gly Trp Glu Ile Tyr Pro 325 330 335 Glu Gly Leu Arg Asn
Leu Leu Val Arg Met Ala Arg Asp Tyr Val Gly 340 345 350 Asp Leu Pro
Met Val Val Thr Glu Asn Gly Met Ala Trp Ala Asp Glu 355 360 365 Val
Ala Asp Gly Ala Val Arg Asp Thr Ile Arg Thr Glu Tyr Val Ala 370 375
380 Ala His Leu Asn Ala Thr Arg Glu Ala Leu Ala Gly Gly Ala Asn Ile
385 390 395 400 Glu Gly Phe Phe Tyr Trp Ser Leu Leu Asp Asn Tyr Glu
Trp Ala Phe 405 410 415 Gly Tyr Ala Lys Arg Phe Gly Leu Val His Val
Asp Phe Asp Thr Met 420 425 430 Ala Arg Thr Pro Lys Ala Ser Tyr His
Ala Leu Arg Ala Ala Leu Gln 435 440 445 Gly
127774DNAUnknownObtained from environmental sample 127atggacttgc
agctaggcgg aaagcgcgtg ctgatcacgg gtgcgtccaa aggcatcggc 60ctggcctgcg
ccgtcgcctt tgcgcgcgag ggtgccgacc cgattctggt ggcgcgcgat
120gatgcggcgt tgcatcacgc cacgtccgcc atccgcgaac aaagcggccg
cgcggcacat 180gccatcacgc tggacctggc cctgcctggc gcggcggaaa
agctggccaa ggaaaccggc 240cccatcgaca tactggtcaa caacgcgggc
gcggtgcccg gcggcgcgct ggaccaggtg 300caagacgaac gctggcgcgc
gggctgggaa ttgaaagtgc acggctacat cagcctggcg 360cgctgctact
acccgcacat gcgcgaagcg ggcgcgggcg tcatcgccaa catcatcggc
420atggcgggcg cggcgccccg cgccgactac atctgcggcg cggcggccaa
tgcctcactg 480attgccttta cccgcgcgct gggtggcgaa gcgccccgcc
acggcgtgcg cgtctttggc 540gtcaacccct cgcgcacgcg gaccgaccgc
gtgctgaccc tggcccggca acgcgcgcag 600gcgcgctggg gcgacgaaac
gcgttggcag gaaacgctgt cggacctgcc cttcaaccgg 660ctgatggaac
ccgccgaagt ggccgacatg attgtgttcg gcgcctcgcc acgcgcgggt
720tacctgagcg gcacggtcat cgacctggac ggcggcgaac agtacgcgaa atag
774128257PRTUnknownObtained from environmental sample 128Met Asp
Leu Gln Leu Gly Gly Lys Arg Val Leu Ile Thr Gly Ala Ser 1 5 10 15
Lys Gly Ile Gly Leu Ala Cys Ala Val Ala Phe Ala Arg Glu Gly Ala 20
25 30 Asp Pro Ile Leu Val Ala Arg Asp Asp Ala Ala Leu His His Ala
Thr 35 40 45 Ser Ala Ile Arg Glu Gln Ser Gly Arg Ala Ala His Ala
Ile Thr Leu 50 55 60 Asp Leu Ala Leu Pro Gly Ala Ala Glu Lys Leu
Ala Lys Glu Thr Gly 65 70 75 80 Pro Ile Asp Ile Leu Val Asn Asn Ala
Gly Ala Val Pro Gly Gly Ala 85 90 95 Leu Asp Gln Val Gln Asp Glu
Arg Trp Arg Ala Gly Trp Glu Leu Lys 100 105 110 Val His Gly Tyr Ile
Ser Leu Ala Arg Cys Tyr Tyr Pro His Met Arg 115 120 125 Glu Ala Gly
Ala Gly Val Ile Ala Asn Ile Ile Gly Met Ala Gly Ala 130 135 140 Ala
Pro Arg Ala Asp Tyr Ile Cys Gly Ala Ala Ala Asn Ala Ser Leu 145 150
155 160 Ile Ala Phe Thr Arg Ala Leu Gly Gly Glu Ala Pro Arg His Gly
Val 165 170 175 Arg Val Phe Gly Val Asn Pro Ser Arg Thr Arg Thr Asp
Arg Val Leu 180 185 190 Thr Leu Ala Arg Gln Arg Ala Gln Ala Arg Trp
Gly Asp Glu Thr Arg 195 200 205 Trp Gln Glu Thr Leu Ser Asp Leu Pro
Phe Asn Arg Leu Met Glu Pro 210 215 220 Ala Glu Val Ala Asp Met Ile
Val Phe Gly Ala Ser Pro Arg Ala Gly 225 230 235 240 Tyr Leu Ser Gly
Thr Val Ile Asp Leu Asp Gly Gly Glu Gln Tyr Ala 245 250 255 Lys
129747DNAUnknownObtained from environmental sample 129atgcccaaag
tcatgctcgt taccggcggc agccgtggca tcggcgccgc cgtcgccaag 60ctggccgcgc
gccgcggcta cgcggtcggc atcaactacc gcacccattc cgacgccgcc
120gacgccgtcg tggccgaaat ccagcaggcg ggcggcaccg cgctggccat
ccaggccgac 180gtgtcgcagg aagacgatgt gctgcacatg ttccgcacgc
tggacgagcg cctgggccgc 240atcgacgcgc tggtcaataa cgccggcatc
ctggaaacgc agatgcgcct ggaccagatg 300gaagccgacc gcctgctgcg
cgtgctgtcc accaacgtca tcggcgcttt cctatgtgcg 360cgcgaagccg
tgcgcaggat gtcgacgcgc catggcggcg tgggcggcgc catcgtcaac
420gtgtcttcgg cggcggcgcg cctgggctcg cccaatgaat acgtggatta
cgcggcctcc 480aagggcgcgc tggacacgat gaccatcggc ctgtcgaaag
aggtggcgcc cgaaggtatc 540cgcgtgaatg gcgtgcgccc cggcaccatc
tacaccgaca tgcacgcaag cggcggcgag 600ccgggccggg tggatcgcct
gaaaagcgtg atcccgctgc ggcgcggcgg ctcggtggaa 660gaagtggcgg
gcgccgtcat gtggctgttt tccgaagaag ccggctatac cagcggttcg
720ttcatcgacg tgtccggcgg tagttga 747130248PRTUnknownObtained from
environmental sample 130Met Pro Lys Val Met Leu Val Thr Gly Gly Ser
Arg Gly Ile Gly Ala 1 5 10 15 Ala Val Ala Lys Leu Ala Ala Arg Arg
Gly Tyr Ala Val Gly Ile Asn 20 25 30 Tyr Arg Thr His Ser Asp Ala
Ala Asp Ala Val Val Ala Glu Ile Gln 35 40 45 Gln Ala Gly Gly Thr
Ala Leu Ala Ile Gln Ala Asp Val Ser Gln Glu 50 55 60 Asp Asp Val
Leu His Met Phe Arg Thr Leu Asp Glu Arg Leu Gly Arg 65 70 75 80 Ile
Asp Ala Leu Val Asn Asn Ala Gly Ile Leu Glu Thr Gln Met Arg 85 90
95 Leu Asp Gln Met Glu Ala Asp Arg Leu Leu Arg Val Leu Ser Thr Asn
100 105 110 Val Ile Gly Ala Phe Leu Cys Ala Arg Glu Ala Val Arg Arg
Met Ser 115 120 125 Thr Arg His Gly Gly Val Gly Gly Ala Ile Val Asn
Val Ser Ser Ala 130 135 140 Ala Ala Arg Leu Gly Ser Pro Asn Glu Tyr
Val Asp Tyr Ala Ala Ser 145 150 155 160 Lys Gly Ala Leu Asp Thr Met
Thr Ile Gly Leu Ser Lys Glu Val Ala 165 170 175 Pro Glu Gly Ile Arg
Val Asn Gly Val Arg Pro Gly Thr Ile Tyr Thr 180 185 190 Asp Met His
Ala Ser Gly Gly Glu Pro Gly Arg Val Asp Arg Leu Lys 195 200 205 Ser
Val Ile Pro Leu Arg Arg Gly Gly Ser Val Glu Glu Val Ala Gly 210 215
220 Ala Val Met Trp Leu Phe Ser Glu Glu Ala Gly Tyr Thr Ser Gly Ser
225 230 235 240 Phe Ile Asp Val Ser Gly Gly Ser 245
1311041DNAUnknownObtained from environmental sample 131gtggaaacct
attttcccct gcaccgcggg atcaacatga gccactggct ttcgcaagtg 60aatgaaaaca
ttcccgaccg ttccacctat gtgacggagc gggacctgca atttttgcgg
120gcagcgggct tcgaccatgt gcgtctgccg atcgatgaga tcgaactctg
ggatgaggag 180ggccatcaga tcgaggaggc ctggcaatac atgcacaact
ttatgcgctg gagccgaaag 240aatgacctcc gggttattct cgacctgcac
acggtattgt cccaccactt caacgcgatc 300aacatgggag aggtcaacac
cctctttaat gatcccaagg aacaggaaaa attcctcaat 360ctctgggagc
aaatcatgga tgccgtaggg caccacccca acgagtttct cgcttatgaa
420atgctcaatg aggcggtcgc ggaagatgat gaagactgga acctgctcct
caaccgtgcg 480attgaacgca tccgggaacg tgagccgcat cgcgttctga
ttgccggggc caactggtgg 540cagcatgccg cccgcgttcc caacctgagg
cttccccctg gtgatcccaa catcatcatc 600agttttcact tttactcacc
ctttctcttc acgcactatc gcagcagctg gactgccatg 660cgggcatacc
agggtttcgt ccaatacccc ggcattacca ttcccgccat ccatctcgaa
720ggaatgaact atccggagtc ctttgtccaa atgtgggaag agcacaatca
gtattacgac 780atccattcaa tgtatgccga aatggtcccg gcggtgcgtt
ttgccgaaaa gctgggcctt 840cggctctatt gcggcgaatt tggagccatg
aagaccgttg atcgtgccca aatgctgcag 900tggtatcggg atgtggtcag
agtctttgaa atgttggaca ttccctacac tgcctgggat 960tatcagggaa
cctttggaat ccgcgatgag ctgaccggtg agcctgatca tgaactgatc
1020gacattctcc tcggccgcta a 1041132346PRTUnknownObtained from
environmental sample 132Met Glu Thr Tyr Phe Pro Leu His Arg Gly Ile
Asn Met Ser His Trp 1 5 10 15 Leu Ser Gln Val Asn Glu Asn Ile Pro
Asp Arg Ser Thr Tyr Val Thr 20 25 30 Glu Arg Asp Leu Gln Phe Leu
Arg Ala Ala Gly Phe Asp His Val Arg 35 40 45 Leu Pro Ile Asp Glu
Ile Glu Leu Trp Asp Glu Glu Gly His Gln Ile 50 55 60 Glu Glu Ala
Trp Gln Tyr Met His Asn Phe Met Arg Trp Ser Arg Lys 65 70 75 80 Asn
Asp Leu Arg Val Ile Leu Asp Leu His Thr Val Leu Ser His His 85 90
95 Phe Asn Ala Ile Asn Met Gly Glu Val Asn Thr Leu Phe Asn Asp Pro
100 105 110 Lys Glu Gln Glu Lys Phe Leu Asn Leu Trp Glu Gln Ile Met
Asp Ala 115 120 125 Val Gly His His Pro Asn Glu Phe Leu Ala Tyr Glu
Met Leu Asn Glu 130 135 140 Ala Val Ala Glu Asp Asp Glu Asp Trp Asn
Leu Leu Leu Asn Arg Ala 145 150 155 160 Ile Glu Arg Ile Arg Glu Arg
Glu Pro His Arg Val Leu Ile Ala Gly 165 170 175 Ala Asn Trp Trp Gln
His Ala Ala Arg Val Pro Asn Leu Arg Leu Pro 180 185 190 Pro Gly Asp
Pro Asn Ile Ile Ile Ser Phe His Phe Tyr Ser Pro Phe 195 200 205 Leu
Phe Thr His Tyr Arg Ser Ser Trp Thr Ala Met Arg Ala Tyr Gln 210 215
220 Gly Phe Val Gln Tyr Pro Gly Ile Thr Ile Pro Ala Ile His Leu Glu
225 230 235 240 Gly Met Asn Tyr Pro Glu Ser Phe Val Gln Met Trp Glu
Glu His Asn 245 250 255 Gln Tyr Tyr Asp Ile His Ser Met Tyr Ala Glu
Met Val Pro Ala Val 260 265 270 Arg Phe Ala Glu Lys Leu Gly Leu Arg
Leu Tyr Cys Gly Glu Phe Gly 275 280 285 Ala Met Lys Thr Val Asp Arg
Ala Gln Met Leu Gln Trp Tyr Arg Asp 290 295 300 Val Val Arg Val Phe
Glu Met Leu Asp Ile Pro Tyr Thr Ala Trp Asp 305 310 315 320 Tyr Gln
Gly Thr Phe Gly Ile Arg Asp Glu Leu Thr Gly Glu Pro Asp 325 330 335
His Glu Leu Ile Asp Ile Leu Leu Gly Arg 340 345
1331377DNAUnknownObtained from environmental sample 133atgacacaac
tggcttttcc atctaacttc atctggggaa cagctacttc cgcttaccaa 60atcgaaggcg
cctggaacgc agacggcaag ggcgaatcta tttgggatcg cttttcccat
120acgcagggga agatcattga cggcagcaac ggcgatgtgg cctgcgatca
ctaccaccgc 180tggcgcgagg acgtggccct catgagagac ttgggtatgc
aggcatatcg cttctccatc 240tcctggccac gcatcctgcc caccggtcat
ggacagatca atcaggctgg gctggacttt 300tacaatcgcc tggtggacgg
gttgctggaa gctggcatca agccctttgc caccctctac 360cactgggacc
tgccgctggc gctacaggct gacggcggct ggccggagcg ctccacggcc
420aaggcctttg tcgaatacgc cgacgtggtc agccgcgcgc tgggcgatcg
ggtgaagagc 480tggatcaccc ataacgaacc gtggtgcatc agcatgctga
gccatcaaat tggggagcat 540gcgcccggct ggcgggactg gcaggctgcg
ttggcggccg cgcaccacgt cctcctttcg 600catggttggg ccgtgccgga
actgcgtcgc aacagccgcg atgcagaaat cggcatcacg 660ttgaacttta
ccccggcgga gccagcttcg aacagcgcag ccgatttcaa ggcctatcgc
720cagttcgatg gctacttcaa ccgctggttc ctggacccgc tctatggccg
ccactatccg 780gcagatatgg tgcacgatta catcgcgcaa ggctacctgc
catcacaggg tttgactttc 840gtggaagctg gtgacctgga cgcgatcgcg
acgcgcaccg atttcctggg tgtgaactat 900tacacgcgcg aagtggtccg
tagccaggaa atcccagaga gtgagaacgc gccgcgcaca 960gtcttgcgcg
cgccacagga agagtggaca gagatgggct gggaagtgta tcctgagggc
1020ctctacaggt tgctcaatcg gttgcacttt gaataccagc cgcgcaagct
ctacgtgacc 1080gagagcggtt gcagctactc cgatggaccc ggccccaacg
gtcggatacc ggaccaacgc 1140cgtatcaact acctgcgcga tcacttcgca
gcggcgcatc aggcgataca atgcggcgtc 1200ccgctggccg gctacttcgt
ctggtcgttc atggacaact tcgagtgggc caaagggtac 1260acccaacgtt
ttggtatcgt atgggtggat tatcaatcgc aacgacggat accgaaagac
1320agcgcctact ggtatcgcga tgtcgtcgcc gccaacgcgg tgcaagttcc tgattag
1377134458PRTUnknownObtained from environmental sample 134Met Thr
Gln Leu Ala Phe Pro Ser Asn Phe Ile Trp Gly Thr Ala Thr 1 5 10 15
Ser Ala Tyr Gln Ile Glu Gly Ala Trp Asn Ala Asp Gly Lys Gly Glu 20
25 30 Ser Ile Trp Asp Arg Phe Ser His Thr Gln Gly Lys Ile Ile Asp
Gly 35 40 45 Ser Asn Gly Asp Val Ala Cys Asp His Tyr His Arg Trp
Arg Glu Asp 50 55 60 Val Ala Leu Met Arg Asp Leu Gly Met Gln Ala
Tyr Arg Phe Ser Ile 65 70 75 80 Ser Trp Pro Arg Ile Leu Pro Thr Gly
His Gly Gln Ile Asn Gln Ala 85 90 95 Gly Leu Asp Phe Tyr Asn Arg
Leu Val Asp Gly Leu Leu Glu Ala Gly 100 105 110 Ile Lys Pro Phe Ala
Thr Leu Tyr His Trp Asp Leu Pro Leu Ala Leu 115 120 125 Gln Ala Asp
Gly Gly Trp Pro Glu Arg Ser Thr Ala Lys Ala Phe Val 130 135 140 Glu
Tyr Ala Asp Val Val Ser Arg Ala Leu Gly Asp Arg Val Lys Ser 145 150
155 160 Trp Ile Thr His Asn Glu Pro Trp Cys Ile Ser Met Leu Ser His
Gln 165 170 175 Ile Gly Glu His Ala Pro Gly Trp Arg Asp Trp Gln Ala
Ala Leu Ala 180 185 190 Ala Ala His His Val Leu Leu Ser His Gly Trp
Ala Val Pro Glu Leu 195 200 205 Arg Arg Asn Ser Arg Asp Ala Glu Ile
Gly Ile Thr Leu Asn Phe Thr 210 215 220 Pro Ala Glu Pro Ala Ser Asn
Ser Ala Ala Asp Phe
Lys Ala Tyr Arg 225 230 235 240 Gln Phe Asp Gly Tyr Phe Asn Arg Trp
Phe Leu Asp Pro Leu Tyr Gly 245 250 255 Arg His Tyr Pro Ala Asp Met
Val His Asp Tyr Ile Ala Gln Gly Tyr 260 265 270 Leu Pro Ser Gln Gly
Leu Thr Phe Val Glu Ala Gly Asp Leu Asp Ala 275 280 285 Ile Ala Thr
Arg Thr Asp Phe Leu Gly Val Asn Tyr Tyr Thr Arg Glu 290 295 300 Val
Val Arg Ser Gln Glu Ile Pro Glu Ser Glu Asn Ala Pro Arg Thr 305 310
315 320 Val Leu Arg Ala Pro Gln Glu Glu Trp Thr Glu Met Gly Trp Glu
Val 325 330 335 Tyr Pro Glu Gly Leu Tyr Arg Leu Leu Asn Arg Leu His
Phe Glu Tyr 340 345 350 Gln Pro Arg Lys Leu Tyr Val Thr Glu Ser Gly
Cys Ser Tyr Ser Asp 355 360 365 Gly Pro Gly Pro Asn Gly Arg Ile Pro
Asp Gln Arg Arg Ile Asn Tyr 370 375 380 Leu Arg Asp His Phe Ala Ala
Ala His Gln Ala Ile Gln Cys Gly Val 385 390 395 400 Pro Leu Ala Gly
Tyr Phe Val Trp Ser Phe Met Asp Asn Phe Glu Trp 405 410 415 Ala Lys
Gly Tyr Thr Gln Arg Phe Gly Ile Val Trp Val Asp Tyr Gln 420 425 430
Ser Gln Arg Arg Ile Pro Lys Asp Ser Ala Tyr Trp Tyr Arg Asp Val 435
440 445 Val Ala Ala Asn Ala Val Gln Val Pro Asp 450 455
135987DNAUnknownObtained from environmental sample 135atggttgagc
ctgccgatca gagtcatttt tcagatgctt ttcaggtaaa tcgcactctt 60ggaaaaggca
tcaatcttgg taacacactg gaggctccaa atgaaggcga gtggggattg
120acaattcgcg aggagtattt tgatgaagtg aaacaagccg gatttgaatc
cgtgcgtatt 180ccgatacgat ggaatgctca tgctctggaa ggttttccat
atacgataga tgaatctttt 240tttgaccggg ttgatgaagt tattggctgg
gcttttgatc gtgatcttgc agtcatgatt 300aacattcatc actacaacga
attgatggag cagccacagg atcaccggga tcgctttttg 360aaactttggg
agcaaattgc tgcgcactat aaagagtacc cggaagaact ggtattcgag
420attttaaacg aaccccacga taatctgacc ccggctatct ggaatagctt
tttggctgat 480gctctcggta ttatacgcca aaccaatcca ggaagggtta
ttgcagtcgg aacagctgaa 540tggggcggtt tcgggagttt gcaggatctt
gagctgcctg ataatgaccg ccagataatc 600accaccgttc attactataa
cccatttcat ttcacgcatc agggggcaga ttgggttgga 660gatgaagcgg
atcagtggct tggaaccgaa tgggatggag cagatcatga aaaagctgaa
720gttgacagcg attttgactc tgtggaacag tgggcccgaa atcatgaccg
gccaatacac 780gtgggagagt tcggagcttt cagcgccgca gatgatttgt
cacgtgaaca gtggacggca 840tacgtacgtg agtcttcgga gaaccggcag
tttagctggg cgtattggga gtttgggtca 900gggttcggtg cctatgatcc
cggttccgga gaatggcgtg aatatttact ccgggcgtta 960atccccgaca
gtccggtgat tgattaa 987136328PRTUnknownObtained from environmental
sample 136Met Val Glu Pro Ala Asp Gln Ser His Phe Ser Asp Ala Phe
Gln Val 1 5 10 15 Asn Arg Thr Leu Gly Lys Gly Ile Asn Leu Gly Asn
Thr Leu Glu Ala 20 25 30 Pro Asn Glu Gly Glu Trp Gly Leu Thr Ile
Arg Glu Glu Tyr Phe Asp 35 40 45 Glu Val Lys Gln Ala Gly Phe Glu
Ser Val Arg Ile Pro Ile Arg Trp 50 55 60 Asn Ala His Ala Leu Glu
Gly Phe Pro Tyr Thr Ile Asp Glu Ser Phe 65 70 75 80 Phe Asp Arg Val
Asp Glu Val Ile Gly Trp Ala Phe Asp Arg Asp Leu 85 90 95 Ala Val
Met Ile Asn Ile His His Tyr Asn Glu Leu Met Glu Gln Pro 100 105 110
Gln Asp His Arg Asp Arg Phe Leu Lys Leu Trp Glu Gln Ile Ala Ala 115
120 125 His Tyr Lys Glu Tyr Pro Glu Glu Leu Val Phe Glu Ile Leu Asn
Glu 130 135 140 Pro His Asp Asn Leu Thr Pro Ala Ile Trp Asn Ser Phe
Leu Ala Asp 145 150 155 160 Ala Leu Gly Ile Ile Arg Gln Thr Asn Pro
Gly Arg Val Ile Ala Val 165 170 175 Gly Thr Ala Glu Trp Gly Gly Phe
Gly Ser Leu Gln Asp Leu Glu Leu 180 185 190 Pro Asp Asn Asp Arg Gln
Ile Ile Thr Thr Val His Tyr Tyr Asn Pro 195 200 205 Phe His Phe Thr
His Gln Gly Ala Asp Trp Val Gly Asp Glu Ala Asp 210 215 220 Gln Trp
Leu Gly Thr Glu Trp Asp Gly Ala Asp His Glu Lys Ala Glu 225 230 235
240 Val Asp Ser Asp Phe Asp Ser Val Glu Gln Trp Ala Arg Asn His Asp
245 250 255 Arg Pro Ile His Val Gly Glu Phe Gly Ala Phe Ser Ala Ala
Asp Asp 260 265 270 Leu Ser Arg Glu Gln Trp Thr Ala Tyr Val Arg Glu
Ser Ser Glu Asn 275 280 285 Arg Gln Phe Ser Trp Ala Tyr Trp Glu Phe
Gly Ser Gly Phe Gly Ala 290 295 300 Tyr Asp Pro Gly Ser Gly Glu Trp
Arg Glu Tyr Leu Leu Arg Ala Leu 305 310 315 320 Ile Pro Asp Ser Pro
Val Ile Asp 325 137702DNAUnknownObtained from environmental sample
137atgagccacc gatcgcagga attcaacggc cagccactga tggtgtccga
agacggccac 60ttcgtgctcg gattcgggcg cgacgacgag gccacccacc gactgcgcgt
tcagctaccg 120gatgagcgag tctgggagaa gaatctgcgt ccggaatcgc
gcgagttcga tattcagcgg 180atcgacggct tgccgcaaga ccaggtcacc
ccaccccact ccgtgctggc gagaatccga 240gaggacgctt cgctgtcgcg
ccgtgcccgc gaacgacgcg atccgcggac cgactggacc 300gatggctgga
tctggccggc cgagggccgc atttccggcg tgtacggcag ccagcgcatc
360ctcaacggtg agcctcgcaa cccgcactgg gggctggata tcgccgcgcc
aaccggcagc 420ccggtcgtgg cgcctgccgg cggcatcgtc agcctgactc
atccggacat gtatttttcc 480ggcggcaccc tgttaatcga ccacggtcac
ggcctggtgt ctgcgttcct ccacctgagt 540gaaatcctgg tcgaggaagg
gcagcgggtc gagcaggggg atctgatcgc acgcattggc 600gccaccggtc
gtgccaccgg gccgcacctg gactggcgga tcaatctcgg cgatgtacgc
660gtggacccac agctgctgct gccgccgatg gacgcgcagt ga
702138233PRTUnknownObtained from environmental sample 138Met Ser
His Arg Ser Gln Glu Phe Asn Gly Gln Pro Leu Met Val Ser 1 5 10 15
Glu Asp Gly His Phe Val Leu Gly Phe Gly Arg Asp Asp Glu Ala Thr 20
25 30 His Arg Leu Arg Val Gln Leu Pro Asp Glu Arg Val Trp Glu Lys
Asn 35 40 45 Leu Arg Pro Glu Ser Arg Glu Phe Asp Ile Gln Arg Ile
Asp Gly Leu 50 55 60 Pro Gln Asp Gln Val Thr Pro Pro His Ser Val
Leu Ala Arg Ile Arg 65 70 75 80 Glu Asp Ala Ser Leu Ser Arg Arg Ala
Arg Glu Arg Arg Asp Pro Arg 85 90 95 Thr Asp Trp Thr Asp Gly Trp
Ile Trp Pro Ala Glu Gly Arg Ile Ser 100 105 110 Gly Val Tyr Gly Ser
Gln Arg Ile Leu Asn Gly Glu Pro Arg Asn Pro 115 120 125 His Trp Gly
Leu Asp Ile Ala Ala Pro Thr Gly Ser Pro Val Val Ala 130 135 140 Pro
Ala Gly Gly Ile Val Ser Leu Thr His Pro Asp Met Tyr Phe Ser 145 150
155 160 Gly Gly Thr Leu Leu Ile Asp His Gly His Gly Leu Val Ser Ala
Phe 165 170 175 Leu His Leu Ser Glu Ile Leu Val Glu Glu Gly Gln Arg
Val Glu Gln 180 185 190 Gly Asp Leu Ile Ala Arg Ile Gly Ala Thr Gly
Arg Ala Thr Gly Pro 195 200 205 His Leu Asp Trp Arg Ile Asn Leu Gly
Asp Val Arg Val Asp Pro Gln 210 215 220 Leu Leu Leu Pro Pro Met Asp
Ala Gln 225 230 139351DNAUnknownObtained from environmental sample
139atggaaaaaa ttctcgttat cggatgcgcg ggccagatag gctcagagct
tacgctcgaa 60cttcgtaaga tttatggtga tgacaatgtg gtggctactg acattaagcc
ggccagcaag 120gaaattaccg agggcggccc ctttgaaatt cttgatgtgc
tcgacaccca ccggcttttt 180ggcactgtaa gccgcaacaa gatcacccag
atttatcacc ttgcagccat cctttcgggc 240aatgccgaga aaaaaccact
tgcaagctgg cacattaaca tggagagttt gctcaacgtg 300cttgaactgg
cccgtgaact gaagcttcat aaaattttct ggccaagctc a
351140117PRTUnknownObtained from environmental sample 140Met Glu
Lys Ile Leu Val Ile Gly Cys Ala Gly Gln Ile Gly Ser Glu 1 5 10 15
Leu Thr Leu Glu Leu Arg Lys Ile Tyr Gly Asp Asp Asn Val Val Ala 20
25 30 Thr Asp Ile Lys Pro Ala Ser Lys Glu Ile Thr Glu Gly Gly Pro
Phe 35 40 45 Glu Ile Leu Asp Val Leu Asp Thr His Arg Leu Phe Gly
Thr Val Ser 50 55 60 Arg Asn Lys Ile Thr Gln Ile Tyr His Leu Ala
Ala Ile Leu Ser Gly 65 70 75 80 Asn Ala Glu Lys Lys Pro Leu Ala Ser
Trp His Ile Asn Met Glu Ser 85 90 95 Leu Leu Asn Val Leu Glu Leu
Ala Arg Glu Leu Lys Leu His Lys Ile 100 105 110 Phe Trp Pro Ser Ser
115 1411350DNAUnknownObtained from environmental sample
141atgctgtcct atacgagtcc gttcccaaag aactttgtct ggggtgtggc
gacggcggcg 60ccgcagatcg agggcgctgc gcgagaagac ggaaagggcg aatcgatatg
ggatcgcttt 120tgccgcgtgc ccggaaaggt ccacaatggc gatactctcg
atgttgcgtg cgaccactac 180caccggttcc gggaggattt cgcgctcatg
cgagacttgg gcgtgcgcca ctaccggctt 240tcgcttgcct ggccccgcat
attcccggac ggcgacggcg cattgaacca gcgcggagtg 300gatttctacc
accggctctt tgaggccatg atcgagcacg ggattacgcc ttgggtgacg
360ctctttcact gggatttgcc gcaggcgctc gaggaccgcg gcggctggtg
tgagcgtctc 420accgtcgatg cattcgggcg ctacgctgac accgtggtga
aggcgtttgg cgatcgcgtg 480aagaattgga tcaccctgaa cgaaatccgc
tgcttcacgt tgctcgctta cgatctctgc 540atcaaggccc cgggccgcaa
ggtctcgcgg gcgcagctca accagaccta tcatcacgcg 600ctgatctgcc
atgggcatgg cgtccgggcg gtccgcgaac acggcgggcg aggcgctcgc
660gtcgggctta ccgacaacag cgacgtatgc gtgcccgtca ccgagaccgc
gcccgacatc 720attgcggcca gatcctggta tgcgtcgcga aatattcatc
tgctcgatcc gatctatcgc 780ggcgagtatg cgccggaata cctcgaacgc
tgcggtgcgg acgcgcccca ggtggccgag 840gacgatttcg cgctgatttc
aatgccgacg gattttctcg ggctgaatgt atatacggcg 900acctttgtgc
gtgccgacgc ggagggcagg ccggaggaga ttaaactgcc gcggaattac
960ccgcgcgcgg atagcgcgtg gttgaatatt gtgccccagt cgatgtactg
ggccacacgg 1020ctggcgcggg aaacctacgg cgtgagatca atctacatca
ccgaaaacgg ctgcggctac 1080gacgacgagc ccgtcgacgg cggcgaggtg
ctcgacctgc atcgacgcga ttttctgcgc 1140aaccaccttc gggaattgca
tcgcgccata ggcgacggcg tgcccgttga cgggtatttt 1200ctctggtcct
tcatggacaa ctacgagtgg gaggacgggt atgcgcggcg gttcggcatc
1260gttcacgtcg acttcgaaag ccagaaacgg actccaaaac tctcggcgcg
ctattacgcg 1320caggtaatga aagaaaaccg gatcctgtga
1350142449PRTUnknownObtained from environmental sample 142Met Leu
Ser Tyr Thr Ser Pro Phe Pro Lys Asn Phe Val Trp Gly Val 1 5 10 15
Ala Thr Ala Ala Pro Gln Ile Glu Gly Ala Ala Arg Glu Asp Gly Lys 20
25 30 Gly Glu Ser Ile Trp Asp Arg Phe Cys Arg Val Pro Gly Lys Val
His 35 40 45 Asn Gly Asp Thr Leu Asp Val Ala Cys Asp His Tyr His
Arg Phe Arg 50 55 60 Glu Asp Phe Ala Leu Met Arg Asp Leu Gly Val
Arg His Tyr Arg Leu 65 70 75 80 Ser Leu Ala Trp Pro Arg Ile Phe Pro
Asp Gly Asp Gly Ala Leu Asn 85 90 95 Gln Arg Gly Val Asp Phe Tyr
His Arg Leu Phe Glu Ala Met Ile Glu 100 105 110 His Gly Ile Thr Pro
Trp Val Thr Leu Phe His Trp Asp Leu Pro Gln 115 120 125 Ala Leu Glu
Asp Arg Gly Gly Trp Cys Glu Arg Leu Thr Val Asp Ala 130 135 140 Phe
Gly Arg Tyr Ala Asp Thr Val Val Lys Ala Phe Gly Asp Arg Val 145 150
155 160 Lys Asn Trp Ile Thr Leu Asn Glu Ile Arg Cys Phe Thr Leu Leu
Ala 165 170 175 Tyr Asp Leu Cys Ile Lys Ala Pro Gly Arg Lys Val Ser
Arg Ala Gln 180 185 190 Leu Asn Gln Thr Tyr His His Ala Leu Ile Cys
His Gly His Gly Val 195 200 205 Arg Ala Val Arg Glu His Gly Gly Arg
Gly Ala Arg Val Gly Leu Thr 210 215 220 Asp Asn Ser Asp Val Cys Val
Pro Val Thr Glu Thr Ala Pro Asp Ile 225 230 235 240 Ile Ala Ala Arg
Ser Trp Tyr Ala Ser Arg Asn Ile His Leu Leu Asp 245 250 255 Pro Ile
Tyr Arg Gly Glu Tyr Ala Pro Glu Tyr Leu Glu Arg Cys Gly 260 265 270
Ala Asp Ala Pro Gln Val Ala Glu Asp Asp Phe Ala Leu Ile Ser Met 275
280 285 Pro Thr Asp Phe Leu Gly Leu Asn Val Tyr Thr Ala Thr Phe Val
Arg 290 295 300 Ala Asp Ala Glu Gly Arg Pro Glu Glu Ile Lys Leu Pro
Arg Asn Tyr 305 310 315 320 Pro Arg Ala Asp Ser Ala Trp Leu Asn Ile
Val Pro Gln Ser Met Tyr 325 330 335 Trp Ala Thr Arg Leu Ala Arg Glu
Thr Tyr Gly Val Arg Ser Ile Tyr 340 345 350 Ile Thr Glu Asn Gly Cys
Gly Tyr Asp Asp Glu Pro Val Asp Gly Gly 355 360 365 Glu Val Leu Asp
Leu His Arg Arg Asp Phe Leu Arg Asn His Leu Arg 370 375 380 Glu Leu
His Arg Ala Ile Gly Asp Gly Val Pro Val Asp Gly Tyr Phe 385 390 395
400 Leu Trp Ser Phe Met Asp Asn Tyr Glu Trp Glu Asp Gly Tyr Ala Arg
405 410 415 Arg Phe Gly Ile Val His Val Asp Phe Glu Ser Gln Lys Arg
Thr Pro 420 425 430 Lys Leu Ser Ala Arg Tyr Tyr Ala Gln Val Met Lys
Glu Asn Arg Ile 435 440 445 Leu 1431188DNAUnknownObtained from
environmental sample 143atgaccatca ccttccccga cgggttctgg tgggggacgg
cgacggccgc ccaccaggtg 60gagggcggca actggaacac cgactggtgg gcctacgagc
acgccccggg cacccgctgc 120gcggagccgt ccggcgatgc gtgcgaccac
tggcaccgct acccggagga catcgccctc 180ctcgccgcgc tcgggttcag
tgcctaccgc ttctcggtgg aatgggctcg catcgagccc 240gaggaagggc
atttctcccg cgccaccctc gaccactacc ggcgcatgat cgcctgctgc
300cgcgaccacg ggctggcccc ggtggtgacc ttccaccact tcaccacccc
ccgctgggcc 360gcggccgggg gctgctggtc cgacccggtc accgccgagc
gcttcgcccg ttactgcgag 420cgcaccgtgg ccgccctcgg cgacgagatc
gcgatggcct gcacgatcaa cgagccgaac 480atcgtggcca ccctcgggta
cttcctcggc gagttcccgc cggccgtcgc cgaccccgac 540cgctaccggc
aggcgaacga cacgctgatc cgcgcccatc gcctcgccta cgaggcgctg
600aaggccgggc ccggcgagtt ccccgtcggc ctcaccctgt cgatggccga
gttcgtcgcc 660gagcccggcg gcgaggccca cctcgcccag gtccggcaca
cgatggagga catcttcctg 720gaggccgccc ggggcgacga cttcatcggg
gtgcagacct acagccgcat gcgcttcggt 780cccgactcgc cgatcccgct
cgggccggcc gagggcgtcg aggtcgtcca gatggggtac 840gagtactggc
cgtgggcgct cgaggcgacg atccggcgcg ccgccgaggt caccggcacg
900gcggtccacg tcaccgagaa cggcatcggg accgccgacg acacgcagcg
ggtcgcctac 960gtcaccgagg ccctccgggg gctgcggcgc tgcctcgacg
acggcatcga cgtccgcagc 1020tacttctact ggacgctgct cgacaacttc
gagtggacgc gcggctacgt gccgacgttc 1080gggctcgtcg ccgtcgaccg
caccacccag cgccggtcgg tgaagccgag cgcggtgtgg 1140ctcggcgagg
tcgcccgcac gaaccgcctc gagctcccgg accgctga
1188144395PRTUnknownObtained from environmental sample 144Met Thr
Ile Thr Phe Pro Asp Gly Phe Trp Trp Gly Thr Ala Thr Ala 1 5 10 15
Ala His Gln Val Glu Gly Gly Asn Trp Asn Thr Asp Trp Trp Ala Tyr 20
25 30 Glu His Ala Pro Gly Thr Arg Cys Ala Glu Pro Ser Gly Asp Ala
Cys 35 40 45 Asp His Trp His Arg Tyr Pro Glu Asp Ile Ala Leu Leu
Ala Ala Leu 50 55 60 Gly Phe Ser Ala Tyr Arg Phe Ser Val Glu Trp
Ala Arg Ile Glu Pro 65 70 75 80 Glu Glu Gly His Phe Ser Arg Ala Thr
Leu Asp His Tyr Arg Arg Met 85 90 95 Ile Ala Cys Cys Arg Asp His
Gly Leu Ala Pro Val Val Thr Phe His 100 105 110 His Phe Thr Thr Pro
Arg Trp Ala Ala Ala Gly Gly Cys Trp Ser Asp 115 120 125 Pro Val Thr
Ala Glu Arg Phe Ala Arg Tyr Cys Glu Arg Thr Val Ala 130 135 140 Ala
Leu Gly Asp Glu Ile Ala Met Ala Cys Thr Ile Asn Glu Pro Asn 145
150
155 160 Ile Val Ala Thr Leu Gly Tyr Phe Leu Gly Glu Phe Pro Pro Ala
Val 165 170 175 Ala Asp Pro Asp Arg Tyr Arg Gln Ala Asn Asp Thr Leu
Ile Arg Ala 180 185 190 His Arg Leu Ala Tyr Glu Ala Leu Lys Ala Gly
Pro Gly Glu Phe Pro 195 200 205 Val Gly Leu Thr Leu Ser Met Ala Glu
Phe Val Ala Glu Pro Gly Gly 210 215 220 Glu Ala His Leu Ala Gln Val
Arg His Thr Met Glu Asp Ile Phe Leu 225 230 235 240 Glu Ala Ala Arg
Gly Asp Asp Phe Ile Gly Val Gln Thr Tyr Ser Arg 245 250 255 Met Arg
Phe Gly Pro Asp Ser Pro Ile Pro Leu Gly Pro Ala Glu Gly 260 265 270
Val Glu Val Val Gln Met Gly Tyr Glu Tyr Trp Pro Trp Ala Leu Glu 275
280 285 Ala Thr Ile Arg Arg Ala Ala Glu Val Thr Gly Thr Ala Val His
Val 290 295 300 Thr Glu Asn Gly Ile Gly Thr Ala Asp Asp Thr Gln Arg
Val Ala Tyr 305 310 315 320 Val Thr Glu Ala Leu Arg Gly Leu Arg Arg
Cys Leu Asp Asp Gly Ile 325 330 335 Asp Val Arg Ser Tyr Phe Tyr Trp
Thr Leu Leu Asp Asn Phe Glu Trp 340 345 350 Thr Arg Gly Tyr Val Pro
Thr Phe Gly Leu Val Ala Val Asp Arg Thr 355 360 365 Thr Gln Arg Arg
Ser Val Lys Pro Ser Ala Val Trp Leu Gly Glu Val 370 375 380 Ala Arg
Thr Asn Arg Leu Glu Leu Pro Asp Arg 385 390 395
1451386DNAUnknownObtained from environmental sample 145atgtcgtttc
cgagaaattt cctgtgggga tcagccacct cctcctacca aatcgaaggc 60gcctggcaag
aagacggcaa aggcccaaat atctgggacg tgttttcaca caccccgggg
120aaagtcgcca atggcgacac cggtgatatc gccatcgacc actaccaccg
ataccgagac 180gacgttgccc tgatggctga gcttggactt caggcatacc
gtttctcgtt ctcctgggcc 240agaataatgc cggaaggagc aggccccatc
gagcaacggg gtctggactt ctacgaccgc 300ctcattgatg cactgctgga
gaaaaacatc caacccatgg ccaccctcta ccactgggat 360ttaccagccg
cactgcaaga cagagggggg tggactaacc gcgacagcgc gtcctggttt
420gctgactact cagccgttgt tcacgacgct ttttctgacc gggtgggaat
gtgggcaacg 480ttgaacgagc cgtgggtgtc tgcatttttg ggccacggaa
ctggcatcca cgcacctggc 540atcacaagcc cccacgcggc gttcgccgcg
gggcatcacc tgcttctggg gcatggcaag 600gccatccaag cgatgcgcgc
tcaatcgtct agcacccaac tgggaattgt tttgaacctc 660gcccccgtgt
atctcgaagg tgacacccct gctgaccacc cggctcacac ctccgtggca
720ctacacgatg ccattttgaa tgggttgtgg acagagccgc ttctgcgctc
cagatacccc 780gacctgcttc ttcaactagg cgacatggtg acaaaaaaca
tccacgacgg tgacctcgcc 840atcatggccg agccgattga ctggatgggc
atcaactact accaggacat tagatttgtg 900gccactgatg ttgcccccac
ggctaacccg atggcccctc cgggtaacga cctgccgggc 960accgtcgggg
tggagcctgc gccagcaatc ggaaacatca ccagctttgg ctggtccacc
1020acccccgacg gactgcgagt actgttggtg ggcctggatg aggaatacga
caacctcccg 1080ccgatattca ttaccgaaaa cgggtgtgct tacgattacc
ccgtcgagga cggtgtcgtc 1140aacgacaccc ttcgtgtcac atacatgcga
gaacacctca ccgcgttgtc gcaggccatt 1200gaggcgggtg tgaatgtccg
gggctatatg cactggtctc tgttcgacaa cttcgagtgg 1260gccgaagggt
atcgccaacg ctttggcatg gtgcacgtcg actttgagac cttggagcgg
1320actcccaaag cctcagctca ctactattca cgtgtcatca caaataacgc
cctctctgac 1380gactga 1386146461PRTUnknownObtained from
environmental sample 146Met Ser Phe Pro Arg Asn Phe Leu Trp Gly Ser
Ala Thr Ser Ser Tyr 1 5 10 15 Gln Ile Glu Gly Ala Trp Gln Glu Asp
Gly Lys Gly Pro Asn Ile Trp 20 25 30 Asp Val Phe Ser His Thr Pro
Gly Lys Val Ala Asn Gly Asp Thr Gly 35 40 45 Asp Ile Ala Ile Asp
His Tyr His Arg Tyr Arg Asp Asp Val Ala Leu 50 55 60 Met Ala Glu
Leu Gly Leu Gln Ala Tyr Arg Phe Ser Phe Ser Trp Ala 65 70 75 80 Arg
Ile Met Pro Glu Gly Ala Gly Pro Ile Glu Gln Arg Gly Leu Asp 85 90
95 Phe Tyr Asp Arg Leu Ile Asp Ala Leu Leu Glu Lys Asn Ile Gln Pro
100 105 110 Met Ala Thr Leu Tyr His Trp Asp Leu Pro Ala Ala Leu Gln
Asp Arg 115 120 125 Gly Gly Trp Thr Asn Arg Asp Ser Ala Ser Trp Phe
Ala Asp Tyr Ser 130 135 140 Ala Val Val His Asp Ala Phe Ser Asp Arg
Val Gly Met Trp Ala Thr 145 150 155 160 Leu Asn Glu Pro Trp Val Ser
Ala Phe Leu Gly His Gly Thr Gly Ile 165 170 175 His Ala Pro Gly Ile
Thr Ser Pro His Ala Ala Phe Ala Ala Gly His 180 185 190 His Leu Leu
Leu Gly His Gly Lys Ala Ile Gln Ala Met Arg Ala Gln 195 200 205 Ser
Ser Ser Thr Gln Leu Gly Ile Val Leu Asn Leu Ala Pro Val Tyr 210 215
220 Leu Glu Gly Asp Thr Pro Ala Asp His Pro Ala His Thr Ser Val Ala
225 230 235 240 Leu His Asp Ala Ile Leu Asn Gly Leu Trp Thr Glu Pro
Leu Leu Arg 245 250 255 Ser Arg Tyr Pro Asp Leu Leu Leu Gln Leu Gly
Asp Met Val Thr Lys 260 265 270 Asn Ile His Asp Gly Asp Leu Ala Ile
Met Ala Glu Pro Ile Asp Trp 275 280 285 Met Gly Ile Asn Tyr Tyr Gln
Asp Ile Arg Phe Val Ala Thr Asp Val 290 295 300 Ala Pro Thr Ala Asn
Pro Met Ala Pro Pro Gly Asn Asp Leu Pro Gly 305 310 315 320 Thr Val
Gly Val Glu Pro Ala Pro Ala Ile Gly Asn Ile Thr Ser Phe 325 330 335
Gly Trp Ser Thr Thr Pro Asp Gly Leu Arg Val Leu Leu Val Gly Leu 340
345 350 Asp Glu Glu Tyr Asp Asn Leu Pro Pro Ile Phe Ile Thr Glu Asn
Gly 355 360 365 Cys Ala Tyr Asp Tyr Pro Val Glu Asp Gly Val Val Asn
Asp Thr Leu 370 375 380 Arg Val Thr Tyr Met Arg Glu His Leu Thr Ala
Leu Ser Gln Ala Ile 385 390 395 400 Glu Ala Gly Val Asn Val Arg Gly
Tyr Met His Trp Ser Leu Phe Asp 405 410 415 Asn Phe Glu Trp Ala Glu
Gly Tyr Arg Gln Arg Phe Gly Met Val His 420 425 430 Val Asp Phe Glu
Thr Leu Glu Arg Thr Pro Lys Ala Ser Ala His Tyr 435 440 445 Tyr Ser
Arg Val Ile Thr Asn Asn Ala Leu Ser Asp Asp 450 455 460
1471242DNAUnknownObtained from environmental sample 147atgctaaaag
ttttacgtaa acctattatt tctggattag ctttagctct attattgccg 60gcaggggcag
ctggtgccga aactaatatt tcaaagaagc caaatataag tggattaacc
120gcgccgcaat tagaccaaag atataaagat tctttcacca ttggtgctgc
ggttgagccg 180tatcaattat tagatgcaaa agattcacaa atgctaaagc
ggcattttaa tagtatcgta 240gcagagaatg tcatgaagcc tagtagttta
cagccagtag aaggacaatt caactgggag 300ccggctgata aacttgttca
gtttgcgaag gaaaatggaa tggacatgcg aggtcatacg 360cttgtctggc
atagccaggt accggattgg ttctttgaag atgcggcagg aaatccaatg
420gttgtttggg aaaatggcag gcaagtggtt gccgatccat caaagcttca
ggaaaacaaa 480gagctcttac ttagccgatt acaaaatcat attcaggcag
tcgtaacgcg ttataaagat 540gatataaaat cttgggatgt tgtcaatgaa
gtaatcgatg aatggggcgg acattctgaa 600gggctgcgtc aatctccatg
gttcctcatc accggaacgg actatattaa agttgctttt 660gaaactgcaa
gagaatatgc agctccagac gctaagctgt atatcaatga ttacaataca
720gaagtagaac caaaaaggac gcacctttat aacttagtaa aaagtttaaa
agaagaacag 780aacgttccga ttgatggtgt tgggcatcag tctcacattc
aaattggctg gccttcagaa 840aaagaaattg aagatactat taatatgttt
gcagatcttg gtttagataa ccaaatcacc 900gagcttgatg ttagtatgta
tggctggccg gtaaggtcgt atccaactta tgatgcgatc 960ccagaactta
aattcatgga tcaagcagct cgttatgatc gtttatttaa gttatatgag
1020aaattaggag ataaaatcag taatgtgaca ttctggggta ttgcggataa
ccatacatgg 1080ctgaatgacc gcgcagatgt ttactatgat gaaaatggaa
atgttgtatt agatagagaa 1140acaccaagag tagaaagagg agcaggaaaa
gatgcgccat ttgtatttga tcctgaatac 1200aatgtaaaac cagcttattg
ggcaattatc gatcacaaat aa 1242148413PRTUnknownObtained from
environmental sample 148Met Leu Lys Val Leu Arg Lys Pro Ile Ile Ser
Gly Leu Ala Leu Ala 1 5 10 15 Leu Leu Leu Pro Ala Gly Ala Ala Gly
Ala Glu Thr Asn Ile Ser Lys 20 25 30 Lys Pro Asn Ile Ser Gly Leu
Thr Ala Pro Gln Leu Asp Gln Arg Tyr 35 40 45 Lys Asp Ser Phe Thr
Ile Gly Ala Ala Val Glu Pro Tyr Gln Leu Leu 50 55 60 Asp Ala Lys
Asp Ser Gln Met Leu Lys Arg His Phe Asn Ser Ile Val 65 70 75 80 Ala
Glu Asn Val Met Lys Pro Ser Ser Leu Gln Pro Val Glu Gly Gln 85 90
95 Phe Asn Trp Glu Pro Ala Asp Lys Leu Val Gln Phe Ala Lys Glu Asn
100 105 110 Gly Met Asp Met Arg Gly His Thr Leu Val Trp His Ser Gln
Val Pro 115 120 125 Asp Trp Phe Phe Glu Asp Ala Ala Gly Asn Pro Met
Val Val Trp Glu 130 135 140 Asn Gly Arg Gln Val Val Ala Asp Pro Ser
Lys Leu Gln Glu Asn Lys 145 150 155 160 Glu Leu Leu Leu Ser Arg Leu
Gln Asn His Ile Gln Ala Val Val Thr 165 170 175 Arg Tyr Lys Asp Asp
Ile Lys Ser Trp Asp Val Val Asn Glu Val Ile 180 185 190 Asp Glu Trp
Gly Gly His Ser Glu Gly Leu Arg Gln Ser Pro Trp Phe 195 200 205 Leu
Ile Thr Gly Thr Asp Tyr Ile Lys Val Ala Phe Glu Thr Ala Arg 210 215
220 Glu Tyr Ala Ala Pro Asp Ala Lys Leu Tyr Ile Asn Asp Tyr Asn Thr
225 230 235 240 Glu Val Glu Pro Lys Arg Thr His Leu Tyr Asn Leu Val
Lys Ser Leu 245 250 255 Lys Glu Glu Gln Asn Val Pro Ile Asp Gly Val
Gly His Gln Ser His 260 265 270 Ile Gln Ile Gly Trp Pro Ser Glu Lys
Glu Ile Glu Asp Thr Ile Asn 275 280 285 Met Phe Ala Asp Leu Gly Leu
Asp Asn Gln Ile Thr Glu Leu Asp Val 290 295 300 Ser Met Tyr Gly Trp
Pro Val Arg Ser Tyr Pro Thr Tyr Asp Ala Ile 305 310 315 320 Pro Glu
Leu Lys Phe Met Asp Gln Ala Ala Arg Tyr Asp Arg Leu Phe 325 330 335
Lys Leu Tyr Glu Lys Leu Gly Asp Lys Ile Ser Asn Val Thr Phe Trp 340
345 350 Gly Ile Ala Asp Asn His Thr Trp Leu Asn Asp Arg Ala Asp Val
Tyr 355 360 365 Tyr Asp Glu Asn Gly Asn Val Val Leu Asp Arg Glu Thr
Pro Arg Val 370 375 380 Glu Arg Gly Ala Gly Lys Asp Ala Pro Phe Val
Phe Asp Pro Glu Tyr 385 390 395 400 Asn Val Lys Pro Ala Tyr Trp Ala
Ile Ile Asp His Lys 405 410 1491068DNAUnknownObtained from
environmental sample 149atgacccgaa tgcgcgggat aaacatgggc ggctggctca
gccaaattga cgccatacag 60gaaaaagacc ctgatacatt tcccggaaca gacaaacata
tggaaacttt tatccagcag 120aaggattttg ccaatgtcag gagatggggt
ttcgatcatg tgcgaattcc aattgacgcg 180tatctgttct ttaccgaaaa
aggagagccg attgaaaaca ggcttgccaa tcttgaccgc 240gccgtagagt
atgcgctgcc cgccggcctc aacatgatat tggacctcca cgagtgtccg
300gggcacgatt tttcggaagc agtaaaaagc cctgtccaaa aacttttctc
gggagatgac 360acctggataa ggaaaactga aaaaatatgg gcttgccttg
ccgagcgtta ttctcaaaag 420ggccacgtcc tttttgagac gctcaatgag
cctgtcgctc ccaccgcgga gatttggaac 480aatgttaagg acaggctctg
ccgcgaaata cggctccacg ccccctggtc gactataatc 540accggctcca
acatgtggaa ctcagcggca accttcgaca gcctcacgcc ctttgacgac
600gacaacatga tctacagcgt acatttttac gagccgctgc ttttcacgca
ccagaacgca 660ttgtggatcg acaatccgga aatcaggatc gcaaggccgt
atccgggcga ttacggtccc 720ggctttgtcc ccaaagacgg tttgacgctg
tcggacggcg tctggaacag ggatcgtctc 780gccggcgcat tagcgcccgt
gaacgcgttc aggaaaaagt acaatgcgaa gattatctgt 840aacgagttcg
gcgtttacgc gcccgtagac cttcaatcgc agctgcgctg gtatgaagat
900ctgctctcaa tcctcaatga gacggggatc ggtttcacgt actggaacta
taaaaatctc 960gacttcggga taatttccat aggggagaag ctgcacgaag
cccttccgca gtacgacaat 1020agcgatcgaa taaataaatc ggttcttgaa
gtgttaaaaa agtattag 1068150355PRTUnknownObtained from environmental
sample 150Met Thr Arg Met Arg Gly Ile Asn Met Gly Gly Trp Leu Ser
Gln Ile 1 5 10 15 Asp Ala Ile Gln Glu Lys Asp Pro Asp Thr Phe Pro
Gly Thr Asp Lys 20 25 30 His Met Glu Thr Phe Ile Gln Gln Lys Asp
Phe Ala Asn Val Arg Arg 35 40 45 Trp Gly Phe Asp His Val Arg Ile
Pro Ile Asp Ala Tyr Leu Phe Phe 50 55 60 Thr Glu Lys Gly Glu Pro
Ile Glu Asn Arg Leu Ala Asn Leu Asp Arg 65 70 75 80 Ala Val Glu Tyr
Ala Leu Pro Ala Gly Leu Asn Met Ile Leu Asp Leu 85 90 95 His Glu
Cys Pro Gly His Asp Phe Ser Glu Ala Val Lys Ser Pro Val 100 105 110
Gln Lys Leu Phe Ser Gly Asp Asp Thr Trp Ile Arg Lys Thr Glu Lys 115
120 125 Ile Trp Ala Cys Leu Ala Glu Arg Tyr Ser Gln Lys Gly His Val
Leu 130 135 140 Phe Glu Thr Leu Asn Glu Pro Val Ala Pro Thr Ala Glu
Ile Trp Asn 145 150 155 160 Asn Val Lys Asp Arg Leu Cys Arg Glu Ile
Arg Leu His Ala Pro Trp 165 170 175 Ser Thr Ile Ile Thr Gly Ser Asn
Met Trp Asn Ser Ala Ala Thr Phe 180 185 190 Asp Ser Leu Thr Pro Phe
Asp Asp Asp Asn Met Ile Tyr Ser Val His 195 200 205 Phe Tyr Glu Pro
Leu Leu Phe Thr His Gln Asn Ala Leu Trp Ile Asp 210 215 220 Asn Pro
Glu Ile Arg Ile Ala Arg Pro Tyr Pro Gly Asp Tyr Gly Pro 225 230 235
240 Gly Phe Val Pro Lys Asp Gly Leu Thr Leu Ser Asp Gly Val Trp Asn
245 250 255 Arg Asp Arg Leu Ala Gly Ala Leu Ala Pro Val Asn Ala Phe
Arg Lys 260 265 270 Lys Tyr Asn Ala Lys Ile Ile Cys Asn Glu Phe Gly
Val Tyr Ala Pro 275 280 285 Val Asp Leu Gln Ser Gln Leu Arg Trp Tyr
Glu Asp Leu Leu Ser Ile 290 295 300 Leu Asn Glu Thr Gly Ile Gly Phe
Thr Tyr Trp Asn Tyr Lys Asn Leu 305 310 315 320 Asp Phe Gly Ile Ile
Ser Ile Gly Glu Lys Leu His Glu Ala Leu Pro 325 330 335 Gln Tyr Asp
Asn Ser Asp Arg Ile Asn Lys Ser Val Leu Glu Val Leu 340 345 350 Lys
Lys Tyr 355 1511068DNAUnknownObtained from environmental sample
151atgaccagaa tgcgcggaat aaacatgggc ggctggctca gccagattga
cgccatacag 60gaaaaagacc ccgataaatt tcccggaata gacaaacaca tggaaacatt
tatcggttcc 120aatgattttt ccaatgtcag gaaatggggt ttcgatcatg
tgcgaatccc gattgacgcg 180tacctttttt ttaccgatca ggaagccccg
attgaaaaca ggcttgtcca tattgacaac 240gccgtaaaat acgcgcggag
caacggcctc aaggtgatat tggacctcca cgagtgtccg 300gggcatgatt
tttcggacgc ggcaaaaggc cctgtccaga aacttttctc cggagatgac
360acttatataa aaaagaccga aaaaatatgg gcatgtctgg ccgagcgtta
ttcgaaaaac 420gacaacgtcc tctatgagac tctcaacgag cctgtcgccc
ccacgcctga gatttggaac 480actgttaagg acaggctctg ccgggaaata
cgcctgcacg ccccctgggc gacgataatc 540accggttcca atatgtggaa
ttggccgagc acctttgaca gcctgacgcc ctttgacgac 600gacaacgtga
tctacagcgt gcatttttac gagccgctgc tttttacgca ccagaacgcg
660ccctggatca acaattctga aatcaggatc acaaggccgt atccgggcga
ttacggcccc 720ggctttgtcc gcaaatacgg cttaactctg tcagccggcg
tctggaacag ggacaggctg 780gcgaaggaat tcgcgcccgt gaacgcgttc
aggaaaaaat acaaggcgca ggttatatgc 840gacgaattcg gcgtttacgc
gcctgtcgag attgaatcgc agcttcgatg gtatgaggat 900ttgctctcga
tcctcaggga gatgggtata gggttttcgt actggaacta taaaaacctg
960gactttggga taatttccat aggggagaag ctgcacgaaa gccttctgca
gtacggcaac 1020ggcgacagga taaatcatat ggttcttgac ttgctaaaga agtactaa
1068152355PRTUnknownObtained from environmental sample 152Met Thr
Arg Met Arg Gly Ile Asn Met Gly Gly Trp Leu Ser Gln Ile 1 5 10 15
Asp Ala Ile Gln Glu Lys Asp Pro Asp Lys Phe Pro Gly Ile Asp Lys
20
25 30 His Met Glu Thr Phe Ile Gly Ser Asn Asp Phe Ser Asn Val Arg
Lys 35 40 45 Trp Gly Phe Asp His Val Arg Ile Pro Ile Asp Ala Tyr
Leu Phe Phe 50 55 60 Thr Asp Gln Glu Ala Pro Ile Glu Asn Arg Leu
Val His Ile Asp Asn 65 70 75 80 Ala Val Lys Tyr Ala Arg Ser Asn Gly
Leu Lys Val Ile Leu Asp Leu 85 90 95 His Glu Cys Pro Gly His Asp
Phe Ser Asp Ala Ala Lys Gly Pro Val 100 105 110 Gln Lys Leu Phe Ser
Gly Asp Asp Thr Tyr Ile Lys Lys Thr Glu Lys 115 120 125 Ile Trp Ala
Cys Leu Ala Glu Arg Tyr Ser Lys Asn Asp Asn Val Leu 130 135 140 Tyr
Glu Thr Leu Asn Glu Pro Val Ala Pro Thr Pro Glu Ile Trp Asn 145 150
155 160 Thr Val Lys Asp Arg Leu Cys Arg Glu Ile Arg Leu His Ala Pro
Trp 165 170 175 Ala Thr Ile Ile Thr Gly Ser Asn Met Trp Asn Trp Pro
Ser Thr Phe 180 185 190 Asp Ser Leu Thr Pro Phe Asp Asp Asp Asn Val
Ile Tyr Ser Val His 195 200 205 Phe Tyr Glu Pro Leu Leu Phe Thr His
Gln Asn Ala Pro Trp Ile Asn 210 215 220 Asn Ser Glu Ile Arg Ile Thr
Arg Pro Tyr Pro Gly Asp Tyr Gly Pro 225 230 235 240 Gly Phe Val Arg
Lys Tyr Gly Leu Thr Leu Ser Ala Gly Val Trp Asn 245 250 255 Arg Asp
Arg Leu Ala Lys Glu Phe Ala Pro Val Asn Ala Phe Arg Lys 260 265 270
Lys Tyr Lys Ala Gln Val Ile Cys Asp Glu Phe Gly Val Tyr Ala Pro 275
280 285 Val Glu Ile Glu Ser Gln Leu Arg Trp Tyr Glu Asp Leu Leu Ser
Ile 290 295 300 Leu Arg Glu Met Gly Ile Gly Phe Ser Tyr Trp Asn Tyr
Lys Asn Leu 305 310 315 320 Asp Phe Gly Ile Ile Ser Ile Gly Glu Lys
Leu His Glu Ser Leu Leu 325 330 335 Gln Tyr Gly Asn Gly Asp Arg Ile
Asn His Met Val Leu Asp Leu Leu 340 345 350 Lys Lys Tyr 355
1531068DNAUnknownObtained from environmental sample 153atgcaaagaa
tgcgaggctt aaatattggc ggctggctca gccagattga cgccatacag 60gaaaaggacc
ctgagggctt tcccggaata gacaaacaca tggaaacatt cattgtttcc
120ggagattttt acaatatcag gaaatggggt ttcgaccatg tgcggcttcc
cattgactcg 180tacctgttct ttacggaaga cgatgccccc attgagaaca
ggtttgccca tcttgaccgc 240gccgtacaat tcgcgaagag caacagcctc
aagctgatat tggacctcca cgagtgtccg 300ggacacgatt tttccgaagc
cgcgaaagga cccgtccaga aacttttttc gggagatgac 360gtttacataa
aaaaaaccga gaaaatctgg gcctgcctcg ccgagcgtta ttcgaaaaac
420gaccatgtac tctttgagac tctcaacgaa cctgtcgctc ccactgccga
aatttggaac 480aaggttaagg acaggctctg cagagtaatc cgcatccacg
cgccctggtc gaccataatc 540accggctcca atatgtggaa ctcgccgtcc
gccttcgacg gtcttacgcc ctttgacgat 600ggcaacgtga tctacagcgt
gcatttttac gagccgctgc tttttacgca tcagaacgcg 660ccgtggatcg
acaatccgga gatcaggacg gcaaggccct atccgggcga ttacggcccc
720ggccttgtcc gcaaatacgg tatggcgcag tcggccggca tctggaacaa
gaaacggctt 780gcaaaagaat ttgagcccgt ggacgcgttc aggaaaaaat
acaaggcgcg cgttatctgt 840aacgagtttg gcgtgtacgc ccccgccgat
ctggaatcgc agcttcgctg gtatgaggat 900ctgctctcaa tcctcaacgg
gatgcagata ggttactcgt actggaacta caaaaatctg 960gatttcggaa
taatttccat aggggagaaa ctgcacgaaa gactttcgca gtatgacaac
1020gacgagcgga taaaccaccc ggtgctgaat gtgctgaaga aatattaa
1068154355PRTUnknownObtained from environmental sample 154Met Gln
Arg Met Arg Gly Leu Asn Ile Gly Gly Trp Leu Ser Gln Ile 1 5 10 15
Asp Ala Ile Gln Glu Lys Asp Pro Glu Gly Phe Pro Gly Ile Asp Lys 20
25 30 His Met Glu Thr Phe Ile Val Ser Gly Asp Phe Tyr Asn Ile Arg
Lys 35 40 45 Trp Gly Phe Asp His Val Arg Leu Pro Ile Asp Ser Tyr
Leu Phe Phe 50 55 60 Thr Glu Asp Asp Ala Pro Ile Glu Asn Arg Phe
Ala His Leu Asp Arg 65 70 75 80 Ala Val Gln Phe Ala Lys Ser Asn Ser
Leu Lys Leu Ile Leu Asp Leu 85 90 95 His Glu Cys Pro Gly His Asp
Phe Ser Glu Ala Ala Lys Gly Pro Val 100 105 110 Gln Lys Leu Phe Ser
Gly Asp Asp Val Tyr Ile Lys Lys Thr Glu Lys 115 120 125 Ile Trp Ala
Cys Leu Ala Glu Arg Tyr Ser Lys Asn Asp His Val Leu 130 135 140 Phe
Glu Thr Leu Asn Glu Pro Val Ala Pro Thr Ala Glu Ile Trp Asn 145 150
155 160 Lys Val Lys Asp Arg Leu Cys Arg Val Ile Arg Ile His Ala Pro
Trp 165 170 175 Ser Thr Ile Ile Thr Gly Ser Asn Met Trp Asn Ser Pro
Ser Ala Phe 180 185 190 Asp Gly Leu Thr Pro Phe Asp Asp Gly Asn Val
Ile Tyr Ser Val His 195 200 205 Phe Tyr Glu Pro Leu Leu Phe Thr His
Gln Asn Ala Pro Trp Ile Asp 210 215 220 Asn Pro Glu Ile Arg Thr Ala
Arg Pro Tyr Pro Gly Asp Tyr Gly Pro 225 230 235 240 Gly Leu Val Arg
Lys Tyr Gly Met Ala Gln Ser Ala Gly Ile Trp Asn 245 250 255 Lys Lys
Arg Leu Ala Lys Glu Phe Glu Pro Val Asp Ala Phe Arg Lys 260 265 270
Lys Tyr Lys Ala Arg Val Ile Cys Asn Glu Phe Gly Val Tyr Ala Pro 275
280 285 Ala Asp Leu Glu Ser Gln Leu Arg Trp Tyr Glu Asp Leu Leu Ser
Ile 290 295 300 Leu Asn Gly Met Gln Ile Gly Tyr Ser Tyr Trp Asn Tyr
Lys Asn Leu 305 310 315 320 Asp Phe Gly Ile Ile Ser Ile Gly Glu Lys
Leu His Glu Arg Leu Ser 325 330 335 Gln Tyr Asp Asn Asp Glu Arg Ile
Asn His Pro Val Leu Asn Val Leu 340 345 350 Lys Lys Tyr 355
155954DNAUnknownObtained from environmental sample 155atgttaaagg
attccggttt ttataagggc atcaatctcg gcggctggct gtcccagtgc 60gactacagcg
aggagcgcct gaacagcttc atcaccgaaa aggactttga ggtgatcgcc
120tcctggggtt ttgaccacgt ccgcctcccg gtggactata atgtcatcca
ggatgcggaa 180ggccgcatga tggagaaagg ccttgcacgc atcgacgccg
cgcttcggtt ttgtgagaag 240accgggcttc acatggttct cgacctgcat
aagacaccgg gcttttcctt cgacccgcag 300gagcaggaga tgggattctt
ccggtcggcg cccgaccagc agctcttcta cacgatctgg 360gagagccttg
ctgcccggta tgcagacaaa tcggagatac tcatgttcga tcttctgaac
420gagatcacgg agccggcgta tctggaggac tggaaccgga tttccgcgga
atgcatccgc 480cgcatccggc gtacgatgcc ggacgtccga attctggtcg
gaagctatca ccacaatgcc 540gtcagcgcgg taaaggacct gcctgcgccg
gcagacgata aggtttttta cagctttcac 600tgttacgacc ctcacaccta
tacccaccag ggcgcttact ggatgccgga tgactttgac 660atcgatgcaa
gagtttcctt ccgcgacacc ggcgttaccc ccgtcttctt cgaaaagctg
720tttgcctccg ccgttgaaaa ggcgcaggcg gaagggacgg aactgtactg
cggagaatac 780ggcgtcatcg acattgttcc gccggaggat gccgttctct
ggttccggac cattcatgag 840gtctttgaag cattcgggat tgcaagaagc
gtctggagct ataaggaaat ggatttcggt 900ctcgccgacc cccgcatgga
tgcggtccgg gcagagctgc tgacctgtct ctga 954156317PRTUnknownObtained
from environmental sample 156Met Leu Lys Asp Ser Gly Phe Tyr Lys
Gly Ile Asn Leu Gly Gly Trp 1 5 10 15 Leu Ser Gln Cys Asp Tyr Ser
Glu Glu Arg Leu Asn Ser Phe Ile Thr 20 25 30 Glu Lys Asp Phe Glu
Val Ile Ala Ser Trp Gly Phe Asp His Val Arg 35 40 45 Leu Pro Val
Asp Tyr Asn Val Ile Gln Asp Ala Glu Gly Arg Met Met 50 55 60 Glu
Lys Gly Leu Ala Arg Ile Asp Ala Ala Leu Arg Phe Cys Glu Lys 65 70
75 80 Thr Gly Leu His Met Val Leu Asp Leu His Lys Thr Pro Gly Phe
Ser 85 90 95 Phe Asp Pro Gln Glu Gln Glu Met Gly Phe Phe Arg Ser
Ala Pro Asp 100 105 110 Gln Gln Leu Phe Tyr Thr Ile Trp Glu Ser Leu
Ala Ala Arg Tyr Ala 115 120 125 Asp Lys Ser Glu Ile Leu Met Phe Asp
Leu Leu Asn Glu Ile Thr Glu 130 135 140 Pro Ala Tyr Leu Glu Asp Trp
Asn Arg Ile Ser Ala Glu Cys Ile Arg 145 150 155 160 Arg Ile Arg Arg
Thr Met Pro Asp Val Arg Ile Leu Val Gly Ser Tyr 165 170 175 His His
Asn Ala Val Ser Ala Val Lys Asp Leu Pro Ala Pro Ala Asp 180 185 190
Asp Lys Val Phe Tyr Ser Phe His Cys Tyr Asp Pro His Thr Tyr Thr 195
200 205 His Gln Gly Ala Tyr Trp Met Pro Asp Asp Phe Asp Ile Asp Ala
Arg 210 215 220 Val Ser Phe Arg Asp Thr Gly Val Thr Pro Val Phe Phe
Glu Lys Leu 225 230 235 240 Phe Ala Ser Ala Val Glu Lys Ala Gln Ala
Glu Gly Thr Glu Leu Tyr 245 250 255 Cys Gly Glu Tyr Gly Val Ile Asp
Ile Val Pro Pro Glu Asp Ala Val 260 265 270 Leu Trp Phe Arg Thr Ile
His Glu Val Phe Glu Ala Phe Gly Ile Ala 275 280 285 Arg Ser Val Trp
Ser Tyr Lys Glu Met Asp Phe Gly Leu Ala Asp Pro 290 295 300 Arg Met
Asp Ala Val Arg Ala Glu Leu Leu Thr Cys Leu 305 310 315
157954DNAUnknownObtained from environmental sample 157atgttaaagg
attccggttt ttataagggc atcaatctcg gcggctggct gtcccagtgc 60gactacagcg
aggagcgcct gaacagcttc atcaccgaaa aagactttga ggtgatcgcc
120tcctggggtt ttgaccacgt ccgtctgccg gtggactata atgtcatcca
ggatgcggaa 180ggccgcatga tggaggaagg cctcgcacgc atcgacgccg
cgcttcggtt ttgtgaaaag 240accgggcttc acatggttct cgacctgcat
aagacaccgg gcttttcctt cgacccgcag 300gagcaggaga tgggattctt
ccggtcggcg cccgaccagc agcgcttcta cacgatctgg 360gagagccttg
ctgcccggta tgcagacaaa tcggagatgc tcatgttcga tcttctgaac
420gagatcacgg agccggcgta tctgaaggac tggaaccgga tttccgcgga
atgcatccgc 480cgcatccggc gtacgatgcc ggacgtccgg attctggtcg
gaagctatca ccacaatgcc 540gtcagcgcgg taaaggacct gcctgcgccg
gcggacgacc gggtttttta cagctttcac 600tgttacgacc ctcacaccta
tacccaccag ggcgcttact ggatgccgga tgactttgac 660atcgatgcaa
gagtttcctt ccgcgacatc ggcgtcaccc ccgccttctt cgaagagctg
720tttgcatctg ccgttgaaaa ggcgaaggtg gaagggacgg aactgtactg
cggagaatac 780ggcgtcatcg acattgttcc gccggaggat gccgttctct
ggttccggac cattcatgag 840gtctttgaga aatacgggat tgcaagaagc
gtctggagct ataaggaaat ggatttcggt 900ctctccgacc cccgcatgga
cgcggtccgg gcagagctgc tgacctgtct ctga 954158317PRTUnknownObtained
from environmental sample 158Met Leu Lys Asp Ser Gly Phe Tyr Lys
Gly Ile Asn Leu Gly Gly Trp 1 5 10 15 Leu Ser Gln Cys Asp Tyr Ser
Glu Glu Arg Leu Asn Ser Phe Ile Thr 20 25 30 Glu Lys Asp Phe Glu
Val Ile Ala Ser Trp Gly Phe Asp His Val Arg 35 40 45 Leu Pro Val
Asp Tyr Asn Val Ile Gln Asp Ala Glu Gly Arg Met Met 50 55 60 Glu
Glu Gly Leu Ala Arg Ile Asp Ala Ala Leu Arg Phe Cys Glu Lys 65 70
75 80 Thr Gly Leu His Met Val Leu Asp Leu His Lys Thr Pro Gly Phe
Ser 85 90 95 Phe Asp Pro Gln Glu Gln Glu Met Gly Phe Phe Arg Ser
Ala Pro Asp 100 105 110 Gln Gln Arg Phe Tyr Thr Ile Trp Glu Ser Leu
Ala Ala Arg Tyr Ala 115 120 125 Asp Lys Ser Glu Met Leu Met Phe Asp
Leu Leu Asn Glu Ile Thr Glu 130 135 140 Pro Ala Tyr Leu Lys Asp Trp
Asn Arg Ile Ser Ala Glu Cys Ile Arg 145 150 155 160 Arg Ile Arg Arg
Thr Met Pro Asp Val Arg Ile Leu Val Gly Ser Tyr 165 170 175 His His
Asn Ala Val Ser Ala Val Lys Asp Leu Pro Ala Pro Ala Asp 180 185 190
Asp Arg Val Phe Tyr Ser Phe His Cys Tyr Asp Pro His Thr Tyr Thr 195
200 205 His Gln Gly Ala Tyr Trp Met Pro Asp Asp Phe Asp Ile Asp Ala
Arg 210 215 220 Val Ser Phe Arg Asp Ile Gly Val Thr Pro Ala Phe Phe
Glu Glu Leu 225 230 235 240 Phe Ala Ser Ala Val Glu Lys Ala Lys Val
Glu Gly Thr Glu Leu Tyr 245 250 255 Cys Gly Glu Tyr Gly Val Ile Asp
Ile Val Pro Pro Glu Asp Ala Val 260 265 270 Leu Trp Phe Arg Thr Ile
His Glu Val Phe Glu Lys Tyr Gly Ile Ala 275 280 285 Arg Ser Val Trp
Ser Tyr Lys Glu Met Asp Phe Gly Leu Ser Asp Pro 290 295 300 Arg Met
Asp Ala Val Arg Ala Glu Leu Leu Thr Cys Leu 305 310 315
1591023DNAUnknownObtained from environmental sample 159atgaatccaa
cattcagttc cgtaccggca ttaaaggagc tgtttgcggc ggacttcaac 60atcggggcgg
cggtgaatcc gacgacgatc cggacgcagg aggcgttgct ggcttatcat
120tttaacagcc tgactgcgga gaacgagatg aagttcgtca gcgtgcatcc
ggaggagcag 180acctatacct tcgaggcggc ggaccggctg gtcgaattcg
cccgagagca cggcatggcc 240atgcggggac acacgctcgt atggcataac
cagacgtccg attggctgtt ccaggatcgc 300caaggcggga gggtaagcaa
ggaggtgctg ctcggaaggc tccgggagca tattcatacc 360atagtaggcc
ggtacaagaa cgagatctac gcctgggacg tcgtcaacga ggtcatcgcg
420gacgaagggg aggcgctgct gcgcacttcc aaatggacgg aaatcgcggg
acctgaattt 480atcgctaaag cgttcgagta tgcacatgag gcggatccac
aggcgctgtt gttttataac 540gactacaacg aatcgaatcc tctgaaacgc
gataaaattt acacactcgt tcattcgctg 600ctggagcaag gggtgccgat
ccatggcatc ggattacaag cgcactggaa cctgtacgat 660ccatcgttgg
atgagattaa ggcagcgatt gagaagtatg cttcgctggg tttgcagctg
720cagctgacgg agctggatct ctcgatgttc cgcttcgatg accggcgaac
cgatttgacc 780gcgccagagc cggggatgct ggagcaacag gccgagcgtt
atgaagccgt gttccggctg 840ttgctggagt atcgtgacgt catcagcggc
gttaccttct ggggagcggc ggatgattat 900acctggctgg acaattttcc
ggtgcgcggc cggaagaact ggccgtttct gttcgatgcc 960cagcaccagc
cgaaggcagc ttatcaccgt gtggcggcat tggctgcgga gcaacgagca 1020taa
1023160340PRTUnknownObtained from environmental sample 160Met Asn
Pro Thr Phe Ser Ser Val Pro Ala Leu Lys Glu Leu Phe Ala 1 5 10 15
Ala Asp Phe Asn Ile Gly Ala Ala Val Asn Pro Thr Thr Ile Arg Thr 20
25 30 Gln Glu Ala Leu Leu Ala Tyr His Phe Asn Ser Leu Thr Ala Glu
Asn 35 40 45 Glu Met Lys Phe Val Ser Val His Pro Glu Glu Gln Thr
Tyr Thr Phe 50 55 60 Glu Ala Ala Asp Arg Leu Val Glu Phe Ala Arg
Glu His Gly Met Ala 65 70 75 80 Met Arg Gly His Thr Leu Val Trp His
Asn Gln Thr Ser Asp Trp Leu 85 90 95 Phe Gln Asp Arg Gln Gly Gly
Arg Val Ser Lys Glu Val Leu Leu Gly 100 105 110 Arg Leu Arg Glu His
Ile His Thr Ile Val Gly Arg Tyr Lys Asn Glu 115 120 125 Ile Tyr Ala
Trp Asp Val Val Asn Glu Val Ile Ala Asp Glu Gly Glu 130 135 140 Ala
Leu Leu Arg Thr Ser Lys Trp Thr Glu Ile Ala Gly Pro Glu Phe 145 150
155 160 Ile Ala Lys Ala Phe Glu Tyr Ala His Glu Ala Asp Pro Gln Ala
Leu 165 170 175 Leu Phe Tyr Asn Asp Tyr Asn Glu Ser Asn Pro Leu Lys
Arg Asp Lys 180 185 190 Ile Tyr Thr Leu Val His Ser Leu Leu Glu Gln
Gly Val Pro Ile His 195 200 205 Gly Ile Gly Leu Gln Ala His Trp Asn
Leu Tyr Asp Pro Ser Leu Asp 210 215 220 Glu Ile Lys Ala Ala Ile Glu
Lys Tyr Ala Ser Leu Gly Leu Gln Leu 225 230 235 240 Gln Leu Thr Glu
Leu Asp Leu Ser Met Phe Arg Phe Asp Asp Arg Arg 245 250 255 Thr Asp
Leu Thr Ala Pro Glu Pro Gly Met Leu Glu Gln Gln Ala Glu 260 265 270
Arg Tyr Glu Ala Val Phe Arg Leu Leu Leu Glu Tyr Arg Asp Val Ile 275
280 285 Ser Gly Val Thr Phe Trp Gly Ala Ala Asp Asp Tyr Thr Trp Leu
Asp 290 295 300 Asn Phe Pro Val
Arg Gly Arg Lys Asn Trp Pro Phe Leu Phe Asp Ala 305 310 315 320 Gln
His Gln Pro Lys Ala Ala Tyr His Arg Val Ala Ala Leu Ala Ala 325 330
335 Glu Gln Arg Ala 340 1612820DNAUnknownObtained from
environmental sample 161atgtcctgcc gcaccctgat gagtaggcgt gtaggatggg
gacttttatt gtggggaggt 60ttattcctca gaaccggttc ggttacagga caaacttaca
attatgccga agtcctgcag 120aaatctatgt ttttctacga atgtcaggag
tctaaaattg ccccgggcaa tcgggtgaca 180tggcgagcta atgcagccat
gaacgatggg agcgatgttg gaaaagacct gacaggagga 240tggtttgatg
caggtgacca tgtgaaattt aattttccca tggcgtttac cgctacggcg
300ctggcgtggg gagctattga ctttgctcag ggatacatta gttccgggca
aatgcaatac 360ctgaaacgta atctgcgcta cgtcaatgac tatttcatta
aatgtcacac agcccccaac 420gaattgtatg gtcaggtggg taatggaggc
cttgaccatg ccttttgggg accacccgaa 480gtcatgcgca tggctaggcc
tgcctataaa attgatgcgt caaaacccgg atcagatctg 540gctgccgaaa
cagctgctgc aatggctgcc gccagcattg ttttcaaatc cgacgatcct
600acctatagcg ctactttgct gaatcatgca aaacagctgt tttcttttgc
cgaaacctat 660aaaggaaaat attccgacgc tattaccgat gctgcaggat
attataactc ctggagcggc 720tataacgatg aactggtatg gggagctata
tggctttacc gggctaccgg cgatgcaacc 780tatctatcta aggcagaatc
ctattacgac aatctgggta atcagggtca ggaacccgtt 840aaagcctaca
aatggaccat tgcatgggat gacaaatcct atggctgtta tgccctactg
900gccaaattga caggtaagga aaaatacaaa attgacgccg aacgttttct
cgactattgg 960accgatggtt ataatggttc ccggattact tataccccgg
gaggactcgc tttcctcgat 1020atatggggat cgttgcgcta tgctatgaat
actgcctttg ttgctgccta ctatgccgat 1080gcagccactt cagctgctaa
aaccacaaaa tatctcaact ttgctaaaca acaactgcat 1140tatgctcttg
gatccaatcc gagcaacaga agctatgtct gtggctttgg caataatcct
1200cccgttaatc ctcaccatag aggtgcacac ggagcatggt ctaataatgt
tcaaggacct 1260cctaccgaaa cacgacatat cctctacggc gcattagtgg
gtggaccagg cagtaatgac 1320tcctatactg acgaccgatc caattacacc
aataacgaag tagcatgtga ctacaatgct 1380cttttctccg gactgcttgc
aaagttcgtc attgattatg gaggcacacc gttagccaac 1440ttccctgttc
gtgaaacccc aaaagatgaa tatttcgttg aagcaaaagc aaacgctaca
1500ggaaccaatt tctccgaatg gacggtatgg gtatacaacc acactgcatg
gccagcccgt 1560gaaggttctg aatataaatt cagattatac gtaaatattt
cggaaggact ggctgcaggc 1620tatactgcct caaattatgt tgtgcaaacc
aataatgccg gtgtggtaaa ctttacccaa 1680cttttagctg ctgatgcagc
taacggcatc tattataccg aagtaacctt taaacctggt 1740accgaaattt
atcctggcgg gcaacagtat gacaagaagg aagctcagat gcgtattagt
1800ttgcccaatg ctccggcttc tgcatgggat ccgactaacg acccgtcatg
ggcgggaatc 1860acctctacct tgaaacaaat gccgggtata cccatgtatg
tagatggtgt aaaggtattt 1920ggtaatgagc ctgtcccagg tcagacagtt
cccgtcaccg gagtaaccgt atcgcctacc 1980accctgagtc tgactgtagg
acagaccagt acactcaccg ctaccgtatc gccggctaat 2040gctaccaaca
aaaacgtcac ctggagcagc agcaatacca gcgtagccac ggtaagctca
2100acaggcgttg tcacagccgt agcagccggt tcggccacca tcaccgtaac
cacagtcgat 2160ggcgctaaaa cagccacctg cgccgtaacg gtaacaggca
gcaccaacgt tcccgtcacc 2220ggagtaaccg tatcgcccac cacgctgagt
ctgaccgtag ggcagaccgc taccctcacc 2280gctaccgtat cgccggctaa
tgctaccaac aagaacgtta cctggagcag cagcaatacc 2340agcgtagcca
cggtaagttc aacaggcgta gttactgccg tagcggccgg ttcggccacc
2400atcaccgtaa ccaccgtcga tggagctaaa accgctacct gcaccgtaac
ggtaacgggc 2460agcactaccg tacccgtcac cggcgtaact gtatcgccta
ccaccctgag tctgaccgtt 2520ggacaaaccg ctaccctgac cgctaccgta
tcgccagctg atgctaccaa caagaacgtc 2580acctggagca gcagcaatac
cagcgtagcc acggtaagct caacaggcgt agtcactgcc 2640gtagcggccg
gttcagctac catcaccgtg accacagtcg atggggctaa aactgctacc
2700tgtgccgtga ccgtaaccgc cggaggttcc accaccccct gcagtaatcc
ggtaagcaaa 2760accctacctc tggtacagga tggtgccggc gaattcaggt
tgagtaatag ttttaattaa 2820162939PRTUnknownObtained from
environmental sample 162Met Ser Cys Arg Thr Leu Met Ser Arg Arg Val
Gly Trp Gly Leu Leu1 5 10 15 Leu Trp Gly Gly Leu Phe Leu Arg Thr
Gly Ser Val Thr Gly Gln Thr 20 25 30 Tyr Asn Tyr Ala Glu Val Leu
Gln Lys Ser Met Phe Phe Tyr Glu Cys 35 40 45 Gln Glu Ser Lys Ile
Ala Pro Gly Asn Arg Val Thr Trp Arg Ala Asn 50 55 60 Ala Ala Met
Asn Asp Gly Ser Asp Val Gly Lys Asp Leu Thr Gly Gly65 70 75 80 Trp
Phe Asp Ala Gly Asp His Val Lys Phe Asn Phe Pro Met Ala Phe 85 90
95 Thr Ala Thr Ala Leu Ala Trp Gly Ala Ile Asp Phe Ala Gln Gly Tyr
100 105 110 Ile Ser Ser Gly Gln Met Gln Tyr Leu Lys Arg Asn Leu Arg
Tyr Val 115 120 125 Asn Asp Tyr Phe Ile Lys Cys His Thr Ala Pro Asn
Glu Leu Tyr Gly 130 135 140 Gln Val Gly Asn Gly Gly Leu Asp His Ala
Phe Trp Gly Pro Pro Glu145 150 155 160 Val Met Arg Met Ala Arg Pro
Ala Tyr Lys Ile Asp Ala Ser Lys Pro 165 170 175 Gly Ser Asp Leu Ala
Ala Glu Thr Ala Ala Ala Met Ala Ala Ala Ser 180 185 190 Ile Val Phe
Lys Ser Asp Asp Pro Thr Tyr Ser Ala Thr Leu Leu Asn 195 200 205 His
Ala Lys Gln Leu Phe Ser Phe Ala Glu Thr Tyr Lys Gly Lys Tyr 210 215
220 Ser Asp Ala Ile Thr Asp Ala Ala Gly Tyr Tyr Asn Ser Trp Ser
Gly225 230 235 240 Tyr Asn Asp Glu Leu Val Trp Gly Ala Ile Trp Leu
Tyr Arg Ala Thr 245 250 255 Gly Asp Ala Thr Tyr Leu Ser Lys Ala Glu
Ser Tyr Tyr Asp Asn Leu 260 265 270 Gly Asn Gln Gly Gln Glu Pro Val
Lys Ala Tyr Lys Trp Thr Ile Ala 275 280 285 Trp Asp Asp Lys Ser Tyr
Gly Cys Tyr Ala Leu Leu Ala Lys Leu Thr 290 295 300 Gly Lys Glu Lys
Tyr Lys Ile Asp Ala Glu Arg Phe Leu Asp Tyr Trp305 310 315 320 Thr
Asp Gly Tyr Asn Gly Ser Arg Ile Thr Tyr Thr Pro Gly Gly Leu 325 330
335 Ala Phe Leu Asp Ile Trp Gly Ser Leu Arg Tyr Ala Met Asn Thr Ala
340 345 350 Phe Val Ala Ala Tyr Tyr Ala Asp Ala Ala Thr Ser Ala Ala
Lys Thr 355 360 365 Thr Lys Tyr Leu Asn Phe Ala Lys Gln Gln Leu His
Tyr Ala Leu Gly 370 375 380 Ser Asn Pro Ser Asn Arg Ser Tyr Val Cys
Gly Phe Gly Asn Asn Pro385 390 395 400 Pro Val Asn Pro His His Arg
Gly Ala His Gly Ala Trp Ser Asn Asn 405 410 415 Val Gln Gly Pro Pro
Thr Glu Thr Arg His Ile Leu Tyr Gly Ala Leu 420 425 430 Val Gly Gly
Pro Gly Ser Asn Asp Ser Tyr Thr Asp Asp Arg Ser Asn 435 440 445 Tyr
Thr Asn Asn Glu Val Ala Cys Asp Tyr Asn Ala Leu Phe Ser Gly 450 455
460 Leu Leu Ala Lys Phe Val Ile Asp Tyr Gly Gly Thr Pro Leu Ala
Asn465 470 475 480 Phe Pro Val Arg Glu Thr Pro Lys Asp Glu Tyr Phe
Val Glu Ala Lys 485 490 495 Ala Asn Ala Thr Gly Thr Asn Phe Ser Glu
Trp Thr Val Trp Val Tyr 500 505 510 Asn His Thr Ala Trp Pro Ala Arg
Glu Gly Ser Glu Tyr Lys Phe Arg 515 520 525 Leu Tyr Val Asn Ile Ser
Glu Gly Leu Ala Ala Gly Tyr Thr Ala Ser 530 535 540 Asn Tyr Val Val
Gln Thr Asn Asn Ala Gly Val Val Asn Phe Thr Gln545 550 555 560 Leu
Leu Ala Ala Asp Ala Ala Asn Gly Ile Tyr Tyr Thr Glu Val Thr 565 570
575 Phe Lys Pro Gly Thr Glu Ile Tyr Pro Gly Gly Gln Gln Tyr Asp Lys
580 585 590 Lys Glu Ala Gln Met Arg Ile Ser Leu Pro Asn Ala Pro Ala
Ser Ala 595 600 605 Trp Asp Pro Thr Asn Asp Pro Ser Trp Ala Gly Ile
Thr Ser Thr Leu 610 615 620 Lys Gln Met Pro Gly Ile Pro Met Tyr Val
Asp Gly Val Lys Val Phe625 630 635 640 Gly Asn Glu Pro Val Pro Gly
Gln Thr Val Pro Val Thr Gly Val Thr 645 650 655 Val Ser Pro Thr Thr
Leu Ser Leu Thr Val Gly Gln Thr Ser Thr Leu 660 665 670 Thr Ala Thr
Val Ser Pro Ala Asn Ala Thr Asn Lys Asn Val Thr Trp 675 680 685 Ser
Ser Ser Asn Thr Ser Val Ala Thr Val Ser Ser Thr Gly Val Val 690 695
700 Thr Ala Val Ala Ala Gly Ser Ala Thr Ile Thr Val Thr Thr Val
Asp705 710 715 720 Gly Ala Lys Thr Ala Thr Cys Ala Val Thr Val Thr
Gly Ser Thr Asn 725 730 735 Val Pro Val Thr Gly Val Thr Val Ser Pro
Thr Thr Leu Ser Leu Thr 740 745 750 Val Gly Gln Thr Ala Thr Leu Thr
Ala Thr Val Ser Pro Ala Asn Ala 755 760 765 Thr Asn Lys Asn Val Thr
Trp Ser Ser Ser Asn Thr Ser Val Ala Thr 770 775 780 Val Ser Ser Thr
Gly Val Val Thr Ala Val Ala Ala Gly Ser Ala Thr785 790 795 800 Ile
Thr Val Thr Thr Val Asp Gly Ala Lys Thr Ala Thr Cys Thr Val 805 810
815 Thr Val Thr Gly Ser Thr Thr Val Pro Val Thr Gly Val Thr Val Ser
820 825 830 Pro Thr Thr Leu Ser Leu Thr Val Gly Gln Thr Ala Thr Leu
Thr Ala 835 840 845 Thr Val Ser Pro Ala Asp Ala Thr Asn Lys Asn Val
Thr Trp Ser Ser 850 855 860 Ser Asn Thr Ser Val Ala Thr Val Ser Ser
Thr Gly Val Val Thr Ala865 870 875 880 Val Ala Ala Gly Ser Ala Thr
Ile Thr Val Thr Thr Val Asp Gly Ala 885 890 895 Lys Thr Ala Thr Cys
Ala Val Thr Val Thr Ala Gly Gly Ser Thr Thr 900 905 910 Pro Cys Ser
Asn Pro Val Ser Lys Thr Leu Pro Leu Val Gln Asp Gly 915 920 925 Ala
Gly Glu Phe Arg Leu Ser Asn Ser Phe Asn 930 935
1632733DNAUnknownObtained from environmental sample 163atgcaaactt
acaattatgc cgaagtcctg cagaaatcta tgtttttcta cgaatgtcag 60gagtctaaaa
ttgccccggg caatcgggtg acatggcgag ctaatgcagc catgaacgat
120gggagcgatg ttggaaaaga cctgacagga ggatggtttg atgcaggtga
ccatgtgaaa 180tttaattttc ccatggcgtt taccgctacg gcgctggcgt
ggggagctat tgactttgct 240cagggataca ttagttccgg gcaaatgcaa
tacctgaaac gtaatctgcg ctacgtcaat 300gactatttca ttaaatgtca
cacagccccc aacgaattgt atggtcaggt gggtaatgga 360ggccttgacc
atgccttttg gggaccaccc gaagtcatgc gcatggctag gcctgcctat
420aaaattgatg cgtcaaaacc cggatcagat ctggctgccg aaacagctgc
tgcaatggct 480gccgccagca ttgttttcaa atccgacgat cctacctata
gcgctacttt gctgaatcat 540gcaaaacagc tgttttcttt tgccgaaacc
tataaaggaa aatattccga cgctattacc 600gatgctgcag gatattataa
ctcctggagc ggctataacg atgaactggt atggggagct 660atatggcttt
accgggctac cggcgatgca acctatctat ctaaggcaga atcctattac
720gacaatctgg gtaatcaggg tcaggaaccc gttaaagcct acaaatggac
cattgcatgg 780gatgacaaat cctatggctg ttatgcccta ctggccaaat
tgacaggtaa ggaaaaatac 840aaaattgacg ccgaacgttt tctcgactat
tggaccgatg gttataatgg ttcccggatt 900acttataccc cgggaggact
cgctttcctc gatatatggg gatcgttgcg ctatgctatg 960aatactgcct
ttgttgctgc ctactatgcc gatgcagcca cttcagctgc taaaaccaca
1020aaatatctca actttgctaa acaacaactg cattatgctc ttggatccaa
tccgagcaac 1080agaagctatg tctgtggctt tggcaataat cctcccgtta
atcctcacca tagaggtgca 1140cacggagcat ggtctaataa tgttcaagga
cctcctaccg aaacacgaca tatcctctac 1200ggcgcattag tgggtggacc
aggcagtaat gactcctata ctgacgaccg atccaattac 1260accaataacg
aagtagcatg tgactacaat gctcttttct ccggactgct tgcaaagttc
1320gtcattgatt atggaggcac accgttagcc aacttccctg ttcgtgaaac
cccaaaagat 1380gaatatttcg ttgaagcaaa agcaaacgct acaggaacca
atttctccga atggacggta 1440tgggtataca accacactgc atggccagcc
cgtgaaggtt ctgaatataa attcagatta 1500tacgtaaata tttcggaagg
actggctgca ggctatactg cctcaaatta tgttgtgcaa 1560accaataatg
ccggtgtggt aaactttacc caacttttag ctgctgatgc agctaacggc
1620atctattata ccgaagtaac ctttaaacct ggtaccgaaa tttatcctgg
cgggcaacag 1680tatgacaaga aggaagctca gatgcgtatt agtttgccca
atgctccggc ttctgcatgg 1740gatccgacta acgacccgtc atgggcggga
atcacctcta ccttgaaaca aatgccgggt 1800atacccatgt atgtagatgg
tgtaaaggta tttggtaatg agcctgtccc aggtcagaca 1860gttcccgtca
ccggagtaac cgtatcgcct accaccctga gtctgactgt aggacagacc
1920agtacactca ccgctaccgt atcgccggct aatgctacca acaaaaacgt
cacctggagc 1980agcagcaata ccagcgtagc cacggtaagc tcaacaggcg
ttgtcacagc cgtagcagcc 2040ggttcggcca ccatcaccgt aaccacagtc
gatggcgcta aaacagccac ctgcgccgta 2100acggtaacag gcagcaccaa
cgttcccgtc accggagtaa ccgtatcgcc caccacgctg 2160agtctgaccg
tagggcagac cgctaccctc accgctaccg tatcgccggc taatgctacc
2220aacaagaacg ttacctggag cagcagcaat accagcgtag ccacggtaag
ttcaacaggc 2280gtagttactg ccgtagcggc cggttcggcc accatcaccg
taaccaccgt cgatggagct 2340aaaaccgcta cctgcaccgt aacggtaacg
ggcagcacta ccgtacccgt caccggcgta 2400actgtatcgc ctaccaccct
gagtctgacc gttggacaaa ccgctaccct gaccgctacc 2460gtatcgccag
ctgatgctac caacaagaac gtcacctgga gcagcagcaa taccagcgta
2520gccacggtaa gctcaacagg cgtagtcact gccgtagcgg ccggttcagc
taccatcacc 2580gtgaccacag tcgatggggc taaaactgct acctgtgccg
tgaccgtaac cgccggaggt 2640tccaccaccc cctgcagtaa tccggtaagc
aaaaccctac ctctggtaca ggatggtgcc 2700ggcgaattca ggttgagtaa
tagttttaat taa 2733164910PRTUnknownObtained from environmental
sample 164Met Gln Thr Tyr Asn Tyr Ala Glu Val Leu Gln Lys Ser Met
Phe Phe1 5 10 15Tyr Glu Cys Gln Glu Ser Lys Ile Ala Pro Gly Asn Arg
Val Thr Trp 20 25 30Arg Ala Asn Ala Ala Met Asn Asp Gly Ser Asp Val
Gly Lys Asp Leu 35 40 45Thr Gly Gly Trp Phe Asp Ala Gly Asp His Val
Lys Phe Asn Phe Pro 50 55 60Met Ala Phe Thr Ala Thr Ala Leu Ala Trp
Gly Ala Ile Asp Phe Ala65 70 75 80Gln Gly Tyr Ile Ser Ser Gly Gln
Met Gln Tyr Leu Lys Arg Asn Leu 85 90 95Arg Tyr Val Asn Asp Tyr Phe
Ile Lys Cys His Thr Ala Pro Asn Glu 100 105 110Leu Tyr Gly Gln Val
Gly Asn Gly Gly Leu Asp His Ala Phe Trp Gly 115 120 125Pro Pro Glu
Val Met Arg Met Ala Arg Pro Ala Tyr Lys Ile Asp Ala 130 135 140Ser
Lys Pro Gly Ser Asp Leu Ala Ala Glu Thr Ala Ala Ala Met Ala145 150
155 160Ala Ala Ser Ile Val Phe Lys Ser Asp Asp Pro Thr Tyr Ser Ala
Thr 165 170 175Leu Leu Asn His Ala Lys Gln Leu Phe Ser Phe Ala Glu
Thr Tyr Lys 180 185 190Gly Lys Tyr Ser Asp Ala Ile Thr Asp Ala Ala
Gly Tyr Tyr Asn Ser 195 200 205Trp Ser Gly Tyr Asn Asp Glu Leu Val
Trp Gly Ala Ile Trp Leu Tyr 210 215 220Arg Ala Thr Gly Asp Ala Thr
Tyr Leu Ser Lys Ala Glu Ser Tyr Tyr225 230 235 240Asp Asn Leu Gly
Asn Gln Gly Gln Glu Pro Val Lys Ala Tyr Lys Trp 245 250 255Thr Ile
Ala Trp Asp Asp Lys Ser Tyr Gly Cys Tyr Ala Leu Leu Ala 260 265
270Lys Leu Thr Gly Lys Glu Lys Tyr Lys Ile Asp Ala Glu Arg Phe Leu
275 280 285Asp Tyr Trp Thr Asp Gly Tyr Asn Gly Ser Arg Ile Thr Tyr
Thr Pro 290 295 300Gly Gly Leu Ala Phe Leu Asp Ile Trp Gly Ser Leu
Arg Tyr Ala Met305 310 315 320Asn Thr Ala Phe Val Ala Ala Tyr Tyr
Ala Asp Ala Ala Thr Ser Ala 325 330 335Ala Lys Thr Thr Lys Tyr Leu
Asn Phe Ala Lys Gln Gln Leu His Tyr 340 345 350Ala Leu Gly Ser Asn
Pro Ser Asn Arg Ser Tyr Val Cys Gly Phe Gly 355 360 365Asn Asn Pro
Pro Val Asn Pro His His Arg Gly Ala His Gly Ala Trp 370 375 380Ser
Asn Asn Val Gln Gly Pro Pro Thr Glu Thr Arg His Ile Leu Tyr385 390
395 400Gly Ala Leu Val Gly Gly Pro Gly Ser Asn Asp Ser Tyr Thr Asp
Asp 405 410 415Arg Ser Asn Tyr Thr Asn Asn Glu Val Ala Cys Asp Tyr
Asn Ala Leu 420 425 430Phe Ser Gly Leu Leu Ala Lys Phe Val Ile Asp
Tyr Gly Gly Thr Pro 435 440 445Leu Ala Asn Phe Pro Val Arg Glu Thr
Pro Lys Asp Glu Tyr Phe Val 450 455 460Glu Ala Lys Ala Asn Ala Thr
Gly Thr Asn Phe Ser Glu Trp Thr Val465 470 475 480Trp Val
Tyr Asn His Thr Ala Trp Pro Ala Arg Glu Gly Ser Glu Tyr 485 490
495Lys Phe Arg Leu Tyr Val Asn Ile Ser Glu Gly Leu Ala Ala Gly Tyr
500 505 510Thr Ala Ser Asn Tyr Val Val Gln Thr Asn Asn Ala Gly Val
Val Asn 515 520 525Phe Thr Gln Leu Leu Ala Ala Asp Ala Ala Asn Gly
Ile Tyr Tyr Thr 530 535 540Glu Val Thr Phe Lys Pro Gly Thr Glu Ile
Tyr Pro Gly Gly Gln Gln545 550 555 560Tyr Asp Lys Lys Glu Ala Gln
Met Arg Ile Ser Leu Pro Asn Ala Pro 565 570 575Ala Ser Ala Trp Asp
Pro Thr Asn Asp Pro Ser Trp Ala Gly Ile Thr 580 585 590Ser Thr Leu
Lys Gln Met Pro Gly Ile Pro Met Tyr Val Asp Gly Val 595 600 605Lys
Val Phe Gly Asn Glu Pro Val Pro Gly Gln Thr Val Pro Val Thr 610 615
620Gly Val Thr Val Ser Pro Thr Thr Leu Ser Leu Thr Val Gly Gln
Thr625 630 635 640Ser Thr Leu Thr Ala Thr Val Ser Pro Ala Asn Ala
Thr Asn Lys Asn 645 650 655Val Thr Trp Ser Ser Ser Asn Thr Ser Val
Ala Thr Val Ser Ser Thr 660 665 670Gly Val Val Thr Ala Val Ala Ala
Gly Ser Ala Thr Ile Thr Val Thr 675 680 685Thr Val Asp Gly Ala Lys
Thr Ala Thr Cys Ala Val Thr Val Thr Gly 690 695 700Ser Thr Asn Val
Pro Val Thr Gly Val Thr Val Ser Pro Thr Thr Leu705 710 715 720Ser
Leu Thr Val Gly Gln Thr Ala Thr Leu Thr Ala Thr Val Ser Pro 725 730
735Ala Asn Ala Thr Asn Lys Asn Val Thr Trp Ser Ser Ser Asn Thr Ser
740 745 750Val Ala Thr Val Ser Ser Thr Gly Val Val Thr Ala Val Ala
Ala Gly 755 760 765Ser Ala Thr Ile Thr Val Thr Thr Val Asp Gly Ala
Lys Thr Ala Thr 770 775 780Cys Thr Val Thr Val Thr Gly Ser Thr Thr
Val Pro Val Thr Gly Val785 790 795 800Thr Val Ser Pro Thr Thr Leu
Ser Leu Thr Val Gly Gln Thr Ala Thr 805 810 815Leu Thr Ala Thr Val
Ser Pro Ala Asp Ala Thr Asn Lys Asn Val Thr 820 825 830Trp Ser Ser
Ser Asn Thr Ser Val Ala Thr Val Ser Ser Thr Gly Val 835 840 845Val
Thr Ala Val Ala Ala Gly Ser Ala Thr Ile Thr Val Thr Thr Val 850 855
860Asp Gly Ala Lys Thr Ala Thr Cys Ala Val Thr Val Thr Ala Gly
Gly865 870 875 880Ser Thr Thr Pro Cys Ser Asn Pro Val Ser Lys Thr
Leu Pro Leu Val 885 890 895Gln Asp Gly Ala Gly Glu Phe Arg Leu Ser
Asn Ser Phe Asn 900 905 9101651347DNAUnknownObtained from
environmental sample 165atgacaatta acaacaaaac tacagcgagt cctagtattc
ccagcaccca caattccctc 60ccgtcgcttc gcacactgtt taccaccagc ctgctcacgc
tggccctgac cgcctgcggt 120ggttcttcca gcagcgacaa ggacccttca
agctccagct ccagtgaatc atcaagttcc 180agcgaatcct cgagctcagc
ttccagcgaa tcctcgagca gtgagtccag cagtagctct 240tccgcgggcc
atttctccat cgagccggac ttccagctct acagcctggc caacttcccg
300gtgggcgtgg cggtctccgc cgccaacgag aacgacagca tcttcaacag
tccggatgcc 360gccgaacgtc aggccgttat tattgagcac ttctctcagc
tcaccgccgg caacatcatg 420aaaatgagct acctgcagcc gagtcaaggc
aacttcacct tcgatgacgc cgacgagttg 480gttaacttcg cccaagccaa
tggcatgacc gtacacggcc actccaccat ctggcacgcg 540gactaccaag
taccgaactt catgagaaac tttgaaggtg accaggagga atgggcagaa
600attctgaccg atcacgtcac taccatcatc gagcacttcc ccgacgatgt
ggtcatcagc 660tgggacgtgg tgaacgaggc tgtcgatcaa ggcacggcga
acggctggcg ccattcggtg 720ttctacaatg cattcgacgc cccggaagaa
ggcgacattc ccgaatacat caaagtcgct 780ttccgcgccg cgcgcgaggc
tgacgccaac gtagacctct actacaacga ctacgacaat 840accgccaatg
cccagcgcct ggccaaaaca ctgcaaattg ccgaggtact ggacgccgaa
900ggcaccattg acggcgtcgg tttccagatg cacgcctaca tggattaccc
gagcctgacc 960cattttgaaa acgccttccg gcaagtcgtc gacctggggc
tcaaagtgaa agttaccgag 1020ctggacgtat ccgtagtcaa cccctacggc
ggcgaagcac ctccacaacc ggaatacgac 1080aaagaactgg ccggcgcgca
aaaactgcgc ttctgccaaa tcgccgaagt ttacatgaac 1140actgtacccg
aggagttacg cggtggcttc accgtctggg gcctgaccga tgatgaaagt
1200tggctgatgc aacagttcag aaacgccacc ggcgccgact acgacgacgt
ctggccgtta 1260ctgttcaatg ccgacaaatc cgccaaaccg gcactgcaag
gcgtggccga cgcctttacc 1320ggacaaacct gcacctccga gttctaa
1347166448PRTUnknownObtained from environmental sample 166Met Thr
Ile Asn Asn Lys Thr Thr Ala Ser Pro Ser Ile Pro Ser Thr1 5 10 15His
Asn Ser Leu Pro Ser Leu Arg Thr Leu Phe Thr Thr Ser Leu Leu 20 25
30Thr Leu Ala Leu Thr Ala Cys Gly Gly Ser Ser Ser Ser Asp Lys Asp
35 40 45Pro Ser Ser Ser Ser Ser Ser Glu Ser Ser Ser Ser Ser Glu Ser
Ser 50 55 60Ser Ser Ala Ser Ser Glu Ser Ser Ser Ser Glu Ser Ser Ser
Ser Ser65 70 75 80Ser Ala Gly His Phe Ser Ile Glu Pro Asp Phe Gln
Leu Tyr Ser Leu 85 90 95Ala Asn Phe Pro Val Gly Val Ala Val Ser Ala
Ala Asn Glu Asn Asp 100 105 110Ser Ile Phe Asn Ser Pro Asp Ala Ala
Glu Arg Gln Ala Val Ile Ile 115 120 125Glu His Phe Ser Gln Leu Thr
Ala Gly Asn Ile Met Lys Met Ser Tyr 130 135 140Leu Gln Pro Ser Gln
Gly Asn Phe Thr Phe Asp Asp Ala Asp Glu Leu145 150 155 160Val Asn
Phe Ala Gln Ala Asn Gly Met Thr Val His Gly His Ser Thr 165 170
175Ile Trp His Ala Asp Tyr Gln Val Pro Asn Phe Met Arg Asn Phe Glu
180 185 190Gly Asp Gln Glu Glu Trp Ala Glu Ile Leu Thr Asp His Val
Thr Thr 195 200 205Ile Ile Glu His Phe Pro Asp Asp Val Val Ile Ser
Trp Asp Val Val 210 215 220Asn Glu Ala Val Asp Gln Gly Thr Ala Asn
Gly Trp Arg His Ser Val225 230 235 240Phe Tyr Asn Ala Phe Asp Ala
Pro Glu Glu Gly Asp Ile Pro Glu Tyr 245 250 255Ile Lys Val Ala Phe
Arg Ala Ala Arg Glu Ala Asp Ala Asn Val Asp 260 265 270Leu Tyr Tyr
Asn Asp Tyr Asp Asn Thr Ala Asn Ala Gln Arg Leu Ala 275 280 285Lys
Thr Leu Gln Ile Ala Glu Val Leu Asp Ala Glu Gly Thr Ile Asp 290 295
300Gly Val Gly Phe Gln Met His Ala Tyr Met Asp Tyr Pro Ser Leu
Thr305 310 315 320His Phe Glu Asn Ala Phe Arg Gln Val Val Asp Leu
Gly Leu Lys Val 325 330 335Lys Val Thr Glu Leu Asp Val Ser Val Val
Asn Pro Tyr Gly Gly Glu 340 345 350Ala Pro Pro Gln Pro Glu Tyr Asp
Lys Glu Leu Ala Gly Ala Gln Lys 355 360 365Leu Arg Phe Cys Gln Ile
Ala Glu Val Tyr Met Asn Thr Val Pro Glu 370 375 380Glu Leu Arg Gly
Gly Phe Thr Val Trp Gly Leu Thr Asp Asp Glu Ser385 390 395 400Trp
Leu Met Gln Gln Phe Arg Asn Ala Thr Gly Ala Asp Tyr Asp Asp 405 410
415Val Trp Pro Leu Leu Phe Asn Ala Asp Lys Ser Ala Lys Pro Ala Leu
420 425 430Gln Gly Val Ala Asp Ala Phe Thr Gly Gln Thr Cys Thr Ser
Glu Phe 435 440 445
* * * * *
References