U.S. patent application number 15/831357 was filed with the patent office on 2018-11-22 for xylanases, nucleic acids encoding them and methods for making and using them.
The applicant listed for this patent is BP Corporation North America Inc.. Invention is credited to Reinhard Dirmeier, Kevin A. Gray.
Application Number | 20180334659 15/831357 |
Document ID | / |
Family ID | 40526901 |
Filed Date | 2018-11-22 |
United States Patent
Application |
20180334659 |
Kind Code |
A1 |
Gray; Kevin A. ; et
al. |
November 22, 2018 |
XYLANASES, NUCLEIC ACIDS ENCODING THEM AND METHODS FOR MAKING AND
USING THEM
Abstract
The invention relates to enzymes having xylanase, mannanase
and/or glucanase activity, e.g., catalyzing hydrolysis of internal
.beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase
linkages; and/or degrading a linear polysaccharide beta-1,4-xylan
into xylose. Thus, the invention provides methods and processes for
breaking down hemicellulose, which is a major component of the cell
wall of plants, including methods and processes for hydrolyzing
hemicelluloses in any plant or wood or wood product, wood waste,
paper pulp, paper product or paper waste or byproduct. In addition,
methods of designing new xylanases, mannanases and/or glucanases
and methods of use thereof are also provided. The xylanases,
mannanases and/or glucanases have increased activity and stability
at increased pH and temperature.
Inventors: |
Gray; Kevin A.; (San Diego,
CA) ; Dirmeier; Reinhard; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BP Corporation North America Inc. |
Houston |
TX |
US |
|
|
Family ID: |
40526901 |
Appl. No.: |
15/831357 |
Filed: |
December 4, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14732217 |
Jun 5, 2015 |
RE46733 |
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15831357 |
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12681604 |
Sep 14, 2010 |
8486680 |
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PCT/US08/72030 |
Aug 1, 2008 |
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14732217 |
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60977348 |
Oct 3, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/02 20130101;
C12P 19/14 20130101; A61P 1/00 20180101; Y10T 428/249921 20150401;
Y02E 50/16 20130101; A61P 3/00 20180101; Y02E 50/10 20130101; C12N
9/248 20130101; D21H 17/22 20130101; Y02T 50/678 20130101 |
International
Class: |
C12N 9/24 20060101
C12N009/24; D21H 17/22 20060101 D21H017/22; C12P 19/02 20060101
C12P019/02; C12P 19/14 20060101 C12P019/14 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. 1435-04-03-CA-70224 awarded by the Department of Energy. The
Government has certain rights in the invention.
Claims
1. An isolated, synthetic, or recombinant polypeptide having a
xylanase activity, wherein the polypeptide comprises an amino acid
sequence having at least 95% sequence identity to SEQ ID NO: 2, and
amino acid residue change at amino acid residue 44 such that amino
acid residue 44 Serine, Ser (or "S") is changed to Threonine, "Thr"
(or "T").
2. A protein preparation, or an immobilized polypeptide comprising
the polypeptide of claim 1, wherein the protein preparation
comprises a liquid, a solid or a gel.
3. (canceled)
4. A method for hydrolyzing, liquefying, breaking up, or disrupting
a xylan-, cellulose- or hemicellulose-comprising composition,
comprising: contacting the polypeptide of claim 1 with the
composition comprising a xylan, a cellulose, or a hemicellulose
under conditions wherein the polypeptide hydrolyze, liquefy, break
up, or disrupt the xylan, cellulose, or hemicellulose-comprising
composition, wherein optionally the composition comprises a plant
cell, or a bacterial cell.
5. A biomass, wood, wood pulp, wood product, paper pulp, paper
product, newspaper or paper waste comprising the polypeptide of
claim 1.
6. An enzyme cocktail comprising the polypeptide of claim 1, and
one or more enzymes selected from a group consisting of: a
xylanase, a mannanase, a glucanase, a cellulose, a lipase, an
esterase, a protease, an endoglycosidase, an
endo-beta.-1,4-glucanase, a beta-glucanase, an
endo-beta-1,3(4)-glucanase, a cutinase, a peroxidase, a catalase, a
laccase, an amylase, a glucoamylase, a pectinase, a reductase, an
oxidase, a phenoloxidase, a ligninase, a pullulanase, an
arabinanase, a hemicellulase, a mannanase, a xyloglucanase, a
xylanase, a mannanase, a glucanase, a pectin acetyl esterase, a
rhamnogalacturonan acetyl esterase, a polygalacturonase, a
rhamnogalacturonase, a galactanase, a pectin lyase, a pectin
methylesterase, a cellobiohydrolase, a transglutaminase, or a
combination thereof.
7. A process for hydrolyzing xylans, celluloses, or hemicelluloses
in any organic compound, plant or wood or wood product or
byproduct, wood waste, paper pulp, paper product or paper waste or
byproduct with the polypeptide of claim 1.
8. A composition comprising the polypeptide of claim 1.
9. The enzyme cocktail of claim 6, further comprising at least one
additional enzyme selected from: an Endoglucanase, an Oligomerase I
(beta glucosidase), a CBHI (GH family 7), a CBH2 (GH family 6), a
Xylanase (GH family 11), an Arabinofuranosidase, a Xylanase (GH
family 10), and an Oligomerase II (beta-xylosidase).
10. A fabric, yarn, cloth or textile comprising the polypeptide of
claim 1 wherein the fabric, yam, cloth or textile comprises a
non-cotton cellulosic fabric, yarn, cloth, or textile.
11. A food, a feed, or a nutritional supplement comprising the
polypeptide of claim 1.
12. The polypeptide of claim 1, further comprising a heterologous
amino acid sequence.
13. The polypeptide of claim 12, wherein the heterologous amino
acid sequence comprises: (a) a heterologous signal sequence, a
heterologous carbohydrate binding module, a heterologous dockerin
domain, a heterologous catalytic domain (CD), or a combination
thereof; (b) the sequence of (a), wherein the heterologous signal
sequence, carbohydrate binding module or heterologous catalytic
domain (CD) is derived from a heterologous enzyme; a tag, an
epitope, a targeting peptide, a cleavable sequence, a detectable
moiety or an enzyme; or (c) the sequence of (a), wherein the
heterologous carbohydrate binding module (CBM) comprises, a xylan
binding module, a cellulose binding module, a lignin binding
module, a xylose binding module, a mannanase binding module, a
xyloglucan-specific module or a arabinofuranosidase binding
module.
14. The polypeptide of claim 1, wherein the amino acid sequence
further comprising a second amino acid change selected from a group
consisting of: (a) a change at amino acid residue 4 such that amino
acid residue 4 Threonine, or "Thr" (or "T") is changed to
Asparagine, or Asn (or "N"); (b) a change at amino acid residue 4
such that amino acid residue 4 Threonine, or "Thr" (or "T") is
changed to Arginine, Arg (or "R"); (c) a change at amino acid
residue 4 such that amino acid residue 4 Threonine, or "Thr" (or
"T") is changed to Histidine, His (or "H"); (d) a change at amino
acid residue 73 such that amino acid residue 73 Glycine, Gly (or
"G") is changed to Tyrosine, "Tyr" (or "Y"); (e) a change at amino
acid residue 63 such that amino acid residue 63 Isoleucine, lie (or
"I") is changed to Valine, "Val" (or "V"); (f) a change at amino
acid residue 17 such that amino acid residue 17 Phenylalanine, Phe
(or "F") is changed to Valine, "Val" (or "V"); (g) a change at
amino acid residue 38 such that amino acid residue 38 Arginine, Arg
(or "R") is changed to Histidine, His (or "H"); (h) a change at
amino acid residue 33 such that amino acid residue 33 Leucine, Leu
(or "L") is changed to Alanine, Ala (or "A"); (i) a change at amino
acid residue 73 such that amino acid residue 73 Glycine, Gly (or
"G") is changed to Glutamate, Glu (or "E"); (j) a change at amino
acid residue 73 such that amino acid residue 73 Glycine, Gly (or
"G") is changed to Valine, "Val" (or "V"); (k) a change at amino
acid residue 125 such that amino acid residue 125 Glutamine, Gin
(or "Q") is changed to Tyrosine, "Tyr" (or "Y"); (l) a change at
amino acid residue 188 such that amino acid residue 188 Serine, Ser
(or "S") is changed to Glutamate, Glu (or "E"); (m) a change at
amino acid residue 9, such that amino acid residue 9 Proline, Pro
(or "P") is changed to Aspartate, Asp (or "D"); (n) a change at
amino acid residue 150, such that amino acid residue 150 Valine,
Val (or "V") is changed to Alanine, Ala (or "A"); (o) a change at
amino acid residue 189, such that amino acid residue 189 Serine,
Ser (or "S") is changed to Glutamine, Gin (or "Q"); (p) a change at
amino acid residue 21, such that amino acid residue 21
Phenylalanine, Phe (or "F") is changed to Tyrosine, "Tyr" (or "Y");
(q) a change at amino acid residue 108, such that amino acid
residue 108 Phenylalanine, Phe (or "F") is changed to Lysine, "Lys"
(or "K"); and (r) any combination of the changes (a) to (q).
15. A method for reducing the amount of lignin (delignification),
or solubilizing a lignin, in a paper or paper product, a wood, wood
pulp or wood product, or a wood or paper recycling composition,
comprising: contacting the paper or paper product, wood, wood pulp
or wood product, or wood or paper recycling composition with the
polypeptide of claim 1 thereby reducing the amount of lignin.
16. A method for an enzymatic decoloring of paper, hemp, or flax
pulp comprising: contacting the paper, hemp, or flax pulp with the
polypeptide of claim 1 and a decoloring agent, wherein optionally
the decoloring agent comprises oxygen or hydrogen peroxide under
conditions suitable for enzymatic decoloring.
17. A method for an enzymatic deinking of paper, paper waste, paper
recycled product, deinking toner from non-contact printed
wastepaper or mixtures of non-contact and contact printed
wastepaper, comprising: contacting the paper, paper waste, paper
recycled product, non-contact printed wastepaper, or contact
printed wastepaper with the polypeptide of claim 1 under conditions
suitable for enzymatic deinking.
18. A method for decoloring a fabric, yarn, cloth, or textile
comprising: contacting the fabric, yam, cloth, or textile with the
polypeptide of claim 1 under conditions suitable to produce a
whitening of the textile, wherein optionally the fabric, yarn,
cloth, or textile comprises a non-cotton cellulosic fabric, yarn,
cloth, or textile, under conditions suitable for enzymatic
decoloring.
19. The polypeptide of claim 1, wherein the amino acid sequence is
a fragment of SEQ ID NO:2 having xylanase activity.
20. A method for hydrolyzing celluloses, hemicelluloses, or xylans
in a biomass, a wood, wood product, paper pulp, paper product or
paper waste comprising contacting the wood, wood product, paper
pulp, paper product or paper waste with the polypeptide of claim
1.
21. A method for using the polypeptide of claim 1 for converting
biomass to methanol, butanol, ethanol or propanol.
22-26. (canceled)
27. The polypeptide of claim 1, produced by the method comprising:
(a) transforming a cell with a vector comprising polynucleotide
sequence encoding the polypeptide of claim 1; and (b) culturing the
transformed cell under conditions to produce the polypeptide.
28. The polypeptide of claim 27 further comprising disrupting the
cells and collecting the resulting extract comprising the
polypeptide.
29. The polypeptide of claim 27, wherein the transformed cell is a
bacterial cell or a fungal cell.
30. The polypeptide of claim 27, wherein the polynucleotide
sequence is operably linked to a promoter.
31. The polypeptide of claim 27, wherein the polynucleotide
sequence has the sequence comprising SEQ ID NO:1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/732,217 filed Jun. 5, 2015, now pending;
which is a reissue application of U.S. application Ser. No.
12/681,604 filed Sep. 14, 2010, now issued as U.S. Pat. No.
8,486,680; which is a 35 USC .sctn. 371 National Stage application
of International Application No. PCT/US2008/072020 filed Aug. 1,
2008, now expired; which claims the benefit under 35 USC .sctn.
119(e) to U.S. Application Ser. No. 60/977,348 filed Oct. 3, 2007,
now expired. The disclosure of each of the prior applications is
considered part of and is incorporated by reference in the
disclosure of this application.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0003] This application was filed electronically via the USPTO
EFS-WEB server as authorized and set forth in MPEP .sctn. 1730
II.B.2.(a)(A), and this electronic filing includes an
electronically submitted sequence (SEQ ID) listing; the entire
content of this sequence listing is herein incorporated by
reference for all purposes. The sequence listing is identified on
the electronically filed .txt file as follows:
TABLE-US-00001 File Name Date of Creation Size BP1200-4_ST25.txt
Dec. 2, 2017 87 KB
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] This invention relates generally to enzymes, polynucleotides
encoding the enzymes, the use of such polynucleotides and
polypeptides and more specifically to enzymes having xylanase
activity, e.g., endoxylanase activity, and/or catalyzing hydrolysis
of internal .beta.-1,4-xylosidic linkages or
endo-.beta.-1,4-glucanase linkages; and/or degrading a linear
polysaccharide beta-1,4-xylan into xylose; or, a glucanase
activity, e.g., an endoglucanase activity, for example, catalyzing
hydrolysis of internal endo-.beta.-1,4- and/or 1,3-glucanase
linkages, a xylanase activity, and/or a mannanase activity. Thus,
the invention provides methods and processes for breaking down
hemicellulose, which is a major component of the cell wall of
plants, including methods and processes for hydrolyzing
hemicelluloses in any organic compound, plant or wood or wood
product or byproduct, wood waste, paper pulp, paper product or
paper waste or byproduct. The invention further provides methods
and processes for breaking down plant matter containing cellulose
and/or hemicellulose into simple sugars using the "cocktails" of
the invention.
Background Information
[0005] Xylanases (e.g., endo-1,4-beta-xylanase, EC 3.2.1.8)
hydrolyze internal .beta.-1,4-xylosidic linkages in xylan to
produce smaller molecular weight xylose and xylo-oligomers. Xylans
are polysaccharides formed from 1,4-.beta.-glycoside-linked
D-xylopyranoses. Xylanases are of considerable commercial value,
being used in the food industry, for baking and fruit and vegetable
processing, breakdown of agricultural waste, in the manufacture of
animal feed and in pulp and paper production. Xylanases are formed
by fungi and bacteria.
[0006] Arabinoxylans are major non-starch polysaccharides of
cereals representing 2.5-7.1% w/w 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, arabinoxylans have high
water-binding capacity and may have a role in protein foam
stability. All of these characteristics present problems for
several industries including brewing, baking, animal nutrition and
paper manufacturing. In brewing applications, the presence of xylan
results in wort filterability and haze formation issues. In baking
applications (especially for cookies and crackers), these
arabinoxylans create sticky doughs that are difficult to machine
and reduce biscuit size. In addition, this carbohydrate is
implicated in rapid rehydration of the baked product resulting in
loss of crispiness and reduced shelf-life. For monogastric animal
feed applications with cereal diets, arabinoxylan is a major
contributing factor to viscosity of gut contents and thereby
adversely affects the digestibility of the feed and animal growth
rate. For ruminant animals, these polysaccharides represent
substantial components of fiber intake and more complete digestion
of arabinoxylans would facilitate higher feed conversion
efficiencies.
[0007] There remains a need in the art for xylanases to be used in
the paper and pulp industry, for example, where the enzyme is
active in the temperature range of 65.degree. C. to 75.degree. C.
and at a pH of approximately 10. Additionally, an enzyme useful in
the paper and pulp industry would decrease the need for bleaching
chemicals, such as chlorine dioxide.
[0008] Additionally, there remains a need to provide efficient, low
cost processes and compositions for producing bioalcohols, biofuels
and/or biofuel- (e.g., bioethanol-, propanol-, butanol- and/or
methanol-) by conversion of biomass. An enzyme or enzyme "cocktail"
could provide a route to convert biomass into sugars that could
then be fermented into biofuels.
SUMMARY OF THE INVENTION
[0009] The invention provides enzymes having: xylanase activity,
e.g., endoxylanase activity, and/or catalyzing hydrolysis of
internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase
linkages; and/or, having a glucanase activity, e.g., an
endoglucanase activity, for example, catalyzing hydrolysis of
internal endo-.beta.-1,4- and/or 1,3-glucanase linkages, a xylanase
activity, and/or a mannanase activity; and, nucleic acids encoding
them, vectors and cells comprising them, probes for amplifying and
identifying these xylanase-encoding nucleic acids, and methods for
making and using these polypeptides and peptides.
[0010] For example, the invention provides enzymes having xylanase
(e.g., endoxylanase activity), and compositions and methods
comprising them, for hydrolyzing internal .beta.-1,4-xylosidic
linkages or endo-.beta.-1,4-glucanase linkages, or hemicelluloses,
in a wood, wood product, paper pulp, paper product or paper waste.
In one aspect, the xylanase activity comprises catalyzing
hydrolysis of xylan, e.g., degrading a linear polysaccharide
beta-1,4-xylan into a xylose. Thus, the invention provides methods
and processes for breaking down a xylan-comprising composition
and/or a hemicellulose, which is a major component of the cell wall
of plants.
[0011] In one aspect, the glucanase activity of a polypeptide or
peptide of the invention (which includes a protein or peptide
encoded by a nucleic acid of the invention) comprises an
endoglucanase activity, e.g., endo-1,4- and/or 1,3-beta-D-glucan
4-glucano hydrolase activity. In one aspect, the endoglucanase
activity comprises catalyzing hydrolysis of 1,4-beta-D-glycosidic
linkages. In one aspect, the glucanase, e.g., endoglucanase,
activity comprises an endo-1,4- and/or 1,3-beta-endoglucanase
activity or endo-.beta.-1,4-glucanase activity. In one aspect, the
glucanase activity (e.g., endo-1,4-beta-D-glucan 4-glucano
hydrolase activity) comprises hydrolysis of 1,4-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 and other
plant material containing cellulosic parts. In one aspect, the
glucanase, xylanase, or mannanase activity comprises hydrolyzing a
glucan or other polysaccharide to produce a smaller molecular
weight polysaccharide or oligomer. In one aspect, the glucan
comprises a beta-glucan, such as a water soluble beta-glucan.
[0012] The invention provides enzymes, compositions, methods and
processes for hydrolyzing hemicelluloses in any organic matter,
including cells, plants and/or wood or wood products, wood waste,
paper pulp, paper products or paper waste or byproducts. The
invention further provides methods and processes for breaking down
plant matter containing cellulose and/or hemicellulose into simple
sugars using the "cocktails" of the invention.
[0013] In another aspect, the invention provides polypeptides
having lignocellulolytic (lignocellulosic) activity, e.g., a
ligninolytic and cellulolytic activity, including, e.g., having a
hydrolase activity, e.g., a glycosyl hydrolase activity, including
cellulase, glucanase, xylanase, and/or mannanase activity, and
nucleic acids encoding them, and methods for making and using them.
The invention provides enzymes for the bioconversion of any
biomass, e.g., a lignocellulosic residue, into fermentable sugars
or polysaccharides; and these sugars or polysaccharides can be used
as a chemical feedstock for the production of alcohols such as
ethanol, propanol, butanol and/or methanol, production of fuels,
e.g., biofuels such as synthetic liquids or gases, such as syngas,
and the production of other fermentation products, e.g., succinic
acid, lactic acid, or acetic acid. Enzymes of the invention can be
added to bioconversion and other industrial processes continuously,
in batches or by fed-batch methods. In another aspect, enzymes of
the invention can be recycled in bioconversion and other industrial
processes, thereby lowering enzyme requirements.
[0014] In one aspect, the enzymes of the invention have an
increased catalytic rate to improve the process of substrate (e.g.,
a lignocellulosic residue, cellulose, bagasse) hydrolysis. This
increased efficiency in catalytic rate leads to an increased
efficiency in producing sugars or polysaccharides, which can be
useful in industrial, agricultural or medical applications, e.g.,
to make a biofuel or an alcohol such as ethanol, propanol, butanol
and/or methanol. In one aspect, sugars produced by hydrolysis using
enzymes of this invention can be used by microorganisms for alcohol
(e.g., ethanol, propanol, butanol and/or methanol) production
and/or fuel (e.g., biofuel) production. Additionally, the sugars
produced by hydrolysis using the enzymes of the invention can be
used by microorganisms for the production of other fermentation
products, e.g., succinic acid, lactic acid, or acetic acid.
[0015] The invention provides industrial, agricultural or medical
applications: e.g., biomass to biofuel, e.g., ethanol, propanol,
butanol and/or methanol, using enzymes of the invention having
decreased enzyme costs, e.g., decreased costs in biomass to biofuel
conversion processes. Thus, the invention provides efficient
processes for producing bioalcohols, biofuels and/or biofuel-
(e.g., bioethanol-, propanol-, butanol- and/or methanol-)
comprising compositions, including synthetic, liquid or gas fuels
comprising a bioalcohol, from any biomass.
[0016] In one aspect, enzymes of the invention, including the
enzyme "cocktails" of the invention ("cocktails" meaning mixtures
of enzymes comprising at least one enzyme of this invention), are
used to hydrolyze the major components of a lignocellulosic
biomass, or any composition comprising cellulose and/or
hemicellulose (lignocellulosic biomass also comprises lignin),
e.g., seeds, grains, tubers, plant waste (such as a hay or straw,
e.g., a rice straw or a wheat straw, or any the dry stalk of any
cereal plant) or byproducts of food processing or industrial
processing (e.g., stalks), corn (including cobs, stover, and the
like), grasses (e.g., Indian grass, such as Sorghastrum nutans; or,
switch grass, e.g., Panicum species, such as Panicum virgatum),
wood (including wood chips, processing waste, such as wood waste),
paper, pulp, recycled paper (e.g., newspaper); also including a
monocot or a dicot, or a monocot corn, sugarcane or parts thereof
(e.g., cane tops), rice, wheat, barley, switchgrass or Miscanthus;
or a dicot oilseed crop, soy, canola, rapeseed, flax, cotton, palm
oil, sugar beet, peanut, tree, poplar or lupine; or, woods or wood
processing byproducts, such as wood waste, 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.
[0017] In one aspect, enzymes of the invention are used to
hydrolyze cellulose comprising a linear chain of .beta.-1,4-linked
glucose moieties, and/or hemicellulose as a complex structure that
varies from plant to plant. In one aspect, enzymes of the invention
are used to hydrolyze hemicelluloses containing a backbone of
.beta.-1,4 linked xylose molecules with intermittent branches of
arabinose, galactose, glucuronic acid and/or mannose. In one
aspect, enzymes of the invention are used to hydrolyze
hemicellulose containing noncarbohydrate constituents such as
acetyl groups on xylose and femlic acid esters on arabinose. In one
aspect, enzymes of the invention are used to hydrolyze
hemicelluloses covalently linked to lignin and/or coupled to other
hemicellulose strands via difemlate crosslinks.
[0018] In one aspect, the compositions and methods of the invention
are used in the enzymatic digestion of biomass and can comprise use
of many different enzymes, including the cellulases and
hemicellulases. Lignocellulosic enzymes used to practice the
invention can digest cellulose to monomeric sugars, including
glucose. In one aspect, compositions used to practice the invention
can include mixtures of enzymes, e.g., glycosyl hydrolases, glucose
oxidases, xylanases, xylosidases (e.g., .beta.-xylosidases),
cellobiohydrolases, and/or arabinofuranosidases or other enzymes
that can digest hemicellulose to monomer sugars. Mixtures of the
invention can comprise, or consist of, only enzymes of this
invention, or can include at least one enzyme of this invention and
another enzyme, which can also be a lignocellulosic enzyme and/or
any other enzyme.
[0019] 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.
[0020] In alternative embodiments, the invention provides
polypeptides (and the nucleic acids that encode them) having at
least one conservative amino acid substitution and retaining its
xylanase, a mannanase and/or a glucanase activity; or, wherein the
at least one conservative amino acid substitution comprises
substituting an amino acid with another amino acid of like
characteristics; or, a 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.
[0021] In alternative embodiments, the invention provides
polypeptides (and the nucleic acids that encode them) having a
xylanase (e.g., an endoxylanase), a mannanase and/or a glucanase
activity but lacking a signal sequence, a prepro domain, a dockerin
domain, and/or a carbohydrate binding module (CBM); and in one
aspect, the carbohydrate binding module (CBM) comprises, or
consists of, a xylan binding module, a cellulose binding module, a
lignin binding module, a xylose binding module, a mannanse binding
module, a xyloglucan-specific module and/or an arabinofuranosidase
binding module.
[0022] In alternative embodiments, the invention provides
polypeptides (and the nucleic acids that encode them) having a
xylanase (e.g., an endoxylanase), a mannanase and/or a glucanase
activity further comprising a heterologous sequence; and in one
aspect, the heterologous sequence comprises, or consists of a
sequence encoding: (i) a heterologous signal sequence, a
heterologous carbohydrate binding module, a heterologous dockerin
domain, a heterologous catalytic domain (CD), or a combination
thereof; (ii) the sequence of (ii), wherein the heterologous signal
sequence, carbohydrate binding module or catalytic domain (CD) is
derived from a heterologous enzyme; or, (iii) a tag, an epitope, a
targeting peptide, a cleavable sequence, a detectable moiety or an
enzyme; and in one aspect, the heterologous carbohydrate binding
module (CBM) comprises, or consists of, a xylan binding module, a
cellulose binding module, a lignin binding module, a xylose binding
module, a mannanse binding module, a xyloglucan-specific module
and/or a arabinofuranosidase binding module; and in one aspect, the
heterologous signal sequence targets the encoded protein to a
vacuole, the endoplasmic reticulum, a chloroplast or a starch
granule.
[0023] The invention provides isolated, synthetic or recombinant
nucleic acids comprising [0024] (a) a nucleic acid (polynucleotide)
encoding at least one polypeptide, wherein the nucleic acid
comprises 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 or complete (100%) sequence
identity to: [0025] (i) the nucleic acid (polynucleotide) sequence
of SEQ ID NO:1 having one or more nucleotide residue changes (or
the equivalent thereof) as set forth in Table 1, or having at least
one, two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve, thirteen, fourteen, fifteen, sixteen, seventeen or
eighteen, or some or all of the following nucleotide residue
changes: the codon encoding amino acid residue 4 changed from ACC
to AAC; the codon encoding amino acid residue 4 changed from ACC to
CGC; the codon encoding amino acid residue 4 changed from ACC to
CAC; the codon encoding amino acid residue 9 changed from CCC to
GAC; the codon encoding amino acid residue 17 changed from TTC to
GTC; the codon encoding amino acid residue 21 changed from TTC to
TAC; the codon encoding amino acid residue 33 changed from CTG to
GCG; the codon encoding amino acid residue 38 changed from CGT to
CAC; the codon encoding amino acid residue 44 changed from AGC to
ACG; the codon encoding amino acid residue 63 changed from ATC to
GTC; the codon encoding amino acid residue 73 changed from GGC to
TAC; the codon encoding amino acid residue 73 changed from GGC to
GAG; the codon encoding amino acid residue 73 changed from GGC to
GTC; the codon encoding amino acid residue 108 changed from TTC to
AAG; the codon encoding amino acid residue 125 changed from CAG to
TAC; the codon encoding amino acid residue 150 changed from GTA to
GCC; the codon encoding amino acid residue 188 changed from AGC to
GAG; and/or, the codon encoding amino acid residue 189 changed from
TCC to CAG; or [0026] (ii) the nucleic acid (polynucleotide)
sequence of 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 or SEQ ID NO:23; [0027] wherein the
nucleic acid of (i) or (ii) encodes at least one polypeptide having
a xylanase, a mannanase and/or a glucanase activity, or encodes a
polypeptide or peptide capable of generating a xylanase, a
mannanase and/or a glucanase specific antibody (a polypeptide or
peptide that acts as an epitope or immunogen), [0028] (b) the
nucleic acid (polynucleotide) of (a), wherein the sequence
identities are determined: (A) by analysis with a sequence
comparison algorithm or by a visual inspection, or (B) 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, or over the full
length of a cDNA, transcript (mRNA) or gene; [0029] (c) the nucleic
acid (polynucleotide) of (a) or (b), 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; [0030] (d) a nucleic acid
(polynucleotide) encoding at least one polypeptide or peptide,
wherein the nucleic acid comprises a sequence that hybridizes under
stringent conditions to a nucleic acid comprising the nucleic acid
(polynucleotide) sequence of SEQ ID NO:1 having one or more
nucleotide residue changes (or the equivalent thereof) as set forth
in Table 1, or having at least one, two, three, four, five, six,
seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,
fifteen, sixteen, seventeen or eighteen, or some or all of the
following nucleotide residue changes: the codon encoding amino acid
residue 4 changed from ACC to AAC; the codon encoding amino acid
residue 4 changed from ACC to CGC; the codon encoding amino acid
residue 4 changed from ACC to CAC; the codon encoding amino acid
residue 9 changed from CCC to GAC; the codon encoding amino acid
residue 17 changed from TTC to GTC; the codon encoding amino acid
residue 21 changed from TTC to TAC; the codon encoding amino acid
residue 33 changed from CTG to GCG; the codon encoding amino acid
residue 38 changed from CGT to CAC; the codon encoding amino acid
residue 44 changed from AGC to ACG; the codon encoding amino acid
residue 63 changed from ATC to GTC; the codon encoding amino acid
residue 73 changed from GGC to TAC; the codon encoding amino acid
residue 73 changed from GGC to GAG; the codon encoding amino acid
residue 73 changed from GGC to GTC; the codon encoding amino acid
residue 108 changed from TTC to AAG; the codon encoding amino acid
residue 125 changed from CAG to TAC; the codon encoding amino acid
residue 150 changed from GTA to GCC; the codon encoding amino acid
residue 188 changed from AGC to GAG; and/or, the codon encoding
amino acid residue 189 changed from TCC to CAG, [0031] wherein the
polypeptide or peptide has a xylanase, a mannanase and/or a
glucanase activity or is capable of generating a xylanase, a
mannanase and/or a glucanase specific antibody (a polypeptide or
peptide that acts as an epitope or immunogen), [0032] and 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; [0033] (e) a nucleic acid (polynucleotide) encoding at
least one polypeptide or peptide, wherein the nucleic acid
comprises a sequence that hybridizes under stringent conditions to
a nucleic acid comprising the sequence of 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 or SEQ ID
NO:23, [0034] and 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; [0035] (f) the nucleic acid
(polynucleotide) of any of (a) to (d) having a length of at least
about 20, 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,
1050, 1100, 1150 or more nucleotide residues, or the full length of
a gene or a transcript; [0036] (g) a nucleic acid (polynucleotide)
encoding at least one polypeptide having a xylanase, a mannanase
and/or a glucanase activity, wherein the polypeptide comprises the
sequence of SEQ ID NO:2, or enzymatically active fragments thereof,
has at least one, two, three, four, five, six, seven, eight, nine,
ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,
seventeen or eighteen or some or all of the following amino acid
residue changes; [0037] (h) a nucleic acid (polynucleotide)
encoding at least one polypeptide having a xylanase, a mannanase
and/or a glucanase activity, wherein the polypeptide comprises the
sequence of 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, or enzymatically active
fragments thereof; [0038] (i) (A) the nucleic acid (polynucleotide)
of any of (a) to (h) and encoding a polypeptide having at least one
conservative amino acid substitution and retaining its xylanase, a
mannanase and/or a glucanase activity; or (B) the nucleic acid of
(i)(A), wherein the at least one conservative amino acid
substitution comprises substituting an amino acid with another
amino acid of like characteristics; or, a 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; [0039] (j) the nucleic acid
(polynucleotide) of any of (a) to (i) encoding a polypeptide having
a xylanase, a mannanase and/or a glucanase activity but lacking a
signal sequence, a prepro domain, a dockerin domain, and/or a
carbohydrate binding module (CBM); [0040] (k) the nucleic acid
(polynucleotide) of (j), wherein the carbohydrate binding module
(CBM) comprises, or consists of, a xylan binding module, a
cellulose binding module, a lignin binding module, a xylose binding
module, a mannanse binding module, a xyloglucan-specific module
and/or a arabinofuranosidase binding module; [0041] (l) the nucleic
acid (polynucleotide) of any of (a) to (k) encoding a polypeptide
having a xylanase, a mannanase and/or a glucanase activity further
comprising a heterologous sequence; [0042] (m) the nucleic acid
(polynucleotide) of (l), wherein the heterologous sequence
comprises, or consists of a sequence encoding: (A) a heterologous
signal sequence, a heterologous carbohydrate binding module, a
heterologous dockerin domain, a heterologous catalytic domain (CD),
or a combination thereof; (B) the sequence of (l), wherein the
heterologous signal sequence, carbohydrate binding module or
catalytic domain (CD) is derived from a heterologous enzyme; or,
(C) a tag, an epitope, a targeting peptide, a cleavable sequence, a
detectable moiety or an enzyme; [0043] (n) the nucleic acid
(polynucleotide) of (l), wherein the heterologous carbohydrate
binding module (CBM) comprises, or consists of, a xylan binding
module, a cellulose binding module, a lignin binding module, a
xylose binding module, a mannanse binding module, a
xyloglucan-specific module and/or a arabinofuranosidase binding
module; [0044] (o) the nucleic acid (polynucleotide) of (l),
wherein the heterologous signal sequence targets the encoded
protein to a vacuole, the endoplasmic reticulum, a chloroplast or a
starch granule; or [0045] (p) a nucleic acid sequence
(polynucleotide) fully (completely) complementary to the sequence
of any of (a) to (o).
[0046] The invention provides isolated, synthetic or recombinant
nucleic acids comprising a nucleic acid encoding at least one
polypeptide having a xylanase (e.g., an endoxylanase), a mannanase
and/or a glucanase activity, wherein the polypeptide has a sequence
as set forth in SEQ ID NO:2 having one or more changes as described
herein and in Table 1, or enzymatically active fragments thereof,
including the sequences described herein and in Table 1, and the
Sequence Listing (all of these sequences are "exemplary
enzymes/polypeptides of the invention"), and enzymatically active
subsequences (fragments) thereof and/or immunologically active
subsequences thereof (such as epitopes or immunogens) (all
"peptides of the invention") and variants thereof (all of these
sequences encompassing polypeptide and peptide sequences of the
invention) (or, hereinafter referred to as the exemplary
polypeptide sequences of the inventions).
[0047] The invention provides isolated, synthetic or recombinant
nucleic acids comprising sequences completely complementary to all
of these nucleic acid sequences of the invention (complementary
(non-coding) and coding sequences also hereinafter collectively
referred to as nucleic acid sequences of the invention).
[0048] In one aspect, the sequence identity is at least about 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 100% (complete)
sequence identity (homology). In one aspect, the sequence identity
is over a region of at least about 150, 175, 200, 225, 250, 275,
300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1050, 1100, 1150 or more residues, or the full length of
a gene or a transcript. For example, the invention provides
isolated, synthetic or recombinant nucleic acids comprising a
nucleic acid sequence of SEQ ID NO:1 having one or more mutations
as described herein, e.g., as described in Table 1 (the exemplary
polynucleotide sequences of this invention). The invention provides
isolated, synthetic or recombinant nucleic acids encoding a
polypeptide comprising a sequence of SEQ ID NO:2 having one or more
amino acid change as described herein, e.g., as set forth in Table
1 (the exemplary polypeptide sequences of this invention), and
enzymatically active fragments thereof.
[0049] The invention provides isolated, synthetic or recombinant
nucleic acids encoding a polypeptide having xylanase (e.g., an
endoxylanase), a mannanase and/or a glucanase activity, wherein the
nucleic acid has at least one sequence modification of an exemplary
sequence of the invention, or, any sequence of the invention.
[0050] The invention provides isolated, synthetic or recombinant
nucleic acids encoding a polypeptide having xylanase (e.g., an
endoxylanase), a mannanase and/or a glucanase activity, wherein the
nucleic acid has at least one sequence modification of an exemplary
nucleic acid of the invention, wherein in one aspect the
modifications (changes) are set forth in Table 1.
[0051] In one aspect, the invention also provides enzyme-encoding
nucleic acids with a common novelty in that they encode a novel
subset of xylanases, or a clade, comprising the "X14 module" (J
Bacteriol. 2002 August; 184(15): 4124-4133). In one aspect, the
invention also provides enzyme-encoding nucleic acids with a common
novelty in that they encode a novel subset of xylanases, or a
clade, comprising the "XI4 module". Thus, in one aspect, the
invention provides a novel genus of xylanases comprising xylanase
members of SEQ ID NO:2 having one or more mutations as described
herein, e.g., in Table 1.
[0052] In one aspect (optionally), the isolated, synthetic or
recombinant nucleic acids of the invention have a xylanase (e.g.,
an endoxylanase), a mannanase and/or a glucanase activity, e.g.,
wherein the xylanase activity comprises catalyzing hydrolysis of
internal .beta.-1,4-xylosidic linkages; comprises an
endo-1,4-beta-xylanase activity; comprises hydrolyzing a xylan or
an arabinoxylan to produce a smaller molecular weight xylose and
xylo-oligomer; comprises hydrolyzing a polysaccharide comprising a
1,4-.beta.-glycoside-linked D-xylopyranose; comprises hydrolyzing a
cellulose or a hemicellulose; comprises hydrolyzing a cellulose or
a hemicellulose in a wood, wood product, paper pulp, paper product
or paper waste; comprises catalyzing hydrolysis of a xylan or an
arabinoxylan in a feed or a food product; or, comprises catalyzing
hydrolysis of a xylan or an arabinoxylan in a microbial cell or a
plant cell. In one aspect, the xylanase activity comprises
hydrolyzing polysaccharides comprising 1,4-.beta.-glycoside-linked
D-xylopyranoses or hydrolyzing hemicelluloses, e.g., hydrolyzing
hemicelluloses in a wood, wood product, paper pulp, paper product
or paper waste. In one aspect, the arabinoxylan is a cereal
arabinoxylan, such as a wheat arabinoxylan.
[0053] In one aspect, the xylanase, a mannanase and/or a glucanase
activity comprises catalyzing hydrolysis of polysaccarides, e.g.,
mannans or xylans, in a feed or a food product, such as a
cereal-based animal feed, a wort or a beer, a milk or a milk
product, a fruit or a vegetable. In one aspect, the xylanase, a
mannanase and/or a glucanase activity comprises catalyzing
hydrolysis of polysaccarides, e.g., mannans or xylans, in a
microbial cell or a plant cell.
[0054] In one aspect, the xylanase, a mannanase and/or a glucanase
activity is thermostable, e.g., wherein the polypeptide retains a
xylanase, a mannanase and/or a glucanase activity under conditions
comprising a temperature range from about -100.degree. C. to about
-80.degree. C., about -80.degree. C. to about -40.degree. C., about
-40.degree. C. to about -20.degree. C., about -20.degree. C. to
about 0.degree. C., about 0.degree. C. to about 5.degree. C., about
5.degree. C. to about 15.degree. C., about 15.degree. C. to about
25.degree. C., about 25.degree. C. to about 37.degree. C., about
37.degree. C. to about 45.degree. C., about 45.degree. C. to about
55.degree. C., about 55.degree. C. to about 70.degree. C., about
70.degree. C. to about 75.degree. C., about 75.degree. C. to about
85.degree. C., about 85.degree. C. to about 90.degree. C., about
90.degree. C. to about 95.degree. C., about 95.degree. C. to about
100.degree. C., about 100.degree. C. to about 105.degree. C., about
105.degree. C. to about 110.degree. C., about 110.degree. C. to
about 120.degree. C., or 95.degree. C., 96.degree. C., 97.degree.
C., 98.degree. C., 99.degree. C., 100.degree. C., 101.degree. C.,
102.degree. C., 103.degree. C., 104.degree. C., 105.degree. C.,
106.degree. C., 107.degree. C., 108.degree. C., 109.degree. C.,
110.degree. C., 111.degree. C., 112.degree. C., 113.degree. C.,
114.degree. C., 115.degree. C. or more. In some embodiments, the
thermostable polypeptides according to the invention retains
activity, e.g., a xylanase, a mannanase and/or a glucanase
activity, at a temperature in the ranges described above, at about
pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0,
about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH
7.5, about pH 8.0, about pH 8.5, about pH 9.0, about pH 9.5, about
pH 10.0, about pH 10.5, about pH 11.0, about pH 11.5, about pH 12.0
or more.
[0055] In one aspect, the xylanase, a mannanase and/or a glucanase
activity is thermotolerant, e.g., wherein the polypeptide retains a
xylanase, a mannanase and/or a glucanase activity after exposure to
a temperature in the range from about -100.degree. C. to about
-80.degree. C., about -80.degree. C. to about -40.degree. C., about
-40.degree. C. to about -20.degree. C., about -20.degree. C. to
about 0.degree. C., about 0.degree. C. to about 5.degree. C., about
5.degree. C. to about 15.degree. C., about 15.degree. C. to about
25.degree. C., about 25.degree. C. to about 37.degree. C., about
37.degree. C. to about 45.degree. C., about 45.degree. C. to about
55.degree. C., about 55.degree. C. to about 70.degree. C., about
70.degree. C. to about 75.degree. C., about 75.degree. C. to about
85.degree. C., about 85.degree. C. to about 90.degree. C., about
90.degree. C. to about 95.degree. C., about 95.degree. C. to about
100.degree. C., about 100.degree. C. to about 105.degree. C., about
105.degree. C. to about 110.degree. C., about 110.degree. C. to
about 120.degree. C., or 95.degree. C., 96.degree. C., 97.degree.
C., 98.degree. C., 99.degree. C., 100.degree. C., 101.degree. C.,
102.degree. C., 103.degree. C., 104.degree. C., 105.degree. C.,
106.degree. C., 107.degree. C., 108.degree. C., 109.degree. C.,
110.degree. C., 111.degree. C., 112.degree. C., 113.degree. C.,
114.degree. C., 115.degree. C. or more. The thermotolerant
polypeptides according to the invention can retain activity, e.g.,
a xylanase, a mannanase and/or a glucanase activity, after exposure
to a temperature in the range from about -100.degree. C. to about
-80.degree. C., about -80.degree. C. to about -40.degree. C., about
-40.degree. C. to about -20.degree. C., about -20.degree. C. to
about 0.degree. C., about 0.degree. C. to about 5.degree. C., about
5.degree. C. to about 15.degree. C., about 15.degree. C. to about
25.degree. C., about 25.degree. C. to about 37.degree. C., about
37.degree. C. to about 45.degree. C., about 45.degree. C. to about
55.degree. C., about 55.degree. C. to about 70.degree. C., about
70.degree. C. to about 75.degree. C., about 75.degree. C. to about
85.degree. C., about 85.degree. C. to about 90.degree. C., about
90.degree. C. to about 95.degree. C., about 95.degree. C. to about
100.degree. C., about 100.degree. C. to about 105.degree. C., about
105.degree. C. to about 110.degree. C., about 110.degree. C. to
about 120.degree. C., or 95.degree. C., 96.degree. C., 97.degree.
C., 98.degree. C., 99.degree. C., 100.degree. C., 101.degree. C.,
102.degree. C., 103.degree. C., 104.degree. C., 105.degree. C.,
106.degree. C., 107.degree. C., 108.degree. C., 109.degree. C.,
110.degree. C., 111.degree. C., 112.degree. C., 113.degree. C.,
114.degree. C., 115.degree. C. or more. In some embodiments, the
thermotolerant polypeptides according to the invention retains
activity, e.g., a xylanase, a mannanase and/or a glucanase
activity, after exposure to a temperature in the ranges described
above, at about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5,
about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH
7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 9.0, about
pH 9.5, about pH 10.0, about pH 10.5, about pH 11.0, about pH 11.5,
about pH 12.0 or more.
[0056] In one aspect, the xylanase, a mannanase and/or a glucanase
activity of polypeptides encoded by nucleic acids of the invention
retain activity under acidic conditions comprising about pH 6.5, pH
6, pH 5.5, pH 5, pH 4.5, pH 4.0, pH 3.5, pH 3.0 or less (more
acidic) pH, or, retain a xylanase, a mannanase and/or a glucanase
activity after exposure to acidic conditions comprising about pH
6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4.0, pH 3.5, pH 3.0 or less
(more acidic) pH; or, retain activity under basic conditions
comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10,
pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (more basic) or,
retain a xylanase, a mannanase and/or a glucanase activity after
exposure to basic conditions comprising about pH 7, pH 7.5 pH 8.0,
pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH
12.5 or more (more basic). In one aspect, xylanase, a mannanase
and/or a glucanase activity of polypeptides encoded by nucleic
acids of the invention retain activity at a temperature of at least
about 80.degree. C., 81.degree. C., 82.degree. C., 83.degree. C.,
84.degree. C., 85.degree. C., 86.degree. C., 87.degree. C.,
88.degree. C., 89.degree. C., 90.degree. C., 91.degree. C.,
92.degree. C., 93.degree. C., 94.degree. C., 95.degree. C.,
96.degree. C., 97.degree. C., 98.degree. C., 99.degree. C.,
100.degree. C., 101.degree. C., 102.degree. C., 103.degree. C.,
103.5.degree. C., 104.degree. C., 105.degree. C., 107.degree. C.,
108.degree. C., 109.degree. C. or 110.degree. C., or more, and a
basic pH of at least about pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH
10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (more
basic).
[0057] The invention provides expression cassettes, cloning
vehicles, or a vector (e.g., expression vectors) comprising a
nucleic acid comprising a sequence of the invention. The cloning
vehicle can comprise 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 an artificial chromosome comprising a
bacterial artificial chromosome (BAC), a bacteriophage P1-derived
vector (PAC), a yeast artificial chromosome (YAC), or a mammalian
artificial chromosome (MAC).
[0058] The invention provides nucleic acid probes for identifying a
nucleic acid encoding a polypeptide with a xylanase, a mannanase
and/or a glucanase activity, wherein the probe comprises at least
about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100,
125, 150, 175, 200, 225, 250, 275, 300 or more consecutive bases of
a nucleic acid comprising an exemplary sequence of the invention,
or, any sequence of the invention (as defined herein), wherein in
one aspect (optionally) the probe comprises an oligonucleotide
comprising between at least about 10 to 300, about 25 to 250, about
10 to 50, about 20 to 60, about 30 to 70, about 40 to 80, about 60
to 100, or about 50 to 150 or more consecutive bases.
[0059] The invention provides amplification primer pairs for
amplifying a nucleic acid encoding a polypeptide having a xylanase,
a mannanase and/or a glucanase activity, wherein the primer pair is
capable of amplifying a nucleic acid comprising an exemplary
sequence of the invention, or, any sequence of the invention (as
defined herein), or a subsequence thereof, wherein optionally a
member of the amplification primer sequence pair comprises an
oligonucleotide comprising at least about 10 to 50 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 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') 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 or more
residues of an exemplary sequence of the invention, or, any
sequence of the invention (as defined herein), and a second member
having a sequence as set forth by about the first (the 5') 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 or more residues of the complementary
strand of the first member.
[0060] The invention provides xylanase- and/or a glucanase-encoding
nucleic acids generated by amplification of a polynucleotide using
an amplification primer pair of the invention, wherein optionally
the amplification is by polymerase chain reaction (PCR). In one
aspect, the nucleic acid is generated by amplification of a gene
library, wherein in one aspect (optionally) the gene library is an
environmental library. The invention provides isolated, synthetic
or recombinant xylanases and/or a glucanases encoded by a xylanase-
and/or a glucanase-encoding nucleic acid generated by amplification
of a polynucleotide using an amplification primer pair of the
invention. The invention provides methods of amplifying a nucleic
acid encoding a polypeptide having a xylanase, a mannanase and/or a
glucanase activity, the methods comprising the step of
amplification of a template nucleic acid with an amplification
primer sequence pair capable of amplifying an exemplary sequence of
the invention, or, any sequence of the invention (as defined
herein), or a subsequence thereof.
[0061] The invention provides expression cassette, a vector or a
cloning vehicle comprising a nucleic acid comprising a sequence of
the invention, 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. The viral vector can
comprise an adenovirus vector, a retroviral vector or an
adeno-associated viral vector, or, the artificial chromosome
comprises a bacterial artificial chromosome (BAC), a bacteriophage
P1-derived vector (PAC), a yeast artificial chromosome (YAC), or a
mammalian artificial chromosome (MAC).
[0062] The invention provides transformed cells comprising a
nucleic acid or vector of the invention, or an expression cassette
or cloning vehicle of the invention. The transformed cell can be a
bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an
insect cell or a plant cell.
[0063] The invention provides transgenic non-human animals
comprising a sequence of the invention. The transgenic non-human
animal can be a mouse, a rat, a rabbit, a sheep, a pig, a chicken,
a goat, a fish, a dog, or a cow. The invention provides transgenic
plants comprising a sequence of the invention, e.g., wherein the
plant is a corn plant, a sorghum 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, a grass, or a tobacco plant.
The invention provides transgenic seeds comprising a sequence of
the invention, e.g., wherein the seed is a corn seed, a wheat
kernel, an oilseed, a rapeseed, a soybean seed, a palm kernel, a
sunflower seed, a sesame seed, a rice, a barley, a peanut or a
tobacco plant seed.
[0064] The invention provides antisense oligonucleotides comprising
a nucleic acid sequence complementary to or capable of hybridizing
under stringent conditions to a sequence of the invention
(including, e.g., exemplary sequences of the invention), or a
subsequence thereof, wherein optionally 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, and in
one aspect (optionally) 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.
[0065] The invention provides methods of inhibiting the translation
of a xylanase, a mannanase and/or a glucanase 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 sequence of the invention (including, e.g.,
exemplary sequences of the invention).
[0066] The invention provides double-stranded inhibitory RNA (RNAi)
molecules comprising a subsequence of a sequence of the invention
(including, e.g., exemplary sequences of the invention). The
double-stranded inhibitory RNA (RNAi) molecule can be about 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, or 30 or more duplex nucleotides in length. The invention
provides methods of inhibiting the expression of a xylanase, a
mannanase and/or a glucanase in a cell comprising administering to
the cell or expressing in the cell a double-stranded inhibitory RNA
(iRNA), wherein the RNA comprises a subsequence of a sequence of
the invention (including, e.g., exemplary sequences of the
invention).
[0067] The invention provides isolated, synthetic or recombinant
polypeptides having a xylanase, a mannanase and/or a glucanase
activity, or polypeptides capable of generating an immune response
specific for a xylanase (e.g., an endoxylanase), a mannanase and/or
a glucanase (e.g., an epitope); and in alternative aspects peptide
and polypeptide of the invention comprise a sequence: [0068] (a)
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 has 100%
(complete) sequence identity to: [0069] (i) the amino acid sequence
of SEQ ID NO:2, or enzymatically active fragments thereof, and
having at least one amino acid residue change (or the equivalent
thereof) as set forth in Table 1, or having at least one, two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, fifteen, sixteen, seventeen or eighteen or some
or all of the following amino acid residue changes: amino acid
residue 4 is changed from a T (or thr, or threonine) to an N (or
asn, or asparagine); amino acid residue 4 is changed from a T (or
thr, or threonine) to an R (or arg, or arginine); amino acid
residue 4 is changed from a T (or thr, or threonine) to an H (or
his, or histidine); amino acid residue 9 is changed from a P (or
pro, or proline) to a D (or asp, or aspartic acid); amino acid
residue 17 is changed from an F (or phe, or phenylalanine) to a V
(or val, or valine); amino acid residue 21 is changed from an F (or
phe, or phenylalanine) to a Y (or tyr, or tyrosine); amino acid
residue 33 is changed from an L (or leu, or leucine) to an A (or
ala, or alanine); amino acid residue 38 is changed from an R (or
arg, or arginine) to an H (or his, or histidine); amino acid
residue 44 is changed from an S (or ser, or serine) to a T (or thr,
or threonine); amino acid residue 63 is changed from an I (or ile,
or isoleucine) to a V (or val, or valine); amino acid residue 73 is
change from a G (or gly, or glycine) to a Y (or tyr, or tyrosine);
amino acid residue 73 is changed from a G (or gly, or glycine) to a
V (or val, or valine); amino acid residue 73 is changed from a G
(or gly, or glycine) to an E (or glu, or glutamic acid); amino acid
residue 108 is changed from an F (or phe, or phenylalanine) to a K
(or lys, or lysine); amino acid residue 125 is change from a Q (or
gin, or glutamine) to a Y (or tyr, or tyrosine); amino acid residue
150 is change from a V (or val, or valine) to an A (or ala, or
alanine); amino acid residue 188 is changed from an S (or ser, or
serine) to an E (or glu, or glutamic acid); and/or amino acid
residue 189 is changed from an S (or ser, or serine) to a Q (or
gin, or glutamine), or [0070] (ii) the amino acid sequence of 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 or SEQ ID NO:24; [0071] wherein the polypeptide or
peptide of (i) or (ii) has a xylanase, a mannanase and/or a
glucanase activity, or the polypeptide or peptide is capable of
generating a xylanase, a mannanase and/or a glucanase specific
antibody (a polypeptide or peptide that acts as an epitope or
immunogen), [0072] (b) the polypeptide or peptide of (a), wherein
the sequence identities are determined: (A) by analysis with a
sequence comparison algorithm or by a visual inspection, or (B)
over a region of at least about 20, 25, 30, 35, 40, 45, 50, 55, 60,
75, 100, 150, 200, 250, 300 or more amino acid residues, or over
the full length of the polypeptide or peptide or enzyme, and/or
enzymatically active subsequences (fragments) thereof, [0073] (c)
the polypeptide or peptide of (a) of (b), wherein 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; [0074] (d) an amino acid
sequence encoded by the nucleic acid of claim 1, wherein the
polypeptide has (i) a xylanase, a mannanase and/or a glucanase
activity, or, (ii) has immunogenic activity in that it is capable
of generating an antibody that specifically binds to a polypeptide
having a sequence of (a), and/or enzymatically active subsequences
(fragments) thereof; [0075] (e) the amino acid sequence of any of
(a) to (d), and comprising at least one amino acid residue
conservative substitution, and the polypeptide or peptide retains
xylanase, a mannanase and/or a glucanase activity; [0076] (f) the
amino acid sequence of (e), wherein the 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, [0077] (g)
the amino acid sequence of (f), wherein 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; [0078] (h) the polypeptide of any of (a) to (g) having a
xylanase, a mannanase and/or a glucanase activity but lacking a
signal sequence, a prepro domain, a dockerin domain, and/or a
carbohydrate binding module (CBM), [0079] (i) the polypeptide of
(h) wherein the carbohydrate binding module (CBM) comprises, or
consists of, a xylan binding module, a cellulose binding module, a
lignin binding module, a xylose binding module, a mannanse binding
module, a xyloglucan-specific module and/or a arabinofuranosidase
binding module; [0080] (j) the polypeptide of any of (a) to (i)
having a xylanase, a mannanase and/or a glucanase activity further
comprising a heterologous sequence; [0081] (k) the polypeptide of
(j), wherein the heterologous sequence comprises, or consists of:
(A) a heterologous signal sequence, a heterologous carbohydrate
binding module, a heterologous dockerin domain, a heterologous
catalytic domain (CD), or a combination thereof; (B) the sequence
of (A), wherein the heterologous signal sequence, carbohydrate
binding module or catalytic domain (CD) is derived from a
heterologous lignocellulosic enzyme; and/or, (C) a tag, an epitope,
a targeting peptide, a cleavable sequence, a detectable moiety or
an enzyme; [0082] (l) the polypeptide of (j) or (k), wherein the
heterologous sequence or the heterologous carbohydrate binding
module (CBM) comprises, or consists of, a xylan binding module, a
cellulose binding module, a lignin binding module, a xylose binding
module, a mannan binding module, a xyloglucan-specific module
and/or a arabinofuranosidase binding module; [0083] (m) polypeptide
of (j), wherein the heterologous signal sequence targets the
encoded protein to a vacuole, the endoplasmic reticulum, a
chloroplast or a starch granule; or [0084] (n) comprising an amino
acid sequence encoded any nucleic acid sequence of this
invention.
[0085] In one aspect, the isolated, synthetic or recombinant
peptides of the invention have a xylanase activity, e.g., wherein
the xylanase activity comprises catalyzing hydrolysis of internal
.beta.-1,4-xylosidic linkages; comprises an endo-1,4-beta-xylanase
activity; comprises hydrolyzing a xylan or an arabinoxylan to
produce a smaller molecular weight xylose and xylo-oligomer;
comprises hydrolyzing a polysaccharide comprising a
1,4-.beta.-glycoside-linked D-xylopyranose; comprises hydrolyzing a
cellulose or a hemicellulose; comprises hydrolyzing a cellulose or
a hemicellulose in a wood, wood product, paper pulp, paper product
or paper waste; comprises catalyzing hydrolysis of a xylan or an
arabinoxylan in a feed or a food product; or, comprises catalyzing
hydrolysis of a xylan or an arabinoxylan in a microbial cell or a
plant cell. The xylan can comprises an arabinoxylan, e.g., a water
soluble arabinoxylan, e.g., a water soluble arabinoxylan in a dough
or a bread product.
[0086] In one aspect, the xylanase, a mannanase and/or a glucanase
activity comprises hydrolyzing polysaccharides, for example,
comprising 1,4-.beta.-glycoside-linked D-xylopyranoses, or
hydrolyzing hemicelluloses, e.g., hydrolyzing hemicelluloses in a
wood, wood product, paper pulp, paper product or paper waste.
[0087] In one aspect, the xylanase, a mannanase and/or a glucanase
activity comprises catalyzing hydrolysis of polysaccharides, e.g.,
xylans, in a feed or a food product, such as a cereal-based animal
feed, a wort or a beer, a milk or a milk product, a fruit or a
vegetable. In one aspect, the xylanase activity comprises
catalyzing hydrolysis of xylans in a microbial cell or a plant
cell.
[0088] The invention provides isolated, synthetic or recombinant
polypeptides comprising a polypeptide of the invention and lacking
a signal sequence or a prepro sequence. The invention provides
isolated, synthetic or recombinant polypeptides comprising a
polypeptide of the invention and having a heterologous signal
sequence or a heterologous prepro sequence.
[0089] In one aspect, a polypeptide of the invention has xylanase,
a mannanase and/or a glucanase activity comprising a specific
activity at about 37.degree. C. in the range from about 100 to
about 1000 units per milligram of protein, from about 500 to about
750 units per milligram of protein, from about 500 to about 1200
units per milligram of protein, or from about 750 to about 1000
units per milligram of protein. In one aspect, units are defined as
0.1 to 20 units/g of pulp, where a unit equals umol of xylose
released per minute per mg of enzyme, using arabinoxylan as a
substrate as described in the Nelson Somogyi assay, described in
detail below. In alternative aspects, polypeptides of the invention
have xylanase, a mannanase and/or a glucanase activity in the range
of between about 0.05 to 20 units per gram of pulp, or 0.05, 0.10,
0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.5, 2.0, 2.5,
3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19 or 20 or more units per gram of pulp (where
a unit equals umol of xylose released per minute per mg of enzyme,
using arabinoxylan as a substrate as described in the Nelson
Somogyi assay).
[0090] In one aspect, the thermotolerance comprises retention of at
least half of the specific activity of the xylanase, a mannanase
and/or a glucanase at 37.degree. C. after being heated to an
elevated temperature, such as a temperature from about 0.degree. C.
to about 20.degree. C., about 20.degree. C. to about 37.degree. C.,
about 37.degree. C. to about 50.degree. C., about 50.degree. C. to
about 70.degree. C., about 70.degree. C. to about 75.degree. C.,
about 75.degree. C. to about 80.degree. C., about 80.degree. C. to
about 85.degree. C., about 85.degree. C. to about 90.degree. C.,
about 90.degree. C. to about 95.degree. C., about 95.degree. C. to
about 100.degree. C., about 100.degree. C. to about 110.degree. C.,
or higher. The thermotolerance can comprise retention of specific
activity at 37.degree. C. in the range from about 500 to about 1200
units per milligram of protein after being heated to an elevated
temperature, such as a temperature from about 0.degree. C. to about
20.degree. C., about 20.degree. C. to about 37.degree. C., about
37.degree. C. to about 50.degree. C., about 50.degree. C. to about
70.degree. C., about 70.degree. C. to about 75.degree. C., about
75.degree. C. to about 80.degree. C., about 80.degree. C. to about
85.degree. C., about 85.degree. C. to about 90.degree. C., about
90.degree. C. to about 95.degree. C., about 95.degree. C. to about
100.degree. C., about 100.degree. C. to about 110.degree. C., or
higher.
[0091] In one aspect, the polypeptides of the invention comprise at
least one glycosylation site or further comprises a polysaccharide.
The glycosylation can be an N-linked glycosylation, e.g., wherein
the polypeptide is glycosylated after being expressed in a P.
pastoris or a S. pombe.
[0092] In one aspect, the xylanase, a mannanase and/or a glucanase
activity of polypeptides of the invention retain activity under
acidic conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH
4.5 or pH 4 or less (more acidic), or, retain a xylanase, a
mannanase and/or a glucanase activity after exposure to acidic
conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or
pH 4 or less (more acidic); or, retain activity under basic
conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH
9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (more
basic) or, retain a xylanase, a mannanase and/or a glucanase
activity after exposure to basic conditions comprising about pH 7,
pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH
11.5, pH 12, pH 12.5 or more (more basic). In one aspect, xylanase,
a mannanase and/or a glucanase activity of polypeptides of the
invention retain activity at a temperature of at least about
80.degree. C., 81.degree. C., 82.degree. C., 83.degree. C.,
84.degree. C., 85.degree. C., 86.degree. C., 87.degree. C.,
88.degree. C., 89.degree. C. or 90.degree. C., and a basic pH of at
least about pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH
11, pH 11.5, pH 12, pH 12.5 or more (more basic).
[0093] The invention provides protein preparation comprising a
polypeptide of the invention, wherein the protein preparation
comprises a liquid, a slurry, a solid or a gel. The invention
provides heterodimers comprising a polypeptide of the invention and
a second domain. The second domain can be a polypeptide and the
heterodimer is a fusion protein. The second domain can be an
epitope or a tag. The invention provides homodimers or heterodimers
comprising a polypeptide of the invention. The invention provides
immobilized polypeptides, wherein the polypeptide comprises a
sequence of the invention, or a subsequence thereof, or a
polypeptide encoded by a nucleic acid of the invention, or a
polypeptide comprising a polypeptide of the invention and a second
domain, e.g., wherein the polypeptide is immobilized on or inside a
cell, a vesicle, a liposome, a film, a membrane, a metal, a resin,
a polymer, a ceramic, a glass, a microelectrode, a graphitic
particle, a bead, a gel, a plate, an array, a capillary tube, a
crystal, a tablet, a pill, a capsule, a powder, an agglomerate, a
surface, a porous structure, or materials such as wood chips,
brownstock, pulp, paper, and materials deriving therefrom.
[0094] The xylanases and/or a glucanases of the invention can be
used or formulated alone or as mixture (a "cocktail") of xylanases
and/or a glucanases, and other hydrolytic enzymes such as
cellulases, mannanases, proteases, lipases, amylases, or redox
enzymes such as laccases, peroxidases, catalases, oxidases, or
reductases. They can be used formulated in a solid form such as a
powder, a lyophilized preparation, a granule, a tablet, a bar, a
crystal, a capsule, a pill, a pellet, or in a liquid form such as
in an aqueous solution, an aerosol, a gel, a paste, a slurry, an
aqueous/oil emulsion, a cream, a capsule, or in a vesicular or
micellar suspension. The formulations of the invention can comprise
any or a combination of the following ingredients: polyols such as
a polyethylene glycol, a polyvinylalcohol, a glycerol, a sugar such
as a sucrose, a sorbitol, a trehalose, a glucose, a fructose, a
maltose, a mannose, a gelling agent such as a guar gum, a
carageenan, an alginate, a dextrans, a cellulosic derivative, a
pectin, a salt such as a sodium chloride, a sodium sulfate, an
ammonium sulfate, a calcium chloride, a magnesium chloride, a zinc
chloride, a zinc sulfate, a salt of a fatty acid and a fatty acid
derivative, a metal chelator such as an EDTA, an EGTA, a sodium
citrate, an antimicrobial agent such as a fatty acid or a fatty
acid derivative, a paraben, a sorbate, a benzoate, an additional
modulating compound to block the impact of an enzyme such as a
protease, a bulk proteins such as a BSA, a wheat hydrolysate, a
borate compound, an amino acid or a peptide, an appropriate pH or
temperature modulating compound, an emulsifier such as a non-ionic
and/or an ionic detergent, a redox agent such as a
cystine/cysteine, a glutathione, an oxidized glutathione, a reduced
or an antioxidant compound such as an ascorbic acid, or a
dispersant. Cross-linking and protein modification such as
pegylation, fatty acid modification, glycosylation can also be used
to improve enzyme stability.
[0095] The invention provides arrays comprising immobilized
polypeptide(s) and/or nucleic acids of the invention, and arrays
comprising an immobilized oligonucleotide of the invention. The
enzymes, fragments thereof and nucleic acids which encode the
enzymes, or probes of the invention, and fragments thereof, can be
affixed to a solid support; and these embodiments can be economical
and efficient in the use of enzymes and nucleic acids of the
invention in industrial, medical, research, pharmaceutical, food
and feed and food and feed supplement processing and other
applications and processes. For example, a consortium or cocktail
of enzymes (or active fragments thereof), which are used in a
specific chemical reaction, can be attached to a solid support and
dunked into a process vat. The enzymatic reaction can occur. Then,
the solid support can be taken out of the vat, along with the
enzymes affixed thereto, for repeated use. In one embodiment of the
invention, the isolated nucleic acid is affixed to a solid support.
In another embodiment of the invention, the solid support is
selected from the group of a gel, a resin, a polymer, a ceramic, a
glass, a microelectrode and any combination thereof.
[0096] For example, solid supports useful in this invention include
gels. Some examples of gels include sepharose, gelatin,
glutaraldehyde, chitosan-treated glutaraldehyde,
albumin-glutaraldehyde, chitosan-Xanthan, toyopearl gel (polymer
gel), alginate, alginate-polylysine, carrageenan, agarose, glyoxyl
agarose, magnetic agarose, dextran-agarose, poly(Carbamoyl
Sulfonate) hydrogel, BSA-PEG hydrogel, phosphorylated polyvinyl
alcohol (PVA), monoaminoethyl-N-aminoethyl (MANA), amino, or any
combination thereof. Another solid support useful in the present
invention are resins or polymers. Some examples of resins or
polymers include cellulose, acrylamide, nylon, rayon, polyester,
anion-exchange resin, AMBERLITE.TM. XAD-7, AMBERLITE.TM. XAD-8,
AMBERLITE.TM. IRA-94, AMBERLITE.TM. IRC-50, polyvinyl, polyacrylic,
polymethacrylate, or any combination thereof. Another type of solid
support useful in the present invention is ceramic. Some examples
include non-porous ceramic, porous ceramic, SiO2, Al2O3. Another
type of solid support useful in the present invention is glass.
Some examples include non-porous glass, porus glass, aminopropyl
glass or any combination thereof. Another type of solid support
which can be used is a microelectrode. An example is a
polyethyleneimine-coated magnetite. Graphitic particles can be used
as a solid support. Another example of a solid support is a cell,
such as a red blood cell.
[0097] There are many methods which would be known to one of skill
in the art for immobilizing enzymes or fragments thereof, or
nucleic acids, onto a solid support. Some examples of such methods
include electrostatic droplet generation, electrochemical means,
via adsorption, via covalent binding, via cross-linking, via a
chemical reaction or process, via encapsulation, via entrapment,
via calcium alginate, or via poly (2-hydroxyethyl methacrylate).
Like methods are described in Methods in Enzymology, Immobilized
Enzymes and Cells, Part C. 1987. Academic Press. Edited by S. P.
Colowick and N. O. Kaplan. Volume 136; and Immobilization of
Enzymes and Cells. 1997. Humana Press. Edited by G. F. Bickerstaff.
Series: Methods in Biotechnology, Edited by J. M. Walker.
[0098] The invention provides isolated, synthetic or recombinant
antibodies that specifically binds to a polypeptide of the
invention. The antibody can be a monoclonal or a polyclonal
antibody, or is a single chained antibody. The invention provides
hybridomas comprising an antibody that specifically binds to a
polypeptide of the invention.
[0099] The invention provides methods of isolating or identifying a
polypeptide with a xylanase, a mannanase and/or a glucanase
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 xylanase, a mannanase and/or a glucanase activity. The invention
provides methods of making an anti-xylanase and/or anti-glucanase
antibody comprising administering to a non-human animal a nucleic
acid of the invention or a subsequence thereof in an amount
sufficient to generate a humoral immune response, thereby making an
anti-xylanase and/or anti-glucanase antibody. The invention
provides methods of making an anti-xylanase and/or anti-glucanase
antibody comprising administering to a non-human animal a
polypeptide of the invention or a subsequence thereof in an amount
sufficient to generate a humoral immune response, thereby making an
anti-xylanase and/or anti-glucanase antibody.
[0100] The invention provides methods of producing a recombinant
polypeptide comprising the steps of: (a) providing a nucleic acid
operably linked to a promoter, wherein the nucleic acid comprises a
sequence of the invention; and (b) expressing the nucleic acid of
step (a) under conditions that allow expression of the polypeptide,
thereby producing a recombinant polypeptide. 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.
[0101] The invention provides methods for identifying a polypeptide
having a xylanase, a mannanase and/or a glucanase activity
comprising: (a) providing a polypeptide of the invention; (b)
providing a xylanase, a mannanase and/or a glucanase substrate; and
(c) contacting the polypeptide 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 xylanase, a mannanase and/or
a glucanase activity.
[0102] The invention provides methods for identifying a xylanase, a
mannanase and/or a glucanase substrate comprising: (a) providing a
polypeptide 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 xylanase, a
mannanase and/or a glucanase substrate.
[0103] The invention provides methods of determining whether a test
compound specifically binds to a polypeptide comprising: (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 has a sequence 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.
[0104] The invention provides methods of determining whether a test
compound specifically binds to a polypeptide comprising: (a)
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.
[0105] The invention provides methods for identifying a modulator
of a xylanase, a mannanase and/or a glucanase activity comprising:
(a) providing a polypeptide 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 xylanase, a
mannanase and/or a glucanase, wherein a change in the xylanase, a
mannanase and/or a glucanase 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 xylanase, a mannanase and/or a glucanase activity.
The xylanase, a mannanase and/or a glucanase activity can be
measured by providing a xylanase, a mannanase and/or a glucanase
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. In one aspect, 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 a xylanase, a mannanase and/or a
glucanase activity. In one aspect, 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 a xylanase, a mannanase and/or a
glucanase activity.
[0106] 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, wherein the polypeptide sequence comprises sequence of
the invention, a polypeptide encoded by a nucleic acid of the
invention. The computer systems 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.
[0107] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having a xylanase, a mannanase
and/or a glucanase 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
xylanase, a mannanase and/or a glucanase 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 xylanase, a
mannanase and/or a glucanase activity from an environmental sample.
One or each member of the amplification primer sequence pair can
comprise an oligonucleotide comprising at least about 10 to 50
consecutive bases of a sequence of the invention. In one aspect,
the amplification primer sequence pair is an amplification pair of
the invention.
[0108] The invention provides methods for isolating or recovering a
nucleic acid encoding a polypeptide having a xylanase, a mannanase
and/or a glucanase 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 xylanase, a mannanase and/or a glucanase 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.
[0109] The invention provides methods of generating a variant of a
nucleic acid encoding a polypeptide having a xylanase, a mannanase
and/or a glucanase 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 xylanase, a mannanase and/or a glucanase 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 (e.g., GeneReassembly, see, e.g., U.S. Pat. No.
6,537,776), Gene Site Saturation Mutagenesis (GSSM), synthetic
ligation reassembly (SLR) 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.
[0110] In one aspect, the method can be iteratively repeated until
a xylanase, a mannanase and/or a glucanase 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 xylanase, a mannanase and/or a glucanase
polypeptide is thermotolerant, and retains some activity after
being exposed to an elevated temperature. In another aspect, the
variant xylanase, a mannanase and/or a glucanase polypeptide has
increased glycosylation as compared to the xylanase, a mannanase
and/or a glucanase encoded by a template nucleic acid.
Alternatively, the variant xylanase, a mannanase and/or a glucanase
polypeptide has a xylanase, a mannanase and/or a glucanase activity
under a high temperature, wherein the xylanase, a mannanase and/or
a glucanase encoded by the template nucleic acid is not active
under the high temperature. In one aspect, the method can be
iteratively repeated until a xylanase, a mannanase and/or a
glucanase 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 xylanase, a mannanase
and/or a glucanase gene having higher or lower level of message
expression or stability from that of the template nucleic acid is
produced. In another aspect, formulation of the final xylanase, a
mannanase and/or a glucanase product enables an increase or
modulation of the performance of the xylanase, a mannanase and/or a
glucanase in the product.
[0111] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having a xylanase, a mannanase
and/or a glucanase activity to increase its expression in a host
cell, the method comprising: (a) providing a nucleic acid of the
invention encoding a polypeptide having a xylanase, a mannanase
and/or a glucanase 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.
[0112] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having a xylanase, a mannanase
and/or a glucanase activity; the method comprising: (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 xylanase, a mannanase
and/or a glucanase.
[0113] The invention provides methods for modifying codons in a
nucleic acid encoding a polypeptide having a xylanase, a mannanase
and/or a glucanase activity to increase its expression in a host
cell, the method comprising: (a) providing a nucleic acid of the
invention encoding a xylanase, a mannanase and/or a glucanase
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.
[0114] The invention provides methods for modifying a codon in a
nucleic acid encoding a polypeptide having a xylanase, a mannanase
and/or a glucanase activity to decrease its expression in a host
cell, the method comprising: (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.
[0115] The invention provides methods for producing a library of
nucleic acids encoding a plurality of modified xylanase, a
mannanase and/or a glucanase 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: (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 sequence of the invention, or a
subsequence thereof, and the nucleic acid encodes a xylanase, a
mannanase and/or a glucanase active site or a xylanase, a mannanase
and/or a glucanase 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 xylanase, a mannanase and/or a glucanase 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), or a synthetic ligation reassembly
(SLR). In one aspect, the method comprises mutagenizing the first
nucleic acid of step (a) or variants 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
(GeneReassembly, U.S. Pat. No. 6,537,776), Gene Site Saturation
Mutagenesis (GSSM), synthetic ligation reassembly (SLR) and a
combination thereof. In one 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.
[0116] The invention provides methods for making a small molecule
comprising: (a) providing a plurality of biosynthetic enzymes
capable of synthesizing or modifying a small molecule, wherein one
of the enzymes comprises a xylanase, a mannanase and/or a glucanase
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: (a) providing a xylanase, a mannanase and/or a
glucanase 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 xylanase, a mannanase and/or a glucanase
enzyme, thereby modifying a small molecule by a xylanase, a
mannanase and/or a glucanase 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 xylanase, a mannanase and/or a glucanase 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.
[0117] The invention provides methods for determining a functional
fragment of a xylanase, a mannanase and/or a glucanase enzyme
comprising the steps of: (a) providing a xylanase, a mannanase
and/or a glucanase 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 xylanase, a mannanase
and/or a glucanase activity, thereby determining a functional
fragment of a xylanase, a mannanase and/or a glucanase enzyme. In
one aspect, the xylanase, a mannanase and/or a glucanase activity
is measured by providing a xylanase, a mannanase and/or a glucanase
substrate and detecting a decrease in the amount of the substrate
or an increase in the amount of a reaction product.
[0118] The invention provides methods for whole cell engineering of
new or modified phenotypes by using real-time metabolic flux
analysis, the method comprising: (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.
[0119] The invention provides isolated, synthetic or recombinant
signal sequences consisting of, or comprising, a sequence as set
forth in residues 1 to 12, 1 to 13, 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 or 1 to 44, of a polypeptide of the
invention, including exemplary polypeptide sequences of the
invention.
[0120] The invention provides chimeric polypeptides comprising at
least a first domain comprising a signal peptide (SP) and at least
a second domain comprising a heterologous polypeptide or peptide
comprising a sequence of the invention, or a subsequence thereof,
wherein the heterologous polypeptide or peptide is not naturally
associated with the signal peptide (SP). In one aspect, the signal
peptide (SP) is not derived from a xylanase, a mannanase and/or a
glucanase. The heterologous polypeptide or peptide can be amino
terminal to, carboxy terminal to or on both ends of the signal
peptide (SP) or a xylanase, a mannanase and/or a glucanase
catalytic domain (CD). The invention provides isolated, synthetic
or recombinant nucleic acids encoding a chimeric polypeptide,
wherein the chimeric polypeptide comprises at least a first domain
comprising signal peptide (SP) and at least a second domain
comprising a heterologous polypeptide or peptide comprising a
sequence of the invention, or a subsequence thereof, wherein the
heterologous polypeptide or peptide is not naturally associated
with the signal peptide (SP).
[0121] The invention provides methods of increasing thermotolerance
or thermostability of a xylanase, a mannanase and/or a glucanase
polypeptide, the method comprising glycosylating a xylanase, a
mannanase and/or a glucanase 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 xylanase, a mannanase and/or a glucanase
polypeptide. In one aspect, the xylanase, a mannanase and/or a
glucanase specific activity can be thermostable or thermotolerant
at a temperature in the range from greater than about 0.degree. C.
to about 20.degree. C., about 20.degree. C. to about 37.degree. C.,
about 37.degree. C. to about 50.degree. C., about 50.degree. C. to
about 70.degree. C., about 70.degree. C. to about 75.degree. C.,
about 75.degree. C. to about 80.degree. C., about 80.degree. C. to
about 85.degree. C., about 85.degree. C. to about 90.degree. C.,
about 90.degree. C. to about 95.degree. C., about 95.degree. C. to
about 100.degree. C., about 100.degree. C. to about 110.degree. C.,
or higher.
[0122] The invention provides methods for overexpressing a
recombinant xylanase, a mannanase and/or a glucanase 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.
[0123] The invention provides methods of making a transgenic plant
and seeds comprising: (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 or seed cell; and (b) producing a
transgenic plant from the transformed cell or seed. 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 potato, corn, rice, wheat, tobacco, or barley cell.
[0124] The invention provides methods of expressing a heterologous
nucleic acid sequence in a plant cell comprising: (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: (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.
[0125] The invention provides methods for hydrolyzing, breaking up
or disrupting a xylan-comprising composition comprising: (a)
providing a polypeptide of the invention having a xylanase, a
mannanase and/or a glucanase activity, or a polypeptide encoded by
a nucleic acid of the invention; (b) providing a composition
comprising a xylan; and (c) contacting the polypeptide of step (a)
with the composition of step (b) under conditions wherein the
xylanase, a mannanase and/or a glucanase hydrolyzes, breaks up or
disrupts the xylan-comprising composition. In one aspect, the
composition comprises a plant cell, a bacterial cell, a yeast cell,
an insect cell, or an animal cell. Thus, the composition can
comprise any plant or plant part, any xylan-containing food or
feed, a waste product and the like.
[0126] The invention provides methods for liquefying or removing a
xylan-comprising composition comprising: (a) providing a
polypeptide of the invention having a xylanase activity, or a
polypeptide encoded by a nucleic acid of the invention; (b)
providing a composition comprising a xylan; and (c) contacting the
polypeptide of step (a) with the composition of step (b) under
conditions wherein the xylanase removes, softens or liquefies the
xylan-comprising composition.
[0127] The invention provides detergent compositions comprising a
polypeptide of the invention, or a polypeptide encoded by a nucleic
acid of the invention, wherein the polypeptide has a xylanase, a
mannanase and/or a glucanase activity. The xylanase can be a
nonsurface-active xylanase, a mannanase and/or a glucanase or a
surface-active xylanase, a mannanase and/or a glucanase. The
xylanase, a mannanase and/or a glucanase can be formulated in a
non-aqueous liquid composition, a cast solid, a granular form, a
particulate form, a compressed tablet, a gel form, a paste or a
slurry form. The invention provides methods for washing an object
comprising: (a) providing a composition comprising a polypeptide of
the invention having a xylanase, a mannanase and/or a glucanase
activity, or a polypeptide encoded by a nucleic acid of the
invention; (b) providing an object; and (c) contacting the
polypeptide of step (a) and the object of step (b) under conditions
wherein the composition can wash the object.
[0128] The invention provides textiles or fabrics, including, e.g.,
threads, comprising a polypeptide of the invention, or a
polypeptide encoded by a nucleic acid of the invention. In one
aspect, the textiles or fabrics comprise xylan-containing fibers.
The invention provides methods for treating a textile or fabric
(e.g., removing a stain from a composition) comprising: (a)
providing a composition comprising a polypeptide of the invention
having a xylanase, a mannanase and/or a glucanase activity, or a
polypeptide encoded by a nucleic acid of the invention; (b)
providing a textile or fabric comprising a xylan; and (c)
contacting the polypeptide of step (a) and the composition of step
(b) under conditions wherein the xylanase, a mannanase and/or a
glucanase can treat the textile or fabric (e.g., remove the stain).
The invention provides methods for improving the finish of a fabric
comprising: (a) providing a composition comprising a polypeptide of
the invention having a xylanase, a mannanase and/or a glucanase
activity, or a polypeptide encoded by a nucleic acid of the
invention; (b) providing a fabric; and (c) contacting the
polypeptide of step (a) and the fabric of step (b) under conditions
wherein the polypeptide can treat the fabric thereby improving the
finish of the fabric. In one aspect, the fabric is a wool or a
silk. In another aspect, the fabric is a cellulosic fiber or a
blend of a natural fiber and a synthetic fiber.
[0129] The invention provides feeds or foods comprising a
polypeptide of the invention, or a polypeptide encoded by a nucleic
acid of the invention. The invention provides methods for
hydrolyzing xylans in a feed or a food prior to consumption by an
animal comprising: (a) obtaining a feed material comprising a
xylanase, a mannanase and/or a glucanase of the invention, or a
xylanase, a mannanase and/or a glucanase encoded by a nucleic acid
of the invention; and (b) adding the polypeptide of step (a) to the
feed or food material in an amount sufficient for a sufficient time
period to cause hydrolysis of the xylan and formation of a treated
food or feed, thereby hydrolyzing the xylans in the food or the
feed prior to consumption by the animal. In one aspect, the
invention provides methods for hydrolyzing xylans in a feed or a
food after consumption by an animal comprising: (a) obtaining a
feed material comprising a xylanase, a mannanase and/or a glucanase
of the invention, or a xylanase, a mannanase and/or a glucanase
encoded by a nucleic acid of the invention; (b) adding the
polypeptide of step (a) to the feed or food material; and (c)
administering the feed or food material to the animal, wherein
after consumption, the xylanase, a mannanase and/or a glucanase
causes hydrolysis of xylans in the feed or food in the digestive
tract of the animal. The food or the feed can be, e.g., a cereal, a
grain, a corn and the like.
[0130] The invention provides dough or bread products comprising a
polypeptide having a xylanase, a mannanase and/or a glucanase
activity, wherein the polypeptide comprises a sequence of the
invention, or the polypeptide is encoded by a nucleic acid
comprising a sequence of the invention, or an enzymatically active
fragment thereof. The invention provides methods of dough
conditioning comprising contacting a dough or a bread product with
at least one polypeptide having a xylanase, a mannanase and/or a
glucanase activity, wherein the polypeptide comprises a sequence of
the invention, or the polypeptide is encoded by a nucleic acid
comprising a sequence of the invention, or an enzymatically active
fragment thereof, under conditions sufficient for conditioning the
dough.
[0131] The invention provides beverages comprising a polypeptide
having a xylanase, a mannanase and/or a glucanase activity, wherein
the polypeptide comprises a sequence of the invention, or the
polypeptide is encoded by a nucleic acid comprising a sequence of
the invention. The invention provides methods of beverage
production comprising administration of at least one polypeptide
having a xylanase, a mannanase and/or a glucanase activity, wherein
the polypeptide comprises a sequence of the invention, or the
polypeptide is encoded by a nucleic acid comprising a sequence of
the invention, or an enzymatically active fragment thereof, to a
beverage or a beverage precursor under conditions sufficient for
decreasing the viscosity of the beverage, wherein in one aspect
(optionally) the beverage or beverage precursor is a wort or a
beer.
[0132] 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. 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 xylanase, a
mannanase and/or a glucanase activity is thermotolerant. In another
aspect, the xylanase, a mannanase and/or a glucanase activity is
thermostable.
[0133] The invention provides a food, a feed or a nutritional
supplement comprising a polypeptide of the invention. The invention
provides methods for utilizing a xylanase, a mannanase and/or a
glucanase as a nutritional supplement in an animal diet, the method
comprising: preparing a nutritional supplement containing a
xylanase, a mannanase and/or a glucanase enzyme comprising at least
thirty contiguous amino acids of a polypeptide of the invention;
and administering the nutritional supplement to an animal to
increase utilization of a xylan contained in a feed or a food
ingested by the animal. The animal can be a human, a ruminant or a
monogastric animal. The xylanase, a mannanase and/or a glucanase
enzyme can be prepared by expression of a polynucleotide encoding
the xylanase, a mannanase and/or a glucanase 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, Pseudomonas sp., E. coli, Streptomyces sp.,
Bacillus sp. and Lactobacillus sp.
[0134] The invention provides edible enzyme delivery matrix
comprising a thermostable recombinant xylanase, a mannanase and/or
a glucanase enzyme, e.g., a polypeptide of the invention. The
invention provides methods for delivering a xylanase, a mannanase
and/or a glucanase 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 xylanase, a mannanase and/or a glucanase enzyme,
wherein the pellets readily disperse the xylanase, a mannanase
and/or a glucanase enzyme contained therein into aqueous media, and
administering the edible enzyme delivery matrix to the animal. The
recombinant xylanase, a mannanase and/or a glucanase enzyme can
comprise a polypeptide of the invention. The granulate edible
carrier can comprise a carrier selected from the group consisting
of a grain germ, a grain germ that is spent of oil, a hay, an
alfalfa, a timothy, a soy hull, a sunflower seed meal and a wheat
midd. The edible carrier can comprise grain germ that is spent of
oil. The xylanase, a mannanase and/or a glucanase 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 xylanase, a mannanase and/or a
glucanase. 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.
[0135] The invention provides methods for improving texture and
flavor of a dairy product comprising: (a) providing a polypeptide
of the invention having a xylanase, a mannanase and/or a glucanase
activity, or a xylanase, a mannanase and/or a glucanase encoded by
a nucleic acid of the invention; (b) providing a dairy product; and
(c) contacting the polypeptide of step (a) and the dairy product of
step (b) under conditions wherein the xylanase, a mannanase and/or
a glucanase can improve the texture or flavor of the dairy product.
In one aspect, the dairy product comprises a cheese or a yogurt.
The invention provides dairy products comprising a xylanase, a
mannanase and/or a glucanase of the invention, or is encoded by a
nucleic acid of the invention.
[0136] The invention provides methods for improving the extraction
of oil from an oil-rich plant material comprising: (a) providing a
polypeptide of the invention having a xylanase, a mannanase and/or
a glucanase activity, or a xylanase, a mannanase and/or a glucanase
encoded by a nucleic acid of the invention; (b) providing an
oil-rich plant material; and (c) contacting the polypeptide of step
(a) and the oil-rich plant material. In one aspect, the oil-rich
plant material comprises an oil-rich seed. The oil can be a soybean
oil, an olive oil, a rapeseed (canola) oil or a sunflower oil.
[0137] The invention provides methods for preparing a fruit or
vegetable juice, syrup, puree or extract comprising: (a) providing
a polypeptide of the invention having a xylanase, a mannanase
and/or a glucanase activity, or a xylanase, a mannanase and/or a
glucanase encoded by a nucleic acid of the invention; (b) providing
a composition or a liquid comprising a fruit or vegetable material;
and (c) contacting the polypeptide of step (a) and the composition,
thereby preparing the fruit or vegetable juice, syrup, puree or
extract.
[0138] The invention provides papers or paper products or paper
pulp comprising a xylanase, a mannanase and/or a glucanase of the
invention, or a polypeptide encoded by a nucleic acid of the
invention. The invention provides methods for treating a biomass,
e.g., any paper or a paper or wood pulp comprising: (a) providing a
polypeptide of the invention having a xylanase, a mannanase and/or
a glucanase activity, or a xylanase, a mannanase and/or a glucanase
encoded by a nucleic acid of the invention; (b) providing a
composition, e.g., a biomass, comprising a paper or a paper or wood
pulp; and (c) contacting the polypeptide of step (a) and the
composition of step (b) under conditions wherein the xylanase, a
mannanase and/or a glucanase can treat the paper or paper or wood
pulp.
[0139] The invention provides methods for reducing the amount of
lignin (delignification), or solubilizing a lignin, in a paper or
paper product, a paper waste, a wood, wood pulp or wood product, or
a wood or paper recycling composition, comprising contacting the
paper or paper product, wood, wood pulp or wood product, or wood or
paper recycling composition with a polypeptide of the invention, or
an enzymatically active fragment thereof.
[0140] The invention provides methods for hydrolyzing
hemicelluloses in a wood, wood product, paper pulp, paper product
or paper waste comprising contacting the wood, wood product, paper
pulp, paper product or paper waste with a polypeptide of the
invention, or an enzymatically active fragment thereof.
[0141] The invention provides methods for enzymatic decoloring
(e.g., bleaching) of paper, hemp or flax pulp comprising contacting
the paper, hemp or flax pulp with a xylanase, a mannanase and/or a
glucanase and a decoloring (e.g., bleaching) agent, wherein the
xylanase, a mannanase and/or a glucanase comprises a polypeptide of
the invention, or an enzymatically active fragment thereof. The
decoloring (e.g., bleaching) agent can comprise oxygen or hydrogen
peroxide.
[0142] The invention provides methods for of decoloring (e.g.,
bleaching) a lignocellulose pulp comprising contacting the
lignocellulose pulp with a xylanase, a mannanase and/or a
glucanase, wherein the xylanase, a mannanase and/or a glucanase
comprises a polypeptide of the invention, or an enzymatically
active fragment thereof.
[0143] The invention provides methods for enzymatic deinking of
paper, paper waste, paper recycled product, deinking toner from
non-contact printed wastepaper or mixtures of non-contact and
contact printed wastepaper, comprising contacting the paper, paper
waste, paper recycled product, non-contact printed wastepaper or
contact printed wastepaper with a xylanase, a mannanase and/or a
glucanase, wherein the xylanase, a mannanase and/or a glucanase
comprises a polypeptide of the invention, or an enzymatically
active fragment thereof.
[0144] The invention provides methods for decoloring (e.g.,
bleaching) a thread, fabric, yarn, cloth or textile comprising
contacting the fabric, yarn, cloth or textile with a xylanase, a
mannanase and/or a glucanase under conditions suitable to produce a
whitening of the textile, wherein the xylanase, a mannanase and/or
a glucanase comprises a polypeptide of the invention, or an
enzymatically active fragment thereof. The thread, fabric, yarn,
cloth or textile can comprise a non-cotton cellulosic thread,
fabric, yarn, cloth or textile. The invention provides fabrics,
yarns, cloths or textiles comprising a polypeptide having a
sequence of the invention, or a polypeptide encoded by a nucleic
acid comprising a sequence of the invention, or an enzymatically
active fragment thereof, wherein in one aspect (optionally) the
fabric, yarn, cloth or textile comprises a non-cotton cellulosic
fabric, yarn, cloth or textile.
[0145] The invention provides methods for decoloring (e.g.,
bleaching) or deinking newspaper comprising contacting the
newspaper, wherein the xylanase, a mannanase and/or a glucanase
comprises a polypeptide of the invention, or an enzymatically
active fragment thereof.
[0146] The invention provides wood, wood chips, wood pulp, wood
products, paper pulps, paper products, newspapers or paper waste
comprising a polypeptide of the invention, or an enzymatically
active fragment thereof. The invention provides thread, fabric,
yarn, cloth or textile comprising a polypeptide of the invention,
or an enzymatically active fragment thereof.
[0147] The invention provides methods for reducing lignin in a wood
or wood product comprising contacting the wood or wood product with
a polypeptide having a xylanase, a mannanase and/or a glucanase
activity, wherein the polypeptide has a sequence of the invention,
or the polypeptide is encoded by a nucleic acid comprising a
sequence of the invention, or an enzymatically active fragment
thereof.
[0148] The invention provides methods for reducing a lignin in a
biomass, e.g., in a wood, a wood pulp, a Kraft pulp, a paper, a
paper product or a paper pulp under high temperature and basic pH
conditions, the method comprising: (a) providing at least one
polypeptide having a xylanase, a mannanase and/or a glucanase
activity, wherein the polypeptide retains xylanase, a mannanase
and/or a glucanase activity under conditions comprising a
temperature of at least about 80.degree. C., 85.degree. C.,
90.degree. C. or more, and a basic pH of at least about pH 10.5, pH
11, pH 12, pH 12.5 or more (basic) wherein the polypeptide
comprises a xylanase, a mannanase and/or a glucanase having a
sequence of the invention, or the xylanase, a mannanase and/or a
glucanase is encoded by a nucleic acid comprising a sequence of the
invention, or an enzymatically active fragment thereof; (b)
providing a lignin-comprising biomass, e.g., a lignin-comprising
wood, wood pulp, Kraft pulp, paper, paper product or paper pulp;
and (c) contacting the wood, wood pulp, Kraft pulp, paper, paper
product or paper pulp with the polypeptide of step (a) under
conditions comprising a temperature of at least about 80.degree.
C., 85.degree. C., 90.degree. C. or more, and a basic pH of at
least about pH 10.5, pH 11, pH 12, pH 12.5 or more (basic), wherein
the polypeptide reduces the lignin-comprising biomass, e.g., the
lignin in the wood, wood pulp, Kraft pulp, paper, paper product or
paper pulp.
[0149] The invention provides methods for treating a
lignin-comprising biomass, e.g., a wood, a wood pulp, a Kraft pulp,
a paper product, a paper or a paper pulp under high temperature and
basic pH conditions, the method comprising: (a) providing at least
one polypeptide having a xylanase, a mannanase and/or a glucanase
activity, wherein the polypeptide retains xylanase, a mannanase
and/or a glucanase activity under conditions comprising a
temperature of at least about 80.degree. C., 85.degree. C.,
90.degree. C. or more, and a basic pH of at least about pH 10.5, pH
11, pH 12, pH 12.5 or more (basic), wherein the polypeptide
comprises a xylanase, a mannanase and/or a glucanase having a
sequence of the invention, or the xylanase, a mannanase and/or a
glucanase is encoded by a nucleic acid comprising a sequence of the
invention, or an enzymatically active fragment thereof; (b)
providing a lignin-comprising biomass, e.g., a wood, a wood pulp, a
Kraft pulp, a paper, a paper product or a paper pulp; and (c)
contacting the wood, wood pulp, Kraft pulp, paper, paper product or
paper pulp with the polypeptide of step (a) under conditions
comprising a temperature of at least about 80.degree. C.,
85.degree. C., 90.degree. C. or more, and a basic pH of at least
about pH 10.5, pH 11, pH 12, pH 12.5 or more (basic), wherein the
polypeptide catalyzes hydrolysis of compounds in the
lignin-comprising biomass, e.g., wood, wood pulp, Kraft pulp,
paper, paper product or paper pulp, and wherein in one aspect
(optionally) the wood, wood pulp, Kraft pulp, paper, paper product
or paper pulp comprises a softwood and hardwood, or the wood, wood
pulp, Kraft pulp, paper or paper pulp is derived from a softwood
and hardwood; and wherein in one aspect (optionally) after the
treatment the pulp has a consistency of at least about 10%, or at
least about 32%.
[0150] The invention provides methods for decoloring a biomass,
e.g., a wood, a wood pulp, a Kraft pulp, a paper, a paper product
or a paper pulp under high temperature and basic pH conditions, the
method comprising: (a) providing at least one polypeptide having a
xylanase, a mannanase and/or a glucanase activity, wherein the
polypeptide retains xylanase, a mannanase and/or a glucanase
activity under conditions comprising a temperature of at least
about 80.degree. C., 85.degree. C., 90.degree. C. or more, and a
basic pH of at least about pH 10.5, pH 11, pH 12, pH 12.5 or more
(basic), wherein the polypeptide comprises a xylanase, a mannanase
and/or a glucanase having a sequence of the invention, or the
xylanase, a mannanase and/or a glucanase is encoded by a nucleic
acid comprising a sequence of the invention, or an enzymatically
active fragment thereof; (b) providing a biomass, e.g., a wood, a
wood pulp, a Kraft pulp, a paper, a paper product or a paper pulp;
and (c) contacting the wood, wood pulp, Kraft pulp, paper, paper
product or paper pulp with the polypeptide of step (a) under
conditions comprising a temperature of at least about 80.degree.
C., 85.degree. C., 90.degree. C., 91.degree. C., 92.degree. C.,
93.degree. C., 94.degree. C., 95.degree. C., 96.degree. C.,
97.degree. C., 98.degree. C., 99.degree. C., 100.degree. C.,
101.degree. C., 102.degree. C., 103.degree. C., 103.5.degree. C.,
104.degree. C., 105.degree. C., 107.degree. C., 108.degree. C.,
109.degree. C. or 110.degree. C., or more, and a basic pH of at
least about pH 9.5, pH 10.0, pH 10.5, pH 11, pH 12, pH 12.5 or more
(basic), wherein the polypeptide catalyzes hydrolysis of compounds
in the biomass, e.g., a wood, wood pulp, Kraft pulp, paper, paper
product or paper pulp, thereby decoloring (e.g., bleaching) the
biomass, e.g., a wood, wood pulp, Kraft pulp, paper, paper product
or paper pulp.
[0151] The invention provides methods for reducing the use of
decoloring (e.g., bleaching) chemicals in a biomass, e.g., a wood,
a wood pulp, a Kraft pulp, a paper, a paper product or a paper pulp
decoloring (e.g., bleaching) process under high temperature and
basic pH conditions, the method comprising: (a) providing at least
one polypeptide having a xylanase, a mannanase and/or a glucanase
activity, wherein the polypeptide retains xylanase, a mannanase
and/or a glucanase activity under conditions comprising a
temperature of at least about 80.degree. C., 85.degree. C.,
90.degree. C., 91.degree. C., 92.degree. C., 93.degree. C.,
94.degree. C., 95.degree. C., 96.degree. C., 97.degree. C.,
98.degree. C., 99.degree. C., 100.degree. C., 101.degree. C.,
102.degree. C., 103.degree. C., 103.5.degree. C., 104.degree. C.,
105.degree. C., 107.degree. C., 108.degree. C., 109.degree. C. or
110.degree. C., or more, and a basic pH of at least about pH 10.5,
pH 11, pH 12, pH 12.5 or more (basic), wherein the polypeptide
comprises a xylanase, a mannanase and/or a glucanase having a
sequence of the invention, or the xylanase, a mannanase and/or a
glucanase is encoded by a nucleic acid comprising a sequence of the
invention, or an enzymatically active fragment thereof; (b)
providing a biomass, e.g., a wood, a wood pulp, a Kraft pulp, a
paper, a paper product or a paper pulp; and (c) contacting the
wood, wood pulp, Kraft pulp, paper, paper product or paper pulp
with the polypeptide of step (a) under conditions comprising a
temperature of at least about 80.degree. C., 85.degree. C.,
86.degree. C., 87.degree. C., 88.degree. C., 89.degree. C.,
90.degree. C., 91.degree. C., 92.degree. C., 93.degree. C.,
94.degree. C., 95.degree. C., 96.degree. C., 97.degree. C.,
98.degree. C., 99.degree. C., 100.degree. C., 101.degree. C.,
102.degree. C., 103.degree. C., 103.5.degree. C., 104.degree. C.,
105.degree. C., 107.degree. C., 108.degree. C., 109.degree. C. or
110.degree. C., or more, and a basic pH of at least about pH 10.5,
pH 11, pH 12, pH 12.5 or more (basic), wherein the polypeptide
catalyzes hydrolysis of compounds in the biomass, e.g., wood, wood
pulp, Kraft pulp, paper, paper product or paper pulp, thereby
biobleaching the biomass, e.g., wood, wood pulp, Kraft pulp, paper,
paper product or paper pulp and reducing the use of decoloring
(e.g., bleaching) chemicals in the decoloring (e.g., bleaching)
process; wherein in one aspect (optionally) the decoloring (e.g.,
bleaching) chemical comprises a chlorine, a chlorine dioxide, a
caustic, a peroxide, or any combination thereof.
[0152] The invention provides methods for paper or pulp deinking
under high temperature and basic pH conditions, the method
comprising: (a) providing at least one polypeptide having a
xylanase, a mannanase and/or a glucanase activity, wherein the
polypeptide retains xylanase, a mannanase and/or a glucanase
activity under conditions comprising a temperature of at least
about 80.degree. C., 85.degree. C., 86.degree. C., 87.degree. C.,
88.degree. C., 89.degree. C., 90.degree. C., 91.degree. C.,
92.degree. C., 93.degree. C., 94.degree. C., 95.degree. C.,
96.degree. C., 97.degree. C., 98.degree. C., 99.degree. C.,
100.degree. C., 101.degree. C., 102.degree. C., 103.degree. C.,
103.5.degree. C., 104.degree. C., 105.degree. C., 107.degree. C.,
108.degree. C., 109.degree. C. or 110.degree. C., or more, and a
basic pH of at least about pH 11, wherein the polypeptide comprises
a xylanase, a mannanase and/or a glucanase having a sequence of the
invention, or the xylanase, a mannanase and/or a glucanase is
encoded by a nucleic acid comprising a sequence of the invention,
or an enzymatically active fragment thereof (b) providing an
ink-comprising biomass, e.g., a wood, wood pulp, Kraft pulp, paper,
a paper product or paper pulp; and (c) contacting the biomass,
e.g., wood, wood pulp, Kraft pulp, paper, paper product or paper
pulp with the polypeptide of step (a) under conditions comprising a
temperature of at least about 85.degree. C. and a basic pH of at
least about pH 11, wherein the polypeptide catalyzes hydrolysis of
compounds in the biomass, e.g., wood, wood pulp, Kraft pulp, paper
or paper pulp, thereby facilitating deinking of the biomass, e.g.,
wood, wood pulp, Kraft pulp, paper, paper product or paper
pulp.
[0153] The invention provides methods for releasing a lignin from a
biomass, e.g., a wood, a wood pulp, a Kraft pulp, a paper, a paper
product or a paper pulp under high temperature and basic pH
conditions, the method comprising: (a) providing at least one
polypeptide having a xylanase, a mannanase and/or a glucanase
activity, wherein the polypeptide retains xylanase, a mannanase
and/or a glucanase activity under conditions comprising a
temperature of at least about 80.degree. C., 85.degree. C.,
86.degree. C., 87.degree. C., 88.degree. C., 89.degree. C.,
90.degree. C., 91.degree. C., 92.degree. C., 93.degree. C.,
94.degree. C., 95.degree. C., 96.degree. C., 97.degree. C.,
98.degree. C., 99.degree. C., 100.degree. C., 101.degree. C.,
102.degree. C., 103.degree. C., 103.5.degree. C., 104.degree. C.,
105.degree. C., 107.degree. C., 108.degree. C., 109.degree. C. or
110.degree. C., or more, and a basic pH of at least about pH 11,
wherein the polypeptide comprises a xylanase, a mannanase and/or a
glucanase having a sequence of the invention, or the xylanase, a
mannanase and/or a glucanase is encoded by a nucleic acid
comprising a sequence of the invention, or an enzymatically active
fragment thereof; (b) providing a lignin-comprising biomass, e.g.,
wood, wood pulp, Kraft pulp, paper, paper product or paper pulp;
and (c) contacting the wood, wood pulp, Kraft pulp, paper, paper
product or a paper pulp of step (b) with the polypeptide of step
(a) under conditions comprising a temperature of at least about
80.degree. C., 85.degree. C., 86.degree. C., 87.degree. C.,
88.degree. C., 89.degree. C., 90.degree. C., 91.degree. C.,
92.degree. C., 93.degree. C., 94.degree. C., 95.degree. C.,
96.degree. C., 97.degree. C., 98.degree. C., 99.degree. C.,
100.degree. C., 101.degree. C., 102.degree. C., 103.degree. C.,
103.5.degree. C., 104.degree. C., 105.degree. C., 107.degree. C.,
108.degree. C., 109.degree. C. or 110.degree. C., or more, and a
basic pH of at least about pH 11, wherein the polypeptide catalyzes
hydrolysis of compounds in the wood, wood pulp, Kraft pulp, paper,
paper product or paper pulp, thereby facilitating release of lignin
from the biomass, e.g., wood, wood pulp, Kraft pulp, paper, paper
product or paper pulp; wherein in one aspect (optionally) after the
treatment the pulp has a consistency of about 10%.
[0154] The invention provides compositions comprising a biomass,
e.g., wood, a wood pulp, a Kraft pulp, a paper, a paper product or
a paper pulp comprising a polypeptide having a xylanase, a
mannanase and/or a glucanase activity, wherein the polypeptide has
a sequence of the invention, or the polypeptide is encoded by a
nucleic acid comprising a sequence of the invention, or an
enzymatically active fragment thereof, wherein in one aspect (e.g.,
optionally) the biomass, e.g., wood, wood pulp, Kraft pulp, paper,
paper product or paper pulp comprises a softwood and hardwood, or
the wood, wood pulp, Kraft pulp, paper, paper product or paper pulp
derived from a softwood and hardwood.
[0155] The invention provides methods for making ethanol comprising
contacting a starch-comprising composition with a polypeptide
having a xylanase, a mannanase and/or a glucanase activity, wherein
the polypeptide has a sequence of the invention, or the polypeptide
is encoded by a nucleic acid comprising a sequence of the
invention, or an enzymatically active fragment thereof. The
invention provides compositions comprising an ethanol and a
polypeptide having a xylanase, a mannanase and/or a glucanase
activity, wherein the polypeptide has a sequence of the invention,
or the polypeptide is encoded by a nucleic acid comprising a
sequence of the invention, or an enzymatically active fragment
thereof. The invention provides methods for making ethanol under
high temperature and basic pH conditions, the method comprising:
(a) providing at least one polypeptide having a xylanase, a
mannanase and/or a glucanase activity, wherein the polypeptide
retains xylanase, a mannanase and/or a glucanase activity under
conditions comprising a temperature of at least about 80.degree.
C., 85.degree. C., 86.degree. C., 87.degree. C., 88.degree. C.,
89.degree. C., 90.degree. C., 91.degree. C., 92.degree. C.,
93.degree. C., 94.degree. C., 95.degree. C., 96.degree. C.,
97.degree. C., 98.degree. C., 99.degree. C., 100.degree. C.,
101.degree. C., 102.degree. C., 103.degree. C., 103.5.degree. C.,
104.degree. C., 105.degree. C., 107.degree. C., 108.degree. C.,
109.degree. C. or 110.degree. C., or more, and a basic pH of at
least about pH 11, wherein the polypeptide comprises a xylanase, a
mannanase and/or a glucanase having a sequence of the invention, or
the xylanase, a mannanase and/or a glucanase is encoded by a
nucleic acid comprising a sequence of the invention, or an
enzymatically active fragment thereof; (b) providing a
starch-comprising composition comprising a wood, wood pulp, Kraft
pulp, paper, a paper product or paper pulp; and (c) contacting the
composition of step (b) with the polypeptide of step (a) under
conditions comprising a temperature of at least about 80.degree.
C., 85.degree. C., 86.degree. C., 87.degree. C., 88.degree. C.,
89.degree. C., 90.degree. C., 91.degree. C., 92.degree. C.,
93.degree. C., 94.degree. C., 95.degree. C., 96.degree. C.,
97.degree. C., 98.degree. C., 99.degree. C., 100.degree. C.,
101.degree. C., 102.degree. C., 103.degree. C., 103.5.degree. C.,
104.degree. C., 105.degree. C., 107.degree. C., 108.degree. C.,
109.degree. C. or 110.degree. C., or more, and a basic pH of at
least about pH 11, wherein the polypeptide catalyzes hydrolysis of
compounds in the wood, wood pulp, Kraft pulp, paper or paper pulp,
thereby generating ethanol from the wood, wood pulp, Kraft pulp,
paper, paper product or paper pulp.
[0156] The invention provides pharmaceutical compositions
comprising a polypeptide having a xylanase, a mannanase and/or a
glucanase activity, wherein the polypeptide comprises a sequence of
the invention, or the polypeptide is encoded by a nucleic acid
comprising a sequence of the invention, or an enzymatically active
fragment thereof. In one aspect, the invention provides methods for
eliminating or protecting animals from a microorganism comprising a
xylan comprising administering a polypeptide of the invention. The
microorganism can be a bacterium comprising a xylan, e.g.,
Salmonella.
[0157] In one aspect, the pharmaceutical composition acts as a
digestive aid or an antimicrobial (e.g., against Salmonella). In
one aspect, the treatment is prophylactic. In one aspect, the
invention provides oral care products comprising a polypeptide of
the invention having a xylanase, a mannanase and/or a glucanase
activity, or a xylanase, a mannanase and/or a glucanase encoded by
a nucleic acid of the invention. The oral care product can comprise
a toothpaste, a dental cream, a gel or a tooth powder, an odontic,
a mouth wash, a pre- or post brushing rinse formulation, a chewing
gum, a lozenge or a candy. The invention provides contact lens
cleaning compositions comprising a polypeptide of the invention
having a xylanase, a mannanase and/or a glucanase activity, or a
xylanase, a mannanase and/or a glucanase encoded by a nucleic acid
of the invention.
[0158] The invention provides chimeric glycosidases, xylanases
and/or glucanases comprising a polypeptide (e.g., xylanase, a
mannanase and/or a glucanase) sequence of the invention and at
least one heterologous carbohydrate-binding module (CBM), wherein
in one aspect (optionally) the CBM comprises a CBM3a, CBM3b, CBM4,
CBM6, CBM22 or X14, or a CBM as listed and discussed, below. The
invention also provides chimeric glycosidases, xylanases and/or
glucanases comprising at least one heterologous
carbohydrate-binding module (CBM), wherein the CBM comprises a
carbohydrate-binding subsequence of a xylanase sequence of the
invention, or a carbohydrate-binding subsequence comprising a X14.
The invention provides methods for designing a chimeric
glycosidase, xylanase, a mannanase and/or a glucanase having a new
carbohydrate-binding specificity or an enhanced
carbohydrate-binding specificity, comprising inserting a
heterologous or an additional endogenous carbohydrate-binding
module (CBM) into a glycosidases, xylanases and/or glucanases,
wherein the CBM comprises a carbohydrate-binding subsequence of a
glycosidase, xylanase, mannanase and/or glucanase sequence of the
invention, or a carbohydrate-binding subsequence comprising a X14,
or alternatively a heterologous CBM, or an additional endogenous
CBM, is inserted into a xylanase, a mannanase and/or a glucanase
sequence of the invention.
[0159] The invention provides enzyme mixtures, or "cocktails"
comprising at least one enzyme of the invention and one or more
other enzyme(s), which can be another xylanase, mannanase and/or
glucanase, or any other enzyme; for example, the "cocktails" of the
invention, in addition to at least one enzyme of this invention,
can comprise any other enzyme, such as xylanase (not of this
invention), cellulases, lipases, esterases, proteases, or
endoglycosidases, endo-beta.-1,4-glucanases, beta-glucanases,
endo-beta-1,3(4)-glucanases, cutinases, peroxidases, catalases,
laccases, amylases, glucoamylases, pectinases, reductases,
oxidases, phenoloxidases, ligninases, pullulanases, arabinanases,
hemicellulases, mannanases, xyloglucanases, xylanases, pectin
acetyl esterases, rhamnogalacturonan acetyl esterases,
polygalacturonases, rhamnogalacturonases, galactanases, pectin
lyases, pectin methylesterases, cellobiohydrolases and/or
transglutaminases, to name just a few examples. In alternative
embodiments, these enzyme mixtures, or "cocktails" comprising at
least one enzyme of the invention can be used in any process or
method of the invention, or composition of the invention, e.g., in
foods or feeds, food or feed supplements, textiles, papers,
processed woods, etc. and methods for making them, and in
compositions and methods for treating paper, pulp, wood, paper,
pulp or wood waste or by-products, and the like, and in the final
products thereof.
[0160] The details of one or more embodiments 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.
[0161] All publications, patents, patent applications, GenBank
sequences and ATCC deposits, cited herein are hereby expressly
incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0162] 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.
[0163] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0164] FIG. 1 is a block diagram of a computer system.
[0165] 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.
[0166] FIG. 3 is a flow diagram illustrating one aspect of a
process in a computer for determining whether two sequences are
homologous.
[0167] FIG. 4 is a flow diagram illustrating one aspect of an
identifier process 300 for detecting the presence of a feature in a
sequence.
[0168] FIG. 5 is a schematic flow diagram of an exemplary routine
screening protocol to determine whether a xylanase of the invention
is useful in pretreating paper pulp, as described in detail in
Example 3, below.
[0169] FIG. 6 illustrates a biobleaching industrial process of the
invention, as described in detail in Example 5, below.
[0170] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION OF THE INVENTION
[0171] The invention provides glycosyl hydrolases, including
xylanases (e.g., endoxylanases) and/or a glucanases, and
polynucleotides encoding them and methods of making and using them.
Glycosyl hydrolase, including xylanase, mannanase and/or glucanase
activity, of the polypeptides of the invention encompasses enzymes
having hydrolase activity, for example, enzymes capable of
hydrolyzing glycosidic linkages in a polysaccharide, for example a
glycosidic linkage present in xylan, e.g., catalyzing hydrolysis of
internal .beta.-1,4-xylosidic linkages. The xylanases and/or a
glucanases of the invention can be used to make and/or process
foods, feeds, nutritional supplements, textiles, detergents and the
like. The xylanases and/or a glucanases of the invention can be
used in pharmaceutical compositions and dietary aids.
[0172] Xylanases and/or a glucanases of the invention are
particularly useful in baking, animal feed, beverage and wood, wood
pulp, Kraft pulp, paper, paper product or paper pulp processes. In
one aspect, an enzyme of the invention is thermotolerant and/or
tolerant of high and/or low pH conditions. For example, in one
aspect, a xylanase, a mannanase and/or a glucanase of the invention
retains activity under conditions comprising a temperature of at
least about 80.degree. C., 85.degree. C., 86.degree. C., 87.degree.
C., 88.degree. C., 89.degree. C., 90.degree. C., 91.degree. C.,
92.degree. C., 93.degree. C., 94.degree. C., 95.degree. C.,
96.degree. C., 97.degree. C., 98.degree. C., 99.degree. C.,
100.degree. C., 101.degree. C., 102.degree. C., 103.degree. C.,
103.5.degree. C., 104.degree. C., 105.degree. C., 107.degree. C.,
108.degree. C., 109.degree. C. or 110.degree. C., or more, and a
basic pH of at least about pH 11, or more.
[0173] The invention provides isolated, synthetic or recombinant
nucleic acids comprising a nucleic acid encoding at least one
polypeptide having a xylanase (e.g., an endoxylanase), a mannanase
and/or a glucanase activity, or other activity as described herein,
wherein the nucleic acid comprises a sequence having at least about
50% to 99%, or more, or complete (100%) sequence identity
(homology) to SEQ ID NO:1 having one or more nucleotide residue
changes (modifications, mutations) as set forth in Table 1, and as
described herein, over a region of between about 10 to 2500, or
more residues, or the full length of a cDNA, transcript (mRNA) or
gene. Nucleic acids of the invention includes those encoding a
polypeptide of this invention, which includes, e.g., SEQ ID NO:2
having one or more amino acid residue changes (mutations) as set
forth in Table 1 and as described herein, and also including a
genus of polypeptides having various sequence identities based on
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 or SEQ ID NO:24.
TABLE-US-00002 TABLE 1 Other Codons Clone Name for same (Xyl 11 and
Xyl Original Changed AA original changed Average Average WT-
Average % of 11 mutants) Residue Codon To change AA to AA A560 A560
WT Xyl 11 WT 100 100 Xyl 11 mutant 1 108 TTC AAG AAA F K 0.655
0.740 87.5 Xyl 11 mutant 2 21 TTC TAC TAT F Y 0.815 0.813 100.3 Xyl
11 mutant 3 189 TCC CAG CAA S Q 0.909 0.906 100.4 Xyl 11 mutant 4
150 GTA GCC GCT, V A 0.634 0.623 101.8 GCA, GCG Xyl 11 mutant 5 9
CCC GAC GAT P D 0.570 0.556 102.5 Xyl 11 mutant 6 188 AGC GAG GAA S
E 0.749 0.722 104.1 Xyl 11 mutant 7 125 CAG TAC TAT Q Y 0.936 0.902
104.2 Xyl 11 mutant 8 73 GGC GTC GTT, G V 0.769 0.736 104.6 GTA,
GTG Xyl 11 mutant 9 73 GGC GAG GAA G E 0.965 0.902 106.6 Xyl 11
mutant 10 33 CTG GCG GCT, L A 0.795 0.736 108.0 GCC, GCA Xyl 11
mutant 11 38 CGT CAC CAT R H 0.969 0.894 108.3 Xyl 11 mutant 12 17
TTC GTC GTT, F V 0.981 0.901 109.0 GTA, GTG Xyl 11 mutant 13 63 ATC
GTC GTT, I V 0.996 0.901 110.5 GTA, GTG Xyl 11 mutant 14 44 AGC ACG
ACT, S T 1.008 0.894 112.7 ACC, ACA Xyl 11 mutant 15 73 GGC TAC TAT
G Y 0.958 0.813 118.2 Xyl 11 mutant 16 4 ACC CAC CAT T H 1.057
0.881 119.9 Xyl 11 mutant 17 4 ACC CGC CGT, T R 1.087 0.906 120.1
CGA, CGG, AGA, AGG Xyl 11 mutant 18 4 ACC AAC AAT T N 1.092 0.881
123.7
[0174] The invention provides variants of polynucleotides or
polypeptides of the invention, which comprise sequences modified at
one or more base pairs, codons, introns, exons, or amino acid
residues (respectively) yet still retain the biological activity of
a xylanase, a mannanase and/or a glucanase 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 (e.g., GeneReassembly,
see, e.g., U.S. Pat. No. 6,537,776), GSSM and any combination
thereof.
[0175] 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.
[0176] 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.
[0177] The term "synthetic ligation reassembly" or "SLR" includes a
method of ligating oligonucleotide fragments in a non-stochastic
fashion, and explained in detail, below.
Generating and Manipulating Nucleic Acids
[0178] The invention provides nucleic acids (e.g., nucleic acids
encoding polypeptides having glycosyl hydrolase activity, e.g., a
xylanase, a mannanase and/or a glucanase activity; including
enzymes having at least one sequence modification of an exemplary
nucleic acid sequence of the invention (as defined above), wherein
the sequence modification comprises one or more nucleotide residue
changes (or the equivalent thereof) as set forth in Table 1, or at
least one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or
eighteen, or some or all of the following nucleotide residue
changes: the codon encoding amino acid residue 4 changed from ACC
to AAC; the codon encoding amino acid residue 4 changed from ACC to
CGC; the codon encoding amino acid residue 4 changed from ACC to
CAC; the codon encoding amino acid residue 9 changed from CCC to
GAC; the codon encoding amino acid residue 17 changed from TTC to
GTC; the codon encoding amino acid residue 21 changed from TTC to
TAC; the codon encoding amino acid residue 33 changed from CTG to
GCG; the codon encoding amino acid residue 38 changed from CGT to
CAC; the codon encoding amino acid residue 44 changed from AGC to
ACG; the codon encoding amino acid residue 63 changed from ATC to
GTC; the codon encoding amino acid residue 73 changed from GGC to
TAC; the codon encoding amino acid residue 73 changed from GGC to
GAG; the codon encoding amino acid residue 73 changed from GGC to
GTC; the codon encoding amino acid residue 108 changed from TTC to
AAG; the codon encoding amino acid residue 125 changed from CAG to
TAC; the codon encoding amino acid residue 150 changed from GTA to
GCC; the codon encoding amino acid residue 188 changed from AGC to
GAG; and/or, the codon encoding amino acid residue 189 changed from
TCC to CAG, including expression cassettes such as expression
vectors, encoding the polypeptides of the invention.
[0179] The invention also includes methods for discovering new
xylanase, mannanase and/or glucanase sequences using the nucleic
acids of the invention. The invention also includes methods for
inhibiting the expression of xylanase, mannanase and/or glucanase
genes, transcripts and polypeptides using the nucleic acids of the
invention. Also provided are methods for modifying the nucleic
acids of the invention by, e.g., synthetic ligation reassembly,
optimized directed evolution system and/or saturation
mutagenesis.
[0180] 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.
[0181] In one aspect, the invention also provides xylanase- and/or
glucanase-encoding nucleic acids with a common novelty in that they
are derived from an environmental source, or a bacterial source, or
an archaeal source.
[0182] 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.
[0183] One aspect of the invention is an isolated nucleic acid
comprising one of the sequences of the invention and sequences
substantially identical thereto, 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 consecutive bases of
one of the sequences of a Sequence of the invention (or the
sequences complementary thereto). The isolated, 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 nucleic acids may comprise
RNA.
[0184] Accordingly, another aspect of the invention is an isolated
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 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 a fragment thereof or
may be different coding sequences which encode one of the
polypeptides of the invention, sequences substantially identical
thereto and fragments having at least 5, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150 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, for example, on page
214 of B. Lewin, Genes VI, Oxford University Press, 1997.
[0185] The isolated nucleic acid which encodes one of the
polypeptides of the invention and sequences substantially identical
thereto, may include, but is not limited to: only the coding
sequence of a nucleic acid of the invention and sequences
substantially identical thereto 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
only the coding sequence for the polypeptide as well as a
polynucleotide which includes additional coding and/or non-coding
sequence.
[0186] Alternatively, the nucleic acid sequences of the invention
and sequences substantially identical thereto, may be 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 of the invention
and sequences substantially identical thereto. 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.
[0187] 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 and sequences substantially identical thereto.
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 and sequences substantially
identical thereto (or the sequences complementary thereto) under
conditions of high, moderate, or low stringency as provided
herein.
General Techniques
[0188] The nucleic acids used to practice this invention, whether
RNA, iRNA, 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.,
glycosyl hydrolases of the invention) 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.
[0189] 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.
[0190] 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).
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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 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 that 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.
[0195] 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.
[0196] 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). "Operably linked" as used herein refers to
a functional relationship between two or more nucleic acid (e.g.,
DNA) segments. Typically, it refers 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.
[0197] 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
xylanase, mannanase and/or glucanase 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, in one aspect, with other sequences, e.g.,
transcription termination signals. Additional factors necessary or
helpful in effecting expression may also be used, e.g., enhancers.
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 that 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 in one
aspect 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.
[0198] As used herein, the term "promoter" includes all sequences
capable of driving transcription of a coding sequence in a cell,
e.g., a plant 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
typically 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.
[0199] "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 that 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.
[0200] As used herein, the term "isolated" means that the material
(e.g., a nucleic acid, a polypeptide, a cell) 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
104-106 fold. However, the term "purified" also includes nucleic
acids that 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, typically two or three orders and
more typically four or five orders of magnitude.
[0201] As used herein, the term "recombinant" means that the
nucleic acid is adjacent to a "backbone" nucleic acid to which it
is not adjacent in its natural environment. Additionally, to be
"enriched" the nucleic acids will represent 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. Typically, the enriched nucleic acids represent
15% or more of the number of nucleic acid inserts in the population
of recombinant backbone molecules. More typically, the enriched
nucleic acids represent 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 90% or more of the
number of nucleic acid inserts in the population of recombinant
backbone molecules.
[0202] "Plasmids" are designated by a lower case "p" preceded
and/or followed by capital letters and/or numbers. The starting
plasmids herein are either commercially available, publicly
available on an unrestricted basis, or can be constructed from
available plasmids in accord with published procedures. In
addition, equivalent plasmids to those described herein are known
in the art and will be apparent to the ordinarily skilled artisan.
"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.
[0203] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37 C are ordinarily used, but may vary in
accordance with the supplier's instructions. After digestion, gel
electrophoresis may be performed to isolate the desired
fragment.
[0204] "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 particular, 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.
[0205] For example, hybridization under high stringency conditions
could occur in about 50% formamide at about 37.degree. C. to
42.degree. C. Hybridization could occur under reduced stringency
conditions in about 35% to 25% formamide at about 30.degree. C. to
35.degree. C. In particular, hybridization could occur under high
stringency conditions at 42.degree. C. in 50% formamide,
5.times.SSPE, 0.3% SDS and 200 ug/ml sheared and denatured salmon
sperm DNA. Hybridization could occur under reduced stringency
conditions as described above, 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.
Transcriptional and Translational Control Sequences
[0206] 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 lad, 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. 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. 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 lacI
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 .A-inverted. 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.
Tissue-Specific Plant Promoters
[0207] The invention provides expression cassettes that can be
expressed in a tissue-specific manner, e.g., that can express a
xylanase, mannanase and/or glucanase of the invention in a
tissue-specific manner. The invention also provides plants or seeds
that express a xylanase, mannanase and/or glucanase 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.
[0208] 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.
[0209] 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).
[0210] Alternatively, the plant promoter may direct expression of
xylanase- and/or glucanase-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).
[0211] Tissue-specific promoters can 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.
[0212] Alternatively, 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).
[0213] 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, wheat, potato or other
crops, inducible at any stage of development of the crop.
[0214] 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, 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.
[0215] 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
xylanase- and/or glucanase-producing nucleic acids of the invention
will allow the grower to select plants with the optimal xylanase,
mannanase and/or glucanase 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).
[0216] 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.
[0217] The term "plant" (e.g., as in a transgenic plant or plant
seed of this invention, or plant promoter used in a vector of the
invention) includes whole plants, plant parts (e.g., leaves, stems,
flowers, roots, etc.), plant protoplasts, seeds and plant cells and
progeny of same; the classes of plants that can be used to practice
this invention (including compositions and methods) can be as broad
as the class of higher plants, including plants amenable to
transformation techniques, including angiosperms (monocotyledonous
and dicotyledonous plants), as well as gymnosperms; also including
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,
such a vectors) of the invention. Transgenic plants of the
invention are also discussed, below.
Expression Vectors and Cloning Vehicles
[0218] The invention provides expression vectors and cloning
vehicles comprising nucleic acids of the invention, e.g., sequences
encoding the xylanases and/or glucanases 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 vectors (Qiagen), pBluescript 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.
[0219] 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.
[0220] 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 TRP1 gene. Promoter regions can be selected from any
desired gene using chloramphenicol transferase (CAT) vectors or
other vectors with selectable markers.
[0221] Vectors for expressing the polypeptide or fragment thereof
in eukaryotic cells can 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 bp 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and the adenovirus
enhancers.
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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,
particularly antibiotic resistance, such as resistance to
kanamycin, G418, bleomycin, hygromycin, or herbicide resistance,
such as resistance to chlorosulfuron or Basta.
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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 preferably
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.
[0230] 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.
[0231] 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 bp 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin and the adenovirus
enhancers.
[0232] In addition, the expression vectors typically 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.
[0233] In some aspects, the nucleic acid encoding one of the
polypeptides of the invention and sequences substantially identical
thereto, or fragments comprising at least about 5, 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof is
assembled in appropriate phase with a leader sequence capable of
directing secretion of the translated polypeptide or fragment
thereof. The nucleic acid can encode a fusion polypeptide in which
one of the polypeptides of the invention and sequences
substantially identical thereto, or fragments comprising at least
5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 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.
[0234] 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.
[0235] 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).
Host Cells and Transformed Cells
[0236] The invention also provides transformed cells comprising a
nucleic acid sequence of the invention, e.g., a sequence encoding a
xylanase, a mannanase and/or a glucanase 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 within the genera Escherichia,
Bacillus, Streptomyces, Salmonella, Pseudomonas and Staphylococcus,
including, e.g., Escherichia coli, Lactococcus lactis, Bacillus
subtilis, Bacillus cereus, Salmonella typhimurium, Pseudomonas
fluorescens. Exemplary fungal cells include any species of
Aspergillus. Exemplary yeast cells include any species of Pichia,
Saccharomyces, Schizosaccharomyces, or Schwanniomyces, including
Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces
pombe. 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.
[0237] 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)).
[0238] 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 are can be
used.
[0239] 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.
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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.
[0244] 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.
[0245] The invention provides a method for overexpressing a
recombinant xylanase, mannanase and/or glucanase 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 a 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, or a subsequence thereof. 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.
[0246] 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.
[0247] Additional details regarding this approach are in the public
literature and/or are known to the skilled artisan. In a particular
non-limiting exemplification, such publicly available literature
includes EP 0659215 (WO 9403612 A1) (Nevalainen et al.); Lapidot,
A., Mechaly, A., Shoham, Y., "Overexpression and single-step
purification of a thermostable xylanase from Bacillus
stearothermophilus T-6," J. Biotechnol. November 51:259-64 (1996);
Luthi, E., Jasmat, N. B., Bergquist, P. L., "Xylanase from the
extremely thermophilic bacterium Caldocellum saccharolyticum:
overexpression of the gene in Escherichia coli and characterization
of the gene product," Appl. Environ. Microbiol. September
56:2677-83 (1990); and Sung, W. L., Luk, C. K., Zahab, D. M.,
Wakarchuk, W., "Overexpression of the Bacillus subtilis and
circulans xylanases in Escherichia coli," Protein Expr. Purif. June
4:200-6 (1993), although these references do not teach the
inventive enzymes of the instant application.
[0248] 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. As representative
examples of appropriate hosts, there may be mentioned: bacterial
cells, such as E. coli, Streptomyces, Bacillus subtilis, Bacillus
cereus, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces and Staphylococcus, fungal cells,
such as Aspergillus, yeast such as any species of Pichia,
Saccharomyces, Schizosaccharomyces, Schwanniomyces, including
Pichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces
pombe, insect cells such as Drosophila S2 and Spodoptera Sf9,
animal cells such as CHO, COS or Bowes melanoma and adenoviruses.
The selection of an appropriate host is within the abilities of
those skilled in the art.
[0249] 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)).
[0250] 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.
[0251] Cells are typically 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.
[0252] 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.
[0253] 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.
[0254] Alternatively, the polypeptides of amino acid sequences 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 synthetically produced by conventional peptide synthesizers. 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.
[0255] Cell-free translation systems can also be employed to
produce one of the polypeptides of amino acid sequences 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 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.
Amplification of Nucleic Acids
[0256] In practicing the invention, nucleic acids of the invention
and nucleic acids encoding the xylanases and/or glucanases of the
invention, or modified nucleic acids of the invention, can be
reproduced by amplification. 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.
[0257] In one aspect, the invention provides a nucleic acid
amplified by a 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 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 residues of the complementary strand.
[0258] The invention provides an amplification primer sequence pair
for amplifying a nucleic acid encoding a polypeptide having a
xylanase, mannanase and/or glucanase 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 consecutive
bases of the sequence, or about 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, or 25 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 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
residues of the complementary strand of the first member. The
invention provides xylanases and/or glucanases generated by
amplification, e.g., polymerase chain reaction (PCR), using an
amplification primer pair of the invention. The invention provides
methods of making a xylanase, mannanase and/or glucanase 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.
[0259] 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.
[0260] 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 the Degree of Sequence Identity
[0261] 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 to an
exemplary nucleic acid of the invention (as defined above) 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. 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.
[0262] 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.
[0263] 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. Typically, the substantial identity exists
over a region of at least about 100 residues and most commonly the
sequences are substantially identical over at least about 150-200
residues. In some aspects, the sequences are substantially
identical over the entire length of the coding regions.
[0264] 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, particularly when such a substitution
occurs at a site that is not the active site of the molecule and
provided that the polypeptide essentially retains its functional
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 xylanase, mannanase and/or glucanase 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
xylanase, mannanase and/or glucanase biological activity can be
removed. Modified polypeptide sequences of the invention can be
assayed for xylanase, mannanase and/or glucanase biological
activity by any number of methods, including contacting the
modified polypeptide sequence with a xylanase, mannanase and/or
glucanase 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 xylanase, mannanase and/or glucanase polypeptide with
the substrate.
[0265] 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 consecutive nucleotides of an exemplary sequence of the
invention and sequences substantially identical thereto. Nucleic
acid sequences of the invention can comprise homologous sequences
and fragments of nucleic acid sequences and sequences substantially
identical thereto, refer to a 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 sequence
identity (homology) to these sequences. Homology 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 and sequences substantially identical
thereto, 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.
[0266] Various sequence comparison programs identified elsewhere in
this patent specification are particularly contemplated for use in
this aspect of the invention. Protein and/or nucleic acid sequence
homologies may be evaluated using any of the variety of sequence
comparison algorithms and programs known in the art. Such
algorithms and programs include, but are by no means limited to,
TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (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 et al., 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).
[0267] Homology or identity is often 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. 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.
[0268] For sequence comparison, typically 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.
[0269] 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. 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 organization and are accessible via the
internet.
[0270] One example of a useful algorithm is BLAST and BLAST 2.0
algorithms, 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.
[0271] 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 preferably less than about 0.01 and
most preferably less than about 0.001.
[0272] 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: [0273] (1) BLASTP and BLAST3 compare an amino acid
query sequence against a protein sequence database; [0274] (2)
BLASTN compares a nucleotide query sequence against a nucleotide
sequence database; [0275] (3) BLASTX compares the six-frame
conceptual translation products of a query nucleotide sequence
(both strands) against a protein sequence database; [0276] (4)
TBLASTN compares a query protein sequence against a nucleotide
sequence database translated in all six reading frames (both
strands); and [0277] (5) TBLASTX compares the six-frame
translations of a nucleotide query sequence against the six-frame
translations of a nucleotide sequence database.
[0278] 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 preferably obtained from a
protein or nucleic acid sequence database. High-scoring segment
pairs are preferably identified (i.e., aligned) by means of a
scoring matrix, many of which are known in the art. Preferably, the
scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science
256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61,
1993). Less preferably, 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.
[0279] 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
[0280] To determine and identify sequence identities, 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.
[0281] Accordingly, 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. 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.
[0282] The polypeptides of the invention include the exemplary
sequences of the invention, and sequences substantially identical
thereto, and fragments of any of the preceding sequences.
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 to an exemplary sequence of the invention, e.g., a
polypeptide sequences of the invention.
[0283] Homology may be determined using any of the computer
programs and parameters described herein, including FASTA version
3.0t78 with the default parameters or with any modified parameters.
The homologous sequences may be obtained using any of the
procedures described herein or may result from the correction of a
sequencing error. The polypeptide fragments comprise at least about
10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300,
350, 400, 450, 500 or more consecutive amino acids of the
polypeptides of the invention and sequences substantially identical
thereto. It will be appreciated that the polypeptide codes of amino
acid sequences of the invention and sequences substantially
identical thereto, can be represented in the traditional single
character format or three letter format (See Stryer, Lubert.
Biochemistry, 3rd supra) or in any other format which relates the
identity of the polypeptides in a sequence.
[0284] 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 and sequences
substantially identical thereto, one or more of the polypeptide
sequences of the invention and sequences substantially identical
thereto. 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 sequences of the invention and sequences substantially
identical thereto.
[0285] Another aspect of the invention is a computer readable
medium having recorded thereon one or more of the nucleic acid
sequences of the invention and sequences substantially identical
thereto. Another aspect of the invention is a computer readable
medium having recorded thereon one or more of the polypeptide
sequences of the invention and sequences substantially identical
thereto. 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 sequences as set forth above.
[0286] 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.
[0287] Aspects of the invention include systems (e.g., internet
based systems), particularly 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 and sequences substantially identical thereto, or a
polypeptide sequence as set forth in the amino acid sequences of
the invention. The computer system 100 typically 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.
[0288] Typically 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.
[0289] In one particular aspect, the computer system 100 includes a
processor 105 connected to a bus which is connected to a main
memory 115 (preferably 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.
[0290] 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.
[0291] 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.
[0292] Software for accessing and processing the nucleotide
sequences of a nucleic acid sequence of the invention and sequences
substantially identical thereto, or a polypeptide sequence of the
invention and sequences substantially identical thereto, (such as
search tools, compare tools and modeling tools etc.) may reside in
main memory 115 during execution.
[0293] In some aspects, the computer system 100 may further
comprise a sequence comparison algorithm for comparing a nucleic
acid sequence of the invention and sequences substantially
identical thereto, or a polypeptide sequence of the invention and
sequences substantially identical thereto, 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 and sequences
substantially identical thereto, or a polypeptide sequence of the
invention and sequences substantially identical thereto, stored on
a computer readable medium to reference sequences stored on a
computer readable medium to identify homologies or structural
motifs.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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 and sequences
substantially identical thereto, or a polypeptide sequence of the
invention and sequences substantially identical thereto, 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 and sequences substantially
identical thereto, or a polypeptide sequence of the invention and
sequences substantially identical thereto 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 of
nucleic acid sequences of the invention and sequences substantially
identical thereto, or a polypeptide sequence of the invention and
sequences substantially identical thereto, 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 and sequences substantially identical thereto, or the
polypeptide sequences of the invention and sequences substantially
identical thereto.
[0301] Another aspect of the invention is a method for determining
the level of homology between a nucleic acid sequence of the
invention and sequences substantially identical thereto, or a
polypeptide sequence of the invention and sequences substantially
identical thereto 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.
[0302] 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
preferably in the single letter amino acid code so that the first
and sequence sequences can be easily compared.
[0303] 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.
[0304] 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%.
[0305] 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 a nucleic acid sequence of the invention and sequences
substantially identical thereto, differs from a reference nucleic
acid sequence at one or more positions. In one aspect 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 and sequences substantially identical thereto. In one
aspect, the computer program may be a program which determines
whether a nucleic acid sequence of the invention and sequences
substantially identical thereto, contains a single nucleotide
polymorphism (SNP) with respect to a reference nucleotide
sequence.
[0306] Another aspect of the invention is a method for determining
whether a nucleic acid sequence of the invention and sequences
substantially identical thereto, 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 sequences
substantially identical thereto 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.
[0307] 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 and sequences substantially identical thereto.
[0308] An "identifier" refers to one or more programs which
identifies certain features within a nucleic acid sequence of the
invention and sequences substantially identical thereto, or a
polypeptide sequence of the invention and sequences substantially
identical thereto. In one aspect, the identifier may comprise a
program which identifies an open reading frame in a nucleic acid
sequence of the invention and sequences substantially identical
thereto.
[0309] 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.
[0310] 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.
[0311] 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.
[0312] Accordingly, another aspect of the invention is a method of
identifying a feature within a nucleic acid sequence of the
invention and sequences substantially identical thereto, or a
polypeptide sequence of the invention and sequences substantially
identical thereto, 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 of the nucleic
acid sequences of the invention and sequences substantially
identical thereto, or the polypeptide sequences of the invention
and sequences substantially identical thereto, through the use of
the computer program and identifying features within the nucleic
acid codes or polypeptide codes with the computer program.
[0313] A nucleic acid sequence of the invention and sequences
substantially identical thereto, or a polypeptide sequence of the
invention and sequences substantially identical thereto, 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 and sequences substantially identical thereto, or a
polypeptide sequence of the invention and sequences substantially
identical thereto, 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 and sequences substantially identical
thereto, or a polypeptide sequence of the invention and sequences
substantially identical thereto. 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 and sequences substantially identical thereto, or the
polypeptide sequences of the invention and sequences substantially
identical thereto.
[0314] The programs and databases which may be used include, but
are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular
Applications Group), GeneMine (Molecular Applications Group), Look
(Molecular Applications Group), MacLook (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 (Molecular Simulations
Inc.), Catalyst/SHAPE (Molecular Simulations Inc.),
Cerius.sup.2.DBAccess (Molecular Simulations Inc.), HypoGen
(Molecular Simulations Inc.), Insight II, (Molecular Simulations
Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular
Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,
(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations
Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular
Simulations Inc.), ISIS (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.
[0315] 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
[0316] The invention provides isolated, synthetic or recombinant
nucleic acids that hybridize under stringent conditions to an
exemplary sequence of the invention. 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.
[0317] 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, iRNA (single or double stranded), antisense
or sequences encoding antibody binding peptides (epitopes), motifs,
active sites and the like.
[0318] 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.
[0319] 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 ug/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% formamide at a reduced temperature of
35.degree. C.
[0320] 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.
[0321] 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.
[0322] All of the foregoing hybridizations would be considered to
be under conditions of high stringency.
[0323] 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.
[0324] Nucleic acids which have hybridized to the probe are
identified by autoradiography or other conventional techniques.
[0325] The above procedure may be modified to identify nucleic
acids having decreasing levels of homology to the probe sequence.
For example, to obtain nucleic acids of decreasing 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.
[0326] 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.
[0327] However, the selection of a hybridization format is not
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.
[0328] These methods may be used to isolate nucleic acids of the
invention. For example, the preceding methods may be used to
isolate nucleic acids having a sequence with at least about 97%, at
least 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% homology to a nucleic acid sequence selected from the group
consisting of one of the sequences of the invention and sequences
substantially identical thereto, 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. 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 or
the sequences complementary thereto.
[0329] 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% homology to a polypeptide having the sequence of one of amino
acid sequences 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
[0330] The invention also provides nucleic acid probes that can be
used, e.g., for identifying nucleic acids encoding a polypeptide
with a xylanase, mannanase and/or glucanase activity or fragments
thereof or for identifying xylanase, mannanase and/or glucanase
genes. In one aspect, the probe comprises at least 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.
[0331] The isolated nucleic acids of the invention and sequences
substantially identical thereto, 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 consecutive bases of
one of the sequences of the invention and sequences substantially
identical thereto, 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.
[0332] 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.
[0333] 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.
[0334] 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).
[0335] 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). Typically,
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.
[0336] Probes derived from sequences near the ends of the sequences
of the invention and sequences substantially identical thereto, may
also be used in chromosome walking procedures to identify clones
containing genomic sequences located adjacent to the sequences of
the invention and sequences substantially identical thereto. Such
methods allow the isolation of genes which encode additional
proteins from the host organism.
[0337] The isolated nucleic acids of the invention and sequences
substantially identical thereto, 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 consecutive bases of
one of the sequences of the invention and sequences substantially
identical thereto, or the sequences complementary thereto may be
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.
[0338] 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:
[0339] 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.
[0340] 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.
[0341] Prehybridization may be carried out in 6.times.SSC,
5.times.Denhardt's reagent, 0.5% SDS, 100 .mu.g/ml denatured
fragmented salmon sperm DNA or 6.times.SSC, 5.times.Denhardt's
reagent, 0.5% SDS, 100 .mu.g/ml denatured fragmented salmon sperm
DNA, 50% formamide. The formulas for SSC and Denhardt's solutions
are listed in Sambrook et al., supra.
[0342] 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. 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. Typically, 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 Glycosyl Hydrolases
[0343] The invention provides nucleic acids complementary to (e.g.,
antisense sequences to) the nucleic acids of the invention, e.g.,
xylanase- and/or glucanase-encoding nucleic acids. Antisense
sequences are capable of inhibiting the transport, splicing or
transcription of xylanase- and/or glucanase-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 particularly useful set of inhibitors provided by the
present invention includes oligonucleotides which are able to
either bind xylanase, mannanase and/or glucanase gene or message,
in either case preventing or inhibiting the production or function
of xylanase, mannanase and/or glucanase. The association can be
through sequence specific hybridization. Another useful class of
inhibitors includes oligonucleotides which cause inactivation or
cleavage of xylanase, mannanase and/or glucanase 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 xylanase, mannanase and/or glucanase expression on a
nucleic acid and/or protein level, e.g., antisense, iRNA and
ribozymes comprising xylanase, mannanase and/or glucanase sequences
of the invention and the anti-xylanase and/or anti-glucanase
antibodies of the invention.
[0344] Inhibition of xylanase, mannanase and/or glucanase
expression can have a variety of industrial, medical,
pharmaceutical, research, food and feed and food and feed
supplement processing and other applications and processes. For
example, inhibition of xylanase, mannanase and/or glucanase
expression can slow or prevent spoilage. Spoilage can occur when
polysaccharides, e.g., structural polysaccharides, are
enzymatically degraded. This can lead to the deterioration, or rot,
of fruits and vegetables. In one aspect, use of compositions of the
invention that inhibit the expression and/or activity of xylanases
and/or glucanases, e.g., antibodies, antisense oligonucleotides,
ribozymes and RNAi, 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 and RNAi 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 xylanase,
mannanase and/or glucanase gene of the invention).
[0345] The compositions of the invention for the inhibition of
xylanase, mannanase and/or glucanase 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.
Antisense Oligonucleotides
[0346] The invention provides antisense oligonucleotides capable of
binding xylanase, mannanase and/or glucanase message which can
inhibit xylan hydrolase activity (e.g., catalyzing hydrolysis of
internal .beta.-1,4-xylosidic linkages) by targeting mRNA.
Strategies for designing antisense oligonucleotides are well
described in the scientific and patent literature, and the skilled
artisan can design such xylanase, mannanase and/or glucanase
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.
[0347] 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.
[0348] 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 xylanase, mannanase and/or glucanase sequences of the
invention (see, e.g., Gold (1995) J. of Biol. Chem.
270:13581-13584).
Inhibitory Ribozymes
[0349] The invention provides ribozymes capable of binding
xylanase, mannanase and/or glucanase message. These ribozymes can
inhibit xylanase, mannanase and/or glucanase activity by, e.g.,
targeting mRNA. Strategies for designing ribozymes and selecting
the xylanase- and/or glucanase-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.
[0350] 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 addition, a ribozyme is typically 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.
[0351] 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.
RNA Interference (RNAi)
[0352] In one aspect, the invention provides an RNA inhibitory
molecule, a so-called "RNAi" molecule, comprising a xylanase,
mannanase and/or glucanase enzyme sequence of the invention. The
RNAi molecule can comprise a double-stranded RNA (dsRNA) molecule,
e.g., siRNA, miRNA and/or short hairpin RNA (shRNA) molecules. The
RNAi molecule, e.g., siRNA (small inhibitory RNA) can inhibit
expression of a xylanase, mannanase and/or glucanase enzyme gene,
and/or miRNA (micro RNA) to inhibit translation of a xylanase,
mannanase and/or glucanase message. In one aspect, the RNAi
molecule, e.g., siRNA and/or miRNA, is about 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 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.
[0353] In one aspect, intracellular introduction of the RNAi is by
internalization of a target cell specific ligand bonded to an RNA
binding protein comprising an RNAi (e.g., microRNA) is adsorbed.
The ligand is specific to a unique target cell surface antigen. The
ligand can be spontaneously internalized after binding to the cell
surface antigen. If the unique cell surface antigen is not
naturally internalized after binding to its ligand, internalization
can be promoted by the incorporation of an arginine-rich peptide,
or other membrane permeable peptide, into the structure of the
ligand or RNA binding protein or attachment of such a peptide to
the ligand or RNA binding protein. See, e.g., U.S. Patent App. Pub.
Nos. 20060030003; 20060025361; 20060019286; 20060019258. In one
aspect, the invention provides lipid-based formulations for
delivering, e.g., introducing nucleic acids of the invention as
nucleic acid-lipid particles comprising an RNAi molecule to a cell,
see e.g., U.S. Patent App. Pub. No. 20060008910.
[0354] 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
[0355] The invention provides methods of generating variants of the
nucleic acids of the invention, e.g., those encoding a xylanase,
mannanase and/or glucanase. These methods can be repeated or used
in various combinations to generate xylanases and/or glucanases
having an altered or different activity or an altered or different
stability from that of a xylanase, mannanase and/or glucanase
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.
[0356] 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.
[0357] 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 (e.g., GeneReassembly,
see, e.g., U.S. Pat. No. 6,537,776), 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, and/or a combination of
these and other methods.
[0358] 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.
[0359] 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" Ann. 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).
[0360] 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.
[0361] 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."
[0362] 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 Ser. No.
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.
[0363] Non-stochastic, or "directed evolution," methods include,
e.g., saturation mutagenesis (GSSM), synthetic ligation reassembly
(SLR), or a combination thereof are used to modify the nucleic
acids of the invention to generate xylanases and/or glucanases 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 xylan 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.
Gene Site Saturation Mutagenesis (GSSM)
[0364] 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 xylanase,
mannanase and/or glucanase 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, in one aspect, 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.
[0365] 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.
[0366] 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 in one aspect 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.
[0367] In one aspect, each saturation mutagenesis reaction vessel
contains polynucleotides encoding at least 20 progeny polypeptide
(e.g., xylanases and/or glucanases) 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 xylan hydrolysis activity under alkaline or acidic
conditions), it can be sequenced to identify the correspondingly
favorable amino acid substitution contained therein.
[0368] 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.
[0369] 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.
[0370] 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 preferably 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.
[0371] 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.
[0372] In a particular exemplification, 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.
[0373] 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.
[0374] It is appreciated, however, that the use of a degenerate
triplet (such as N,N,G/T or an N,N, G/C triplet sequence) as
disclosed in the instant invention 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.
[0375] This invention also provides for the use of nondegenerate
oligos, which can in one aspect 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.
[0376] 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.
[0377] It is appreciated that 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.
[0378] Thus, in a non-limiting exemplification, this 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.
[0379] 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 preferably 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 (preferably a subset
totaling from 15 to 100,000) to mutagenesis. Preferably, 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 are preferably
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).
[0380] In a general sense, saturation mutagenesis is comprised of
mutagenizing a complete set of mutagenic cassettes (wherein each
cassette is preferably about 1-500 bases in length) in defined
polynucleotide sequence to be mutagenized (wherein the sequence to
be mutagenized is preferably 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.
[0381] 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.
[0382] In one exemplification 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.
Synthetic Ligation Reassembly (SLR)
[0383] 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.,
xylanases and/or glucanases, or antibodies of the invention, with
new or altered properties.
[0384] 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: (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.
[0385] 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.
[0386] 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.
[0387] 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 preferably 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 preferably 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.
[0388] 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.
[0389] 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 preferably 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.
Synthetic Gene Reassembly
[0390] In one aspect, the present invention provides a
non-stochastic method termed synthetic gene reassembly (e.g.,
GeneReassembly, see, e.g., U.S. Pat. No. 6,537,776), which differs
from stochastic shuffling in that the nucleic acid building blocks
are not shuffled or concatenated or chimerized randomly, but rather
are assembled non-stochastically.
[0391] 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.
[0392] 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.
[0393] In one aspect, synthetic gene reassembly comprises a method
of: 1) preparing a progeny generation of molecule(s) (including a
molecule comprising a polynucleotide sequence, e.g., a molecule
comprising a polypeptide coding sequence), that is mutagenized to
achieve at least one point mutation, addition, deletion, &/or
chimerization, from one or more ancestral or parental generation
template(s); 2) screening the progeny generation molecule(s), e.g.,
using a high throughput method, for at least one property of
interest (such as an improvement in an enzyme activity); 3) in one
aspect obtaining &/or cataloguing structural &/or and
functional information regarding the parental &/or progeny
generation molecules; and 4) in one aspect repeating any of steps
1) to 3). In one aspect, there is generated (e.g., from a parent
polynucleotide template), in what is termed "codon site-saturation
mutagenesis," a progeny generation of polynucleotides, each having
at least one set of up to three contiguous point mutations (i.e.,
different bases comprising a new codon), such that every codon (or
every family of degenerate codons encoding the same amino acid) is
represented at each codon position. Corresponding to, and encoded
by, this progeny generation of polynucleotides, there is also
generated a set of progeny polypeptides, each having at least one
single amino acid point mutation. In a one aspect, there is
generated, in what is termed "amino acid site-saturation
mutagenesis", one such mutant polypeptide for each of the 19
naturally encoded polypeptide-forming alpha-amino acid
substitutions at each and every amino acid position along the
polypeptide. This yields, for each and every amino acid position
along the parental polypeptide, a total of 20 distinct progeny
polypeptides including the original amino acid, or potentially more
than 21 distinct progeny polypeptides if additional amino acids are
used either instead of or in addition to the 20 naturally encoded
amino acids
[0394] Thus, in another aspect, this approach is also serviceable
for generating mutants containing, in addition to &/or in
combination with the 20 naturally encoded polypeptide-forming
alpha-amino acids, other rare &/or not naturally-encoded amino
acids and amino acid derivatives. In yet another aspect, this
approach is also serviceable for generating mutants by the use of,
in addition to &/or in combination with natural or unaltered
codon recognition systems of suitable hosts, altered, mutagenized,
&/or designer codon recognition systems (such as in a host cell
with one or more altered tRNA molecules.
[0395] In yet another aspect, this invention relates to
recombination and more specifically to a method for preparing
polynucleotides encoding a polypeptide by a method of in vivo
re-assortment of polynucleotide sequences containing regions of
partial homology, assembling the polynucleotides to form at least
one polynucleotide and screening the polynucleotides for the
production of polypeptide(s) having a useful property.
[0396] In yet another aspect, this invention is serviceable for
analyzing and cataloguing, with respect to any molecular property
(e.g., an enzymatic activity) or combination of properties allowed
by current technology, the effects of any mutational change
achieved (including particularly saturation mutagenesis). Thus, a
comprehensive method is provided for determining the effect of
changing each amino acid in a parental polypeptide into each of at
least 19 possible substitutions. This allows each amino acid in a
parental polypeptide to be characterized and catalogued according
to its spectrum of potential effects on a measurable property of
the polypeptide.
[0397] In one aspect, an intron may be introduced into a chimeric
progeny molecule by way of a nucleic acid building block. Introns
often have consensus sequences at both termini in order to render
them operational. In addition to enabling gene splicing, introns
may serve an additional purpose by providing sites of homology to
other nucleic acids to enable homologous recombination. For this
purpose, and potentially others, it may be sometimes desirable to
generate a large nucleic acid building block for introducing an
intron. If the size is overly large easily generating by direct
chemical synthesis of two single stranded oligos, such a
specialized nucleic acid building block may also be generated by
direct chemical synthesis of more than two single stranded oligos
or by using a polymerase-based amplification reaction
[0398] 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.
[0399] Coupling can occur in a manner that does not make use of
every nucleotide in a participating overhang. The coupling is
particularly lively to survive (e.g., in a transformed host) if the
coupling reinforced by treatment with a ligase enzyme to form what
may be referred to as a "gap ligation" or a "gapped ligation". This
type of coupling can contribute to generation of unwanted
background product(s), but it can also be used advantageously
increase the diversity of the progeny library generated by the
designed ligation reassembly. Certain overhangs are able to undergo
self-coupling to form a palindromic coupling. A coupling is
strengthened substantially if it is reinforced by treatment with a
ligase enzyme. Lack of 5' phosphates on these overhangs can be used
advantageously to prevent this type of palindromic self-ligation.
Accordingly, this invention provides that nucleic acid building
blocks can be chemically made (or ordered) that lack a 5' phosphate
group. Alternatively, they can be removed, e.g., by treatment with
a phosphatase enzyme, such as a calf intestinal alkaline
phosphatase (CIAP), in order to prevent palindromic self-ligations
in ligation reassembly processes.
[0400] 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.
[0401] 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 xylanases and/or glucanases of the
present invention can be mutagenized in accordance with the methods
described herein.
[0402] 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.
[0403] Typically 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
preferably at almost all of the progenitor templates. Even more
preferably still a serviceable demarcation point is an area of
homology that is shared by all of the progenitor templates.
[0404] 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.
[0405] 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.
[0406] 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
preferably 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.
[0407] 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).
[0408] 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 in one aspect 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.
[0409] 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).
[0410] Accordingly, 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).
Preferably, 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.
[0411] 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 a 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.
[0412] The synthetic gene reassembly method of the invention
utilizes a plurality of nucleic acid building blocks, each of which
preferably 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 preferably one blunt
end and one overhang, or more preferably still two overhangs.
[0413] 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.
[0414] 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.
[0415] 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).
[0416] 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.
[0417] 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. The codon degeneracy can be 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.
[0418] 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.
[0419] The approach of using recombination within a mixed
population of genes can be useful for the generation of any useful
proteins, for example, interleukin I, antibodies, tPA and growth
hormone. 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
mutate ribozymes or aptamers.
[0420] 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.
Optimized Directed Evolution System
[0421] The invention provides a non-stochastic gene modification
system termed "optimized directed evolution system" to generate
polypeptides, e.g., xylanases and/or glucanases, or antibodies of
the invention, with new or altered properties. 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. 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.
[0422] 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.
[0423] 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.
[0424] One method for creating a chimeric progeny polynucleotide
sequence is to create oligonucleotides corresponding to fragments
or portions of each parental sequence. Each oligonucleotide
preferably 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. Additional
information can also be found, e.g., in U.S. Ser. No. 09/332,835;
U.S. Pat. No. 6,361,974.
[0425] 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.
[0426] 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.
[0427] 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.
[0428] 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 preferably 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. Ser. No. 09/332,835.
Determining Crossover Events
[0429] 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 preferably
performed in MATLAB.TM. (The Mathworks, Natick, Mass.) a
programming language and development environment for technical
computing.
Iterative Processes
[0430] In practicing the invention, these processes can be
iteratively repeated. For example, a nucleic acid (or, the nucleic
acid) responsible for an altered or new xylanase, mannanase and/or
glucanase phenotype is 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., xylanase, mannanase and/or glucanase
activity.
[0431] Similarly, if it is determined that a particular
oligonucleotide has no affect at all on the desired trait (e.g., a
new xylanase, mannanase and/or glucanase 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.
In Vivo Shuffling
[0432] In vivo shuffling of molecules is use in methods of the
invention that provide variants of polypeptides of the invention,
e.g., antibodies, xylanases, 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.
[0433] 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 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.
[0434] In vivo reassortment is focused 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.
[0435] Therefore, 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.
[0436] Repeated or "quasi-repeated" sequences play a role in
genetic instability. In the present invention, "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. 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.
[0437] 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.
[0438] Sequences can be assembled in a head to tail orientation
using any of a variety of methods, including the following: [0439]
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. [0440] 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. [0441] c) The inner
few bases of the primer could be thiolated and an exonuclease used
to produce properly tailed molecules.
[0442] 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:
[0443] 1) The use of vectors only stably maintained when the
construct is reduced in complexity. [0444] 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. [0445] 3) The recovery of vectors containing
interrupted genes which can be selected when insert size decreases.
[0446] 4) The use of direct selection techniques with an expression
vector and the appropriate selection.
[0447] 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.
[0448] The following example demonstrates a 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).
[0449] 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.
[0450] In one aspect (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.
[0451] 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.
[0452] 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'-fluro-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-f]-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.
[0453] 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.
Producing Sequence Variants
[0454] The invention also provides additional methods for making
sequence variants of the nucleic acid (e.g., xylanase) sequences of
the invention. The invention also provides additional methods for
isolating xylanases using the nucleic acids and polypeptides of the
invention. In one aspect, the invention provides for variants of a
xylanase 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.
[0455] 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 new nucleic acids which
encode polypeptides having characteristics which enhance their
value in industrial, medical, laboratory (research),
pharmaceutical, food and feed and food and feed supplement
processing and other applications and processes. 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.
[0456] For example, variants may be created using error prone PCR.
In error prone PCR, 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, D. W.,
et al., Technique, 1:11-15, 1989) and Caldwell, R. C. & Joyce
G. F., PCR Methods Applic., 2:28-33, 1992. 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 fmoles 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 MgCl2, 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.
[0457] Variants may also be 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.
Clones containing the mutagenized DNA are recovered and the
activities of the polypeptides they encode are assessed.
[0458] 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.
[0459] Still another method of generating variants is sexual PCR
mutagenesis. In 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.
[0460] Variants may also be 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".
[0461] 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.
[0462] 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 in Arkin,
A. P. and Youvan, D. C., PNAS, USA, 89:7811-7815, 1992.
[0463] 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 in Delegrave, S. and Youvan, D. C.,
Biotechnology Research, 11:1548-1552, 1993. Random and
site-directed mutagenesis are described in Arnold, F. H., Current
Opinion in Biotechnology, 4:450-455, 1993.
[0464] 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.
[0465] 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 invention are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code.
[0466] Conservative substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Typically seen as conservative substitutions are
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.
[0467] Other variants are those in which one or more of the amino
acid residues of the polypeptides of the invention includes a
substituent group.
[0468] Still 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).
[0469] 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.
[0470] In some aspects, the fragments, derivatives and analogs
retain the same biological function or activity as the polypeptides
of the invention and sequences substantially identical thereto. 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.
Optimizing Codons to Achieve High Levels of Protein Expression in
Host Cells
[0471] The invention provides methods for modifying
xylanase-encoding nucleic acids to modify codon usage. In one
aspect, the invention provides methods for modifying codons in a
nucleic acid encoding a xylanase to increase or decrease its
expression in a host cell. The invention also provides nucleic
acids encoding a xylanase modified to increase its expression in a
host cell, xylanase so modified, and methods of making the modified
xylanases. The method comprises identifying a "non-preferred" or a
"less preferred" codon in xylanase-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.
[0472] 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. 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 and Pseudomonas
fluorescens; gram positive bacteria, such as Lactobacillus gasseri,
Lactococcus lactis, Lactococcus cremoris, Bacillus subtilis.
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.
Other exemplary host cells include bacterial cells, such as E.
coli, Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella
typhimurium and various species within the genera Pseudomonas,
Streptomyces and Staphylococcus, fungal cells, such as Aspergillus,
yeast such as any species of Pichia, Saccharomyces,
Schizosaccharomyces, Schwanniomyces, including Pichia pastoris,
Saccharomyces cerevisiae, or Schizosaccharomyces pombe, insect
cells such as Drosophila S2 and Spodoptera Sf9, animal cells such
as CHO, COS or Bowes melanoma and adenoviruses. The selection of an
appropriate host is within the abilities of those skilled in the
art. Thus, the invention also includes nucleic acids and
polypeptides optimized for expression in these organisms and
species.
[0473] For example, the codons of a nucleic acid encoding a
xylanase 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 xylanase 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
[0474] The invention provides transgenic non-human animals
comprising a nucleic acid, a polypeptide (e.g., a xylanase), 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.
[0475] The transgenic non-human animals can be, e.g., goats,
rabbits, sheep, pigs, cows, rats, horses, dogs, fish and mice,
comprising the nucleic acids of the invention. These animals can be
used, e.g., as in vivo models to study xylanase activity, or, as
models to screen for agents that change the xylanase 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. 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, chickens, goats, fish 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 whose cells express proteins
related to the pathology of Alzheimer's disease. U.S. Pat. No.
6,187,992, describes making and using a transgenic mouse whose
genome comprises a disruption of the gene encoding amyloid
precursor protein (APP).
[0476] "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 xylanase of the
invention, or, a fusion protein comprising a xylanase of the
invention.
Transgenic Plants and Seeds
[0477] The invention provides transgenic plants and seeds
comprising a nucleic acid, a polypeptide (e.g., a xylanase), an
expression cassette or vector or a transfected or transformed cell
of the invention. The invention also provides plant products or
byproducts, e.g., fruits, oils, seeds, leaves, extracts and the
like, including any plant part, comprising a nucleic acid and/or a
polypeptide (e.g., a xylanase) of the invention, e.g., wherein the
nucleic acid or polypeptide of the invention is heterologous to the
plant, plant part, seed etc. The transgenic plant (which includes
plant parts, fruits, seeds etc.) 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.
[0478] 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 xylanase 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.
[0479] 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, 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
xylanase. The can change xylanase activity in a plant.
Alternatively, a xylanase 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.
[0480] 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.
[0481] 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.
[0482] 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.
[0483] In one aspect, making transgenic plants or seeds comprises
incorporating sequences of the invention and, in one aspect
(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.
[0484] 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.
[0485] 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.
[0486] 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.
[0487] 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.
[0488] In one aspect, the third step can involve selection and
regeneration of whole plants capable of transmitting the
incorporated target gene to the next generation. Such regeneration
techniques rely on manipulation of certain phytohormones in a
tissue culture growth medium, typically relying on 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.
[0489] 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 xylanase) of the invention. The desired
effects can be passed to future plant generations by standard
propagation means.
[0490] 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. Transgenic plants and seeds
of the invention can be any monocot or dicot, e.g., a monocot corn,
sugarcane, rice, wheat, barley, switchgrass or Miscanthus; or a
dicot oilseed crop, soy, canola, rapeseed, flax, cotton, palm oil,
sugar beet, peanut, tree, poplar or lupine.
[0491] In alternative embodiments, the nucleic acids of the
invention are expressed in plants (and/or their seeds) 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.
[0492] The invention also provides for transgenic plants (and/or
their seeds) to be used for producing large amounts of the
polypeptides (e.g., a xylanase 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).
[0493] Using known procedures, one of skill can screen for plants
(and/or their seeds) 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
[0494] In one aspect, the invention provides isolated, synthetic or
recombinant polypeptides and peptides having xylanase, a mannanase
and/or a glucanase activity, or polypeptides and peptides capable
of generating an antibody that specifically binds to a xylanase or
a glucanase, including an enzyme of this invention, including the
amino acid sequences of the invention, which include those 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% (complete) sequence identity to an exemplary
polypeptide of the invention (as defined above, including 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 and SEQ ID NO:24), or any polypeptide of this invention,
including for example SEQ ID NO:2 having one or more amino acid
residue changes (mutations) as set forth in Table 1 and as
described herein, also including a genus of polypeptides having
various sequence identities based on 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 or
SEQ ID NO:24; and these exemplary polypeptides have the following
enzymatic activity (e.g., the xylanase of SEQ ID NO:2, is encoded
e.g., by SEQ ID NO:1; the arabinofuranosidase of SEQ ID NO:14, is
encoded e.g., by SEQ ID NO:13, and the like):
TABLE-US-00003 SEQ ID NO: Name Activity 1, 2 xyl 11 Xylanase 11, 12
control xylanase Xylanase 13, 14 Ambinofuranosidase 15, 16 Xylanase
17, 18 Oligomerase 19, 20 B-glucosidase 21, 22 Ambinofuranosidase
23, 24 Beta-xylosidase 3, 4 Endoglucanase 5, 6 Oligomerase 7, 8
Cellobiohydrolase 9, 10 Cellobiohydrolase
[0495] The invention also provides enzyme-encoding nucleic acids
with a common novelty in that they encode a subset of xylanases, or
a clade, comprising the "X14 module". In one aspect, the invention
also provides enzyme-encoding nucleic acids with a common novelty
in that they encode a clade comprising the "X14 module" (see, e.g.,
J. Bacteriol. 2002 August; 184(15):4124-4133). X14-comprising
xylanase members include SEQ ID NO:2 having one or more amino acid
residue changes (mutations) as set forth in Table 1 and as
described herein.
[0496] In one aspect, the invention provides chimeric enzymes,
including xylanases, glucanases and/or glycosidases, having
heterologous carbohydrate-binding modules (CBMs), e.g., for use in
the processes of the invention and in various industrial, medical,
pharmaceutical, research, food and feed and food and feed
supplement processing and other applications. For example, in one
aspect the invention provides enzymes, e.g., hydrolases, including
glycosyl hydrolases (such as xylanases, glucanases) comprising one
or more CBMs of an enzyme of the invention, including the CBM-like
X14 module discussed above. In another aspect, CBMs, e.g., X14
modules, between different enzymes of the invention can be swapped;
or, alternatively, one or more CBMs of one or more enzymes of the
invention can be spliced into an enzyme, e.g., a hydrolase, e.g.,
any glycosyl hydrolase, such as a xylanase.
[0497] Glycosyl hydrolases that utilize insoluble substrates are
modular, usually comprising catalytic modules appended to one or
more non-catalytic carbohydrate-binding modules (CBMs). In nature,
CBMs are thought to promote the interaction of the glycosyl
hydrolase with its target substrate polysaccharide. For example, as
discussed above, X14 is a xylan binding module. Thus, the invention
provides chimeric enzymes having heterologous, non-natural
substrates; including chimeric enzymes having multiple substrates
by nature of their "spliced-in" heterologous CBMs, e.g., a
spliced-in X14 module of the invention--thus giving the chimeric
enzyme new specificity for xylan and galactan, or enhanced binding
to xylan and galactan. The heterologous CBMs of the chimeric
enzymes of the invention can be designed to be modular, i.e., to be
appended to a catalytic module or catalytic domain (e.g., an active
site), which also can be heterologous or can be homologous to the
enzyme.
[0498] Utilization of just the catalytic module of a xylanase or a
glucanase (e.g., an enzyme of the invention) has been shown to be
effective. Thus, the invention provides peptides and polypeptides
consisting of, or comprising, modular CBM/active site modules
(e.g., X14), which can be homologously paired or joined as chimeric
(heterologous) active site-CBM pairs. Thus, these chimeric
polypeptides/peptides of the invention can be used to improve or
alter the performance of an individual enzyme, e.g., a xylanase
enzyme. A chimeric catalytic module of the invention (comprising,
e.g., at least one CBM of the invention, e.g., X14) can be designed
to target the enzyme to particular regions of a substrate, e.g., to
particular regions of a pulp. For example, in one aspect, this is
achieved by making fusions of the xylanase and various CBMs (either
a xylanase of the invention with a heterologous CBM, or, a CBM of
the invention with another enzyme, e.g., a hydrolase, such as a
xylanase. For example, CBM4, CBM6, and CBM22 are known to bind
xylan and may enhance the effectiveness of the xylanase in pulp
biobleaching (see, e.g., Czjzek (2001) J. Biol. Chem.
276(51):48580-7, noting that CBM4, CBM6, and CBM22 are related and
CBM interact primarily with xylan). In another embodiment, fusion
of xylanase and CBM3a or CBM3b, which bind crystalline cellulose,
may help the xylanase penetrate the complex polysaccharide matrix
of pulp and reach inaccessible xylans. Any CBM can be used to
practice the instant invention, e.g., as reviewed by Boraston
(2004) Biochem. J. 382:769-781:
TABLE-US-00004 PDB Family Protein code CBM1 Cellulase 7A
(Trichoderma reesei) 1CBH CBM2 Xylanase 10A (Cellulomonas fimi)
1EXG Xylanase 11A (Cellulomonas fimi) 2XBD Xylanase 11A
(Cellulomonas fimi) 1HEH CBM3 Scaffoldin (Clostridium
cellulolyticum) 1G43 Scaffoldin (Clostridium thermocellum) 1NBC
Cellulase 9A (Thermobifida fusca) 1TF4 CBM4 Laminarinase 16A
(Thermotoga maritima) 1GUI Cellulase 9B (Cellulomonas fimi) 1ULO;
1GU3 Cellulase 9B (Cellulomonas fimi) 1CX1 Xylanase 10A
(Rhodothermus marinus) 1K45 CBM5 Cellulase 5A (Envinia
chrysanthemi) 1AIW Chitinase B (Serratia marcescens) 1E15 CBM6
Xylanase 11A (Clostridium thermocellum) 1UXX Xylanase 11A
(Clostridium stercorarium) 1NAE Xylanase 11A (Clostridium
stercorarium) 1UY4 Endoglucanase 5A (Cellvibrio mixtus) 1UZ0 CBM9
Xylanase 10A (Thermotoga maritima) 1I8A CBM10 Xylanase 10A
(Cellvibrio japonicus) 1QLD CBM12 Chitinase Chi 1 (Bacillus
circulans) 1ED7 CBM13* Xylanase 10A (Streptomyces olivaceoviridis)
1XYF Xylanase 10A (Streptomyces lividans) 1MC9 Ricin toxin B-chain
(Ricinus communis) 2AAI Abrin (Abrus precatorius) 1ABR CBM14
Tachycitin (Tachypleus tridentatus) 1DQC CBM15 Xylanase 10C
(Cellvibrio japonicus) 1GNY CBM17 Cellulase 5A (Clostridium
cellulovorans) 1J83 CBM18* Agglutinin (Triticum aestivum) 1WGC
Antimicrobial peptide (Amaranthus caudatus) 1MMC
Chitinase/agglutinin (Urtica dioica) 1EIS CBM20* Glucoamylase
(Aspergillus niger) 1ACO .beta.-amylase (Bacillus cereus) 1CQY
CBM22 Xylanase 10B (Clostridium thermocellum) 1DYO CBM27 Mannanase
5A (Thermotoga maritima) 1OF4 CBM28 Cellulase 5A (Bacillus sp.
1139) 1UWW CBM29 Non-catalytic protein 1 (Pyromyces equi) 1GWK
CBM32 Sialidase 33A (Micromonospora viridifaciens) 1EUU Galactose
oxidase (Cladobotryum dendroides) 1GOF CBM34* .alpha.-Amylase 13A
(Thermoactinomyces vulgaris) 1UH2 Neopullulanase 1J0H (Geobacillus
stearothermophilus) CBM36 Xylanase 43A (Paenibacillus polymyxa)
1UX7 *These families contain too many structure entries to list
them all so only representatives are given.
[0499] Thus, the invention provides chimeric hydrolases, e.g., a
fusion of a glycosidase with different (e.g., heterologous) CBMs to
target the enzyme to particular insoluble polysaccharides to
enhance performance in an application. In one aspect, the chimeric
glycosidase comprises an enzyme of the invention. In one aspect,
the chimeric enzyme comprises fusions of different CBMs to enhance
pulp biobleaching performance, e.g., to achieve greater percentage
reduction of bleaching chemicals. The invention also provides
methods comprising recombining different CBMs with different
xylanases (e.g., CBMs of the invention and/or xylanases of the
invention) and screening the resultant chimerics to find the best
combination for a particular application or substrate.
[0500] Other variations also are within the scope of this
invention, e.g., where one, two, three, four or five or more
residues are removed from the carboxy- or amino-terminal ends of
any polypeptide of the invention. Another variation includes
modifying any residue to increase or decrease pI of a polypeptide,
e.g., removing or modifying (e.g., to another amino acid) a
glutamate. This method was used as a general scheme for improving
the enzyme's properties without creating regulatory issues since no
amino acids are mutated; and this general scheme can be used with
any polypeptide of the invention.
[0501] The invention provides isolated, synthetic or recombinant
polypeptides having xylanase activity, wherein the polypeptide has
a sequence modification of any polypeptide of the invention,
including any exemplary amino acid sequence of the invention,
including 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, and also including SEQ ID
NO:2 having amino acid residue changes (mutations) as set forth in
Table 1 and as described herein. The sequence change(s) can also
comprise any amino acid modification to change the pI of a
polypeptide, e.g., deletion or modification of a glutamate, or
changing from a glutamate to another residue.
[0502] The invention further provides isolated, synthetic 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) to an exemplary
sequence of the invention.
[0503] In one aspect, the polypeptide has a xylanase or a glucanase
activity; for example, wherein the xylanase activity can comprise
hydrolyzing a glycosidic bond in a polysaccharide, e.g., a xylan.
In one aspect, the polypeptide has a xylanase activity comprising
catalyzing hydrolysis of internal .beta.-1,4-xylosidic linkages. In
one aspect, the xylanase activity comprises an
endo-1,4-beta-xylanase activity. In one aspect, the xylanase
activity comprises hydrolyzing a xylan to produce a smaller
molecular weight xylose and xylo-oligomer. In one aspect, the xylan
comprises an arabinoxylan, such as a water soluble
arabinoxylan.
[0504] The invention provides polypeptides having glucanase
activity. In one aspect, the glucanase activity of a polypeptide or
peptide of the invention (which includes a protein or peptide
encoded by a nucleic acid of the invention) comprises an
endoglucanase activity, e.g., endo-1,4- and/or 1,3-beta-D-glucan
4-glucano hydrolase activity. In one aspect, the endoglucanase
activity comprises catalyzing hydrolysis of 1,4-beta-D-glycosidic
linkages. In one aspect, the glucanase, e.g., endoglucanase,
activity comprises an endo-1,4- and/or 1,3-beta-endoglucanase
activity or endo-.beta.-1,4-glucanase activity. In one aspect, the
glucanase activity (e.g., endo-1,4-beta-D-glucan 4-glucano
hydrolase activity) comprises hydrolysis of 1,4-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 and other
plant material containing cellulosic parts. In one aspect, the
glucanase, xylanase, or mannanase activity comprises hydrolyzing a
glucan or other polysaccharide to produce a smaller molecular
weight polysaccharide or oligomer. In one aspect, the glucan
comprises a beta-glucan, such as a water soluble beta-glucan.
[0505] The invention provides polypeptides having mannanase (e.g.,
endo-1,4-beta-D-mannanase) activity, for example, catalyzing the
hydrolysis of a beta-1,4-mannan, e.g., an unsubstituted linear
beta-1,4-mannan. Mannanase activity determination can be determined
using any known methods, e.g., the Congo Red method, as described
e.g., by Downie (1994) "A new assay for quantifying
endo-beta-mannanase activity using Congo red dye. Phytochemistry,
July 1994, vol. 36, no. 4, p. 829-835; or, as described in U.S.
Pat. No. 6,060,299, e.g., by applying a solution to be tested to 4
mm diameter holes punched out in agar plates containing 0.2% AZCL
galactomannan (carob) or any substrate for the assay of
endo-1,4-beta-D-mannanase.
[0506] Any xylanase, glucanase and/or mannanase assay known in the
art can be used to determine if a polypeptide has xylanase,
glucanase and/or mannanase activity and is within scope of the
invention. For example, reducing sugar assays such as the
Nelson-Somogyi method or the dinitrosalicylic acid (DNS) method can
be used to assay for the product sugars (and thus, xylanase
activity). In one aspect, reactions are carried out by mixing and
incubating a dilution of the enzyme preparation with a known amount
of substrate at a buffered pH and set temperature. Xylanase assays
are similar to cellulase assays except that a solution of xylan
(e.g., oat spelts or birch) is substituted for CMC or filter paper.
The DNS assay is easier to use than the Nelson-Somogyi assay. The
DNS assay is satisfactory for cellulase activities, but tends to
over estimate xylanase activity. The Somogyi-Nelson procedure is
more accurate in the determination of reducing sugars, to measure
specific activities and to quantify the total amount of xylanase
produced in the optimized growth conditions, see, e.g., Breuil
(1985) Comparison of the 3,5-dinitrosalicylic acid and
Nelson-Somogyi methods of assaying for reducing sugars and
determining cellulase activity, Enzyme Microb. Technol. 7:327-332;
Somogyi, M. 1952, Notes on sugar determination, J. Biol. Chem.
195:19-23. The invention incorporates use of any reducing sugar
assay, e.g., by Nelson-Somogyi, e.g., based on references Nelson,
N. (1944) J. Biol. Chem. 153:375-380, and Somogyi, M. (1952) J.
Biol. Chem. 195:19-23.
[0507] The polypeptides of the invention include xylanases 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 xylanases 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
xylanase.
[0508] 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.
[0509] 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, synthetic or recombinant signal
sequences, prepro sequences and catalytic domains (e.g., "active
sites") comprising sequences of the invention.
[0510] 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. 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
xylanase; 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 xylanase of the
invention.
[0511] Peptides of the invention (e.g., a subsequence of an
exemplary polypeptide of the invention) can be useful as, e.g.,
labeling probes, antigens, toleragens, motifs, xylanase active
sites (e.g., "catalytic domains"), signal sequences and/or prepro
domains.
[0512] 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.
[0513] 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.
[0514] "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 that 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 phosphytidylinositol, 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, xylan 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.
[0515] "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" nucleic acids
(including oligonucleotides), polypeptides or proteins of the
invention include those prepared by any chemical synthesis, e.g.,
as described, below. 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, 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 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.
[0516] "Fragments" or "enzymatically active fragments" as used
herein are a portion of an amino acid sequence (encoding a protein)
which retains at least one functional activity of the protein to
which it is related. Fragments can have the same or substantially
the same amino acid sequence as the naturally occurring protein.
"Substantially the same" means that an amino acid sequence is
largely, but not entirely, the same, but retains at least one
functional activity of the sequence to which it is related. In
general two amino acid sequences are "substantially the same" or
"substantially homologous" if they are at least about 85%
identical. 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.
[0517] 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, 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 xylanase activity.
[0518] 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, NY).
[0519] 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-biphenylphenyl alanine; 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.
[0520] 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, preferably
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.
[0521] 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. Thus, 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.
[0522] 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. 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).
[0523] 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.
[0524] The invention includes xylanases of the invention with and
without signal. The polypeptide comprising a signal sequence of the
invention can be a xylanase of the invention or another xylanase or
another enzyme or other polypeptide.
[0525] The invention includes immobilized xylanases, anti-xylanase
antibodies and fragments thereof. The invention provides methods
for inhibiting xylanase activity, e.g., using dominant negative
mutants or anti-xylanase antibodies of the invention. The invention
includes heterocomplexes, e.g., fusion proteins, heterodimers,
etc., comprising the xylanases of the invention.
[0526] Polypeptides of the invention can have a xylanase activity
under various conditions, e.g., extremes in pH and/or temperature,
oxidizing agents, and the like. The invention provides methods
leading to alternative xylanase preparations with different
catalytic efficiencies and stabilities, e.g., towards temperature,
oxidizing agents and changing wash conditions. In one aspect,
xylanase 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 xylanase
variants with alternative specificities and stability.
[0527] The proteins of the invention are also useful as research
reagents to identify xylanase modulators, e.g., activators or
inhibitors of xylanase activity. Briefly, test samples (compounds,
broths, extracts, and the like) are added to xylanase 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 xylanases,
inhibitors can be combined to increase the spectrum of
activity.
[0528] The enzymes of the invention are also useful as research
reagents to digest proteins or in protein sequencing. For example,
the xylanases may be used to break polypeptides into smaller
fragments for sequencing using, e.g., an automated sequencer.
[0529] The invention also provides methods of discovering new
xylanases using the nucleic acids, polypeptides and antibodies of
the invention. In one aspect, phagemid libraries are screened for
expression-based discovery of xylanases. In another aspect, lambda
phage libraries are screened for expression-based discovery of
xylanases. 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.
[0530] 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.
[0531] Another aspect of the invention is an isolated or purified
polypeptide comprising the sequence of one of the invention and
sequences substantially identical thereto, or fragments comprising
at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof. As discussed above, such
polypeptides may be obtained by inserting a nucleic acid encoding
the polypeptide into a vector such that the coding sequence is
operably linked to a sequence capable of driving the expression of
the encoded polypeptide in a suitable host cell. For example, the
expression vector may comprise a promoter, a ribosome binding site
for translation initiation and a transcription terminator. The
vector may also include appropriate sequences for amplifying
expression.
[0532] Another aspect of the invention is polypeptides or fragments
thereof which have at least about 50%, at least about 55%, at least
about 60%, at least about 65%, at least about 70%, at least about
75%, at least about 80%, at least about 85%, at least about 90%, at
least about 95%, or more than about 95% homology to one of the
polypeptides of the invention and sequences substantially identical
thereto, or a fragment comprising at least 5, 10, 15, 20, 25, 30,
35, 40, 50, 75, 100, or 150 consecutive amino acids thereof.
Homology may be determined using any of the programs described
above which aligns the polypeptides or fragments being compared and
determines the extent of amino acid identity or similarity between
them. It will be appreciated that amino acid "homology" includes
conservative amino acid substitutions such as those described
above.
[0533] The polypeptides or fragments having homology to one of the
polypeptides of the invention, or a fragment comprising at least
about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof may be obtained by isolating the
nucleic acids encoding them using the techniques described
above.
[0534] Alternatively, the homologous polypeptides or fragments may
be obtained through biochemical enrichment or purification
procedures. The sequence of potentially homologous polypeptides or
fragments may be determined by xylan hydrolase digestion, gel
electrophoresis and/or microsequencing. The sequence of the
prospective homologous polypeptide or fragment can be compared to
one of the polypeptides of the invention, or a fragment comprising
at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150
consecutive amino acids thereof using any of the programs described
above.
[0535] 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 the polypeptides of the
invention.
[0536] The 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.
[0537] 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 a variety of
applications. For example, the polypeptides or fragments thereof
may be used to catalyze biochemical reactions. In accordance with
one aspect of the invention, there is provided a process for
utilizing the polypeptides of the invention or polynucleotides
encoding such polypeptides for hydrolyzing glycosidic linkages. In
such procedures, a substance containing a glycosidic linkage (e.g.,
a starch) is contacted with one of the polypeptides of the
invention, or sequences substantially identical thereto under
conditions which facilitate the hydrolysis of the glycosidic
linkage.
[0538] 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.
[0539] 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.
[0540] 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.
[0541] Many of the procedural steps are performed using robotic
automation enabling the execution of many thousands of biocatalytic
reactions and screening assays per day as well as ensuring a high
level of accuracy and reproducibility. As a result, a library of
derivative compounds can be produced in a matter of weeks which
would take years to produce using current chemical methods.
[0542] 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 in one aspect (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.
Xylanase Signal Sequences, Prepro and Catalytic Domains
[0543] The invention provides xylanase 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, synthetic 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). In one
aspect, the invention provides a signal sequence comprising a
peptide comprising/consisting of a sequence as set forth in
residues 1 to 12, 1 to 13, 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 39, 1 to 40,
1 to 41, 1 to 42, 1 to 43, 1 to 44, 1 to 45, 1 to 46, 1 to 47, 1 to
48, 1 to 49 or 1 to 50, of a polypeptide of the invention.
[0544] The xylanase signal sequences (SPs) and/or prepro sequences
of the invention can be isolated peptides, or, sequences joined to
another xylanase or a non-xylanase polypeptide, e.g., as a fusion
(chimeric) protein. In one aspect, the invention provides
polypeptides comprising xylanase signal sequences of the invention.
In one aspect, polypeptides comprising xylanase signal sequences
SPs and/or prepro of the invention comprise sequences heterologous
to a xylanase of the invention (e.g., a fusion protein comprising
an SP and/or prepro of the invention and sequences from another
xylanase or a non-xylanase protein). In one aspect, the invention
provides xylanases of the invention with heterologous SPs and/or
prepro sequences, e.g., sequences with a yeast signal sequence. A
xylanase of the invention can comprise a heterologous SP and/or
prepro in a vector, e.g., a pPIC series vector (Invitrogen,
Carlsbad, Calif.).
[0545] In one aspect, SPs and/or prepro sequences of the invention
are identified following identification of novel xylanase
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. More than 100 signal sequences for proteins
in this group have been determined. The signal sequences can vary
in length from between about 11 to 41, or between about 13 to 36
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 xylanase 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; see, e.g., Nielsen (1997) "Identification of prokaryotic and
eukaryotic signal peptides and prediction of their cleavage sites."
Protein Engineering 10:1-6.
[0546] It should be understood that in some aspects xylanases of
the invention may not have SPs and/or prepro sequences, or
"domains." In one aspect, the invention provides the xylanases 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 xylanase
operably linked to a nucleic acid sequence of a different xylanase
or, in one aspect (optionally), a signal sequence (SPs) and/or
prepro domain from a non-xylanase protein may be desired.
[0547] The invention also provides isolated, synthetic 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 xylanase) 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, synthetic 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 xylanase
sequence). Similarly in one aspect, the invention provides
isolated, synthetic or recombinant nucleic acids encoding these
polypeptides. Thus, in one aspect, the isolated, synthetic 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.
Hybrid (Chimeric) Xylanases and Peptide Libraries
[0548] In one aspect, the invention provides hybrid xylanases 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 xylanase 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).
[0549] 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 xylanases of the invention and
other peptides, including known and random peptides. They can be
fused in such a manner that the structure of the xylanases 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.
[0550] 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 xylanase 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 xylanase 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 xylan 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.
[0551] The invention provides xylanases 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., xylanase activity) although variants can be selected to
modify the characteristics of the xylanases as needed.
[0552] In one aspect, xylanases of the invention comprise epitopes
or purification tags, signal sequences or other fusion sequences,
etc. In one aspect, the xylanases 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
xylanase are linked together, in such a manner as to minimize the
disruption to the stability of the xylanase structure, e.g., it
retains xylanase activity. The fusion polypeptide (or fusion
polynucleotide encoding the fusion polypeptide) can comprise
further components as well, including multiple peptides at multiple
loops.
[0553] 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.
[0554] Xylanases are multidomain enzymes that consist in one aspect
(optionally) of a signal peptide, a carbohydrate binding module, a
xylanase catalytic domain, a linker and/or another catalytic
domain.
[0555] The invention provides a means for generating chimeric
polypeptides which may encode biologically active hybrid
polypeptides (e.g., hybrid xylanases). In one aspect, the original
polynucleotides encode biologically active polypeptides. The 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 from the
original biologically active polypeptides. For example, the
original polynucleotides may encode a particular enzyme from
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 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.
[0556] Enzymes encoded by the polynucleotides of the invention
include, but are not limited to, hydrolases, such as xylanases.
Glycosidase hydrolases were first classified into families in 1991,
see, e.g., Henrissat (1991) Biochem. J. 280:309-316. Since then,
the classifications have been continually updated, see, e.g.,
Henrissat (1993) Biochem. J. 293:781-788; Henrissat (1996) Biochem.
J. 316:695-696; Henrissat (2000) Plant Physiology 124:1515-1519.
There are 87 identified families of glycosidase hydrolases. In one
aspect, the xylanases of the invention may be categorized in
families 8, 10, 11, 26 and 30. In one aspect, the invention also
provides xylanase-encoding nucleic acids with a common novelty in
that they are derived from a common family, e.g., 11.
[0557] A hybrid polypeptide resulting from the 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 hydrolase
activities, the resulting hybrid polypeptide encoded by a hybrid
polynucleotide can be screened for specialized hydrolase activities
obtained from each of the original enzymes, i.e., the type of bond
on which the hydrolase acts and the temperature at which the
hydrolase functions. Thus, for example, the hydrolase may be
screened to ascertain those chemical functionalities which
distinguish the hybrid hydrolase from the original hydrolases, such
as: (a) amide (peptide bonds), i.e., xylanases; (b) ester bonds,
i.e., esterases and lipases; (c) acetals, i.e., glycosidases and,
for example, the temperature, pH or salt concentration at which the
hybrid polypeptide functions.
[0558] Sources of the original polynucleotides 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 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.
[0559] "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.
[0560] For example, 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. 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.
[0561] Additionally, subcloning may be 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.
[0562] It should be understood that some of the xylanases of the
invention may or may not contain signal sequences. It may be
desirable to include a nucleic acid sequence encoding a signal
sequence from one xylanase operably linked to a nucleic acid
sequence of a different xylanase or, in one aspect (optionally), a
signal sequence from a non-xylanase protein may be desired.
[0563] The microorganisms from which the polynucleotide may be
prepared include prokaryotic microorganisms, such as Eubacteria and
Archaebacteria and lower eukaryotic microorganisms such as fungi,
some algae and protozoa. Polynucleotides may be isolated 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. Such enzymes may function at
temperatures above 100.degree. C. in terrestrial hot springs and
deep sea thermal vents, at temperatures below 0.degree. C. in
arctic waters, in the saturated salt environment of the Dead Sea,
at pH values around 0 in coal deposits and geothermal sulfur-rich
springs, or at pH values greater than 11 in sewage sludge. For
example, several esterases and lipases cloned and expressed from
extremophilic organisms show high activity throughout a wide range
of temperatures and pHs.
[0564] 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 preferably
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
preferably, 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 (Davis et al., 1986).
[0565] As representative examples of appropriate hosts, there may
be mentioned: 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. The selection
of an appropriate host is deemed to be within the scope of those
skilled in the art from the teachings herein.
[0566] With particular references to various mammalian cell culture
systems that 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 will 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.
[0567] In another aspect, it is envisioned the method of the
present invention can be used 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.
[0568] Gene cluster DNA can be isolated from different organisms
and ligated into vectors, particularly 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 are
particularly 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 of
the invention 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.
[0569] Therefore, in a one aspect, the invention relates to a
method for producing a biologically active hybrid polypeptide and
screening such a polypeptide for enhanced activity by: [0570] 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;
[0571] 2) growing the host cell under conditions which promote
sequence reorganization resulting in a hybrid polynucleotide in
operable linkage; [0572] 3) expressing a hybrid polypeptide encoded
by the hybrid polynucleotide; [0573] 4) screening the hybrid
polypeptide under conditions which promote identification of
enhanced biological activity; and [0574] 5) isolating the a
polynucleotide encoding the hybrid polypeptide.
[0575] Methods for screening for various enzyme activities are
known to those of skill in the art and are discussed throughout the
present specification. Such methods may be employed when isolating
the polypeptides and polynucleotides of the invention.
Screening Methodologies and "On-line" Monitoring Devices
[0576] 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 xylanase activity (e.g., assays such as hydrolysis
of casein in zymograms, the release of fluorescence from gelatin,
or the release of p-nitroanalide from various small peptide
substrates), to screen compounds as potential modulators, e.g.,
activators or inhibitors, of a xylanase 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 No.
20020001809.
Capillary Arrays
[0577] 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.
[0578] 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.
[0579] 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".
[0580] 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.
[0581] 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.
Arrays or "Biochips"
[0582] Nucleic acids and/or polypeptides of the invention can be
immobilized to or applied to an array, e.g., a "biochip". 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
xylanase 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.
[0583] 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.
[0584] 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
[0585] The invention provides isolated, synthetic or recombinant
antibodies that specifically bind to a xylanase of the invention.
These antibodies can be used to isolate, identify or quantify the
xylanases of the invention or related polypeptides. These
antibodies can be used to isolate other polypeptides within the
scope the invention or other related xylanases. The antibodies can
be designed to bind to an active site of a xylanase. Thus, the
invention provides methods of inhibiting xylanases using the
antibodies of the invention (see discussion above regarding
applications for anti-xylanase compositions of the invention).
[0586] The invention provides fragments of the enzymes of the
invention, including immunogenic fragments 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.
[0587] 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.
[0588] 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."
[0589] 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.
[0590] 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.
[0591] 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.
[0592] 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.
[0593] 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 or more 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 will then 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.
[0594] 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).
[0595] 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.
[0596] 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
[0597] 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.,
xylanases) and/or antibodies of the invention. The kits also can
contain instructional material teaching the methodologies and
industrial, research, medical, pharmaceutical, food and feed and
food and feed supplement processing and other applications and
processes of the invention, as described herein.
Whole Cell Engineering and Measuring Metabolic Parameters
[0598] 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 xylanase activity,
by modifying the genetic composition of the cell. 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.
[0599] 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 xylanases of the invention.
[0600] 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: [0601] identity of all
pathway substrates, products and intermediary metabolites, [0602]
identity of all the chemical reactions interconverting the pathway
metabolites, the stoichiometry of the pathway reactions, [0603]
identity of all the enzymes catalyzing the reactions, the enzyme
reaction kinetics, [0604] the regulatory interactions between
pathway components, e.g., allosteric interactions, enzyme-enzyme
interactions, etc., [0605] intracellular compartmentalization of
enzymes or any other supramolecular organization of the enzymes,
and, [0606] the presence of any concentration gradients of
metabolites, enzymes or effector molecules or diffusion barriers to
their movement.
[0607] 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.
[0608] 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.
Monitoring Expression of an mRNA Transcript
[0609] In one aspect of the invention, the engineered phenotype
comprises increasing or decreasing the expression of an mRNA
transcript (e.g., a xylanase message) or generating new (e.g.,
xylanase) transcripts in a cell. This increased or decreased
expression can be traced by testing for the presence of a xylanase
of the invention or by xylanase 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).
[0610] 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.
[0611] 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.
Monitoring Expression of Polypeptides, Peptides and Amino Acids
[0612] In one aspect of the invention, the engineered phenotype
comprises increasing or decreasing the expression of a polypeptide
(e.g., a xylanase) or generating new polypeptides in a cell. This
increased or decreased expression can be traced by determining the
amount of xylanase present or by xylanase 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, Medical, Food Processing and
Other Applications
[0613] Polypeptides of the invention can be used in any industrial,
agricultural, food and feed and food and feed supplement
processing, pharmaceutical, medical, research (laboratory) or other
process. 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 biofuel production, including, e.g., a bioalcohol
such as bioethanol, biomethanol, biobutanol or biopropanol, thus
comprising a "clean" fuel production. Enzymes of the invention can
be added to industrial processes continuously, in batches or by
fed-batch methods. In another aspect, enzymes of the invention can
be recycled in the industrial processes, thereby lowering enzyme
requirements.
[0614] For example, xylanases can be used in the biobleaching and
treatment of chemical pulps, for example, as described in U.S. Pat.
No. 5,202,249; or for biobleaching and treatment of wood or paper
pulps, for example, as described in U.S. Pat. Nos. 5,179,021,
5,116,746, 5,407,827, 5,405,769, 5,395,765, 5,369,024, 5,457,045,
5,434,071, 5,498,534, 5,591,304, 5,645,686, 5,725,732, 5,759,840,
5,834,301, 5,871,730 and 6,057,438; or for reducing lignin in wood
and modifying wood, for example, as described in U.S. Pat. Nos.
5,486,468 and/or 5,770,012; or for use as flour, dough and bread
improvers, for example, as described in U.S. Pat. Nos. 5,108,765
and/or 5,306,633; or for use as feed additives and/or supplements,
for example, as described in U.S. Pat. Nos. 5,432,074; 5,429,828;
5,612,055; 5,720,971; 5,981,233; 5,948,667; 6,099,844; 6,132,727
and/or 6,132,716; or in manufacturing cellulose solutions, for
example, as described in U.S. Pat. No. 5,760,211; or in detergent
compositions; or used for fruit, vegetables and/or mud and clay
compounds, for example, as described in U.S. Pat. No. 5,786,316.
Xylanases of this invention also can be used in hydrolysis of
hemicellulose, for example, as described in U.S. Pat. No.
4,725,544.
[0615] The xylanase enzymes of the invention can be highly
selective catalysts. They can catalyze reactions with exquisite
stereo-, regio- and chemo-selectivities that are unparalleled in
conventional synthetic chemistry. Moreover, enzymes are remarkably
versatile. The xylanase enzymes of the invention can be tailored to
function in organic 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.
Wood, Paper and Pulp Treatments
[0616] The xylanases of the invention can be used in any wood, wood
product, wood waste or by-product, paper, paper product, paper or
wood pulp, Kraft pulp, or wood or paper recycling treatment or
industrial process, e.g., any wood, wood pulp, paper waste, paper
or pulp treatment or wood or paper deinking process. In one aspect,
xylanases of the invention can be used to treat/pretreat paper
pulp, or recycled paper or paper pulp, and the like. In one aspect,
enzyme(s) of the invention are used to increase the "brightness" of
the paper via their use in treating/pretreating paper pulp, or
recycled paper or paper pulp, and the like. The higher the grade of
paper, the greater the brightness; paper brightness can impact the
scan capability of optical scanning equipment; thus, the enzymes
and processes of the invention can be used to make high grade,
"bright" paper for, e.g., use in optical scanning equipment,
including inkjet, laser and photo printing quality paper.
[0617] For example, the enzymes of the invention can be used in any
industrial process using xylanases known in the art, e.g., treating
waste paper, as described in, e.g., U.S. Pat. No. 6,767,728 or
6,426,200; seasoning wood, e.g., for applications in the food
industry, as described in, e.g., U.S. Pat. No. 6,623,953; for the
production of xylose from a paper-grade hardwood pulp, as described
in, e.g., U.S. Pat. No. 6,512,110; treating fibrous lignocellulosic
raw material with a xylanase in an aqueous medium as described in,
e.g., U.S. Pat. No. 6,287,708; dissolving pulp from cellulosic
fiber, as described in, e.g., U.S. Pat. No. 6,254,722; deinking and
decolorizing a printed paper or removing color from wood pulp, as
described in, e.g., U.S. Pat. Nos. 6,241,849, 5,834,301 or
5,582,681; bleaching a chemical paper pulp or lignocellulose pulp
using a xylanase, as described in, e.g., U.S. Pat. No. 5,645,686 or
5,618,386; for treating wood pulp that includes incompletely washed
brownstock, as described in, e.g., U.S. Pat. No. 5,591,304;
purifying and delignifying a waste lignocellulosic material, as
described in, e.g., in U.S. Pat. No. 5,503,709; manufacturing paper
or cardboard from recycled cellulose fibers, as described in, e.g.,
in U.S. Pat. No. 5,110,412; debarking of logs, as described in,
e.g., in U.S. Pat. No. 5,103,883; producing fluff pulp with
improved shredding properties, as described in, e.g., in U.S. Pat.
No. 5,068,009, and the like. The xylanases of the invention can be
used to process or treat any cellulosic material, e.g., fibers from
wood, cotton, hemp, flax or linen.
[0618] In one aspect, the invention provides wood, wood pulp,
paper, paper pulp, paper waste or wood or paper recycling treatment
processes using a xylanase of the invention. In one aspect, the
xylanase of the invention is applicable both in reduction of the
need for a chemical decoloring (e.g., bleaching) agent, such as
chlorine dioxide, and in high alkaline and high temperature
environments. Most lignin is solubilized in the cooking stage of
pulping process. The residual lignin is removed from the pulp in
the bleaching process. In one aspect, xylanase bleaching of pulp
(e.g., using an enzyme of the invention) is based on the partial
hydrolysis of xylan, which is the main component of the
hemicellulose. The enzymatic action (e.g., of an enzyme of the
invention) releases hemicellulose-bound lignin and increases the
extractability of lignin from the pulp in the subsequent bleaching
process, e.g., using chlorine and oxygen chemicals. In one aspect,
xylanases of the invention can be used to increase the final
brightness of the pulp at a fixed level of bleaching chemicals. In
another aspect, xylanases of the invention can be used to decrease
the kappa number of the pulp.
[0619] The invention provides wood, wood pulp, paper, paper pulp,
paper waste or wood or paper recycling treatment processes
(methods) using a xylanase of the invention where the treatment
time (the amount of time the xylanase is in contact with the pulp,
paper, wood, etc.) and/or retention time can be anywhere from
between about 1 minute to 12 hours, or between about 5 minutes to 1
hour, or between about 15 to 30 minutes; or the treatment and/or
retention time can be any time up to about 0.1, 0.25, 0.5, 0.75, 1,
1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours.
[0620] In one aspect, the xylanase of the invention is a
thermostable alkaline endoxylanase which in one aspect can effect a
greater than 25% reduction in the chlorine dioxide requirement of
kraft pulp with a less than 0.5% pulp yield loss. In one aspect,
boundary parameters are pH 10, 65-85.degree. C. and treatment time
of less than 60 minutes at an enzyme loading of less than 0.001 wt
%; in alternative aspects the treatment and/or retention time is
less than about 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11 or 12.
[0621] A pool of xylanases may be tested for the ability to
hydrolyze dye-labeled xylan at, for example, pH 10 and 60.degree.
C. The enzymes that test positive under these conditions may then
be evaluated at, for example pH 10 and 70.degree. C. Alternatively,
enzymes may be tested at pH 8 and pH 10 at 70.degree. C. In
discovery of xylanases desirable in the pulp and paper industry
libraries from high temperature or highly alkaline environments
were targeted. Specifically, these libraries were screened for
enzymes functioning at alkaline pH and a temperature of
approximately 45.degree. C. In another aspect, the xylanases of the
invention are useful in the pulp and paper industry in degradation
of a lignin-hemicellulose linkage, in order to release the
lignin.
[0622] Enzymes of the invention can be used for deinking printed
wastepaper, such as newspaper, or for deinking noncontact-printed
wastepaper, e.g., xerographic and laser-printed paper, and mixtures
of contact and noncontact-printed wastepaper, as described in U.S.
Pat. No. 6,767,728 or 6,426,200; Neo (1986) J. Wood Chem. Tech.
6(2):147. Enzymes of the invention can be used in processes for the
production of xylose from a paper-grade hardwood pulp by extracting
xylan contained in pulp into a liquid phase, subjecting the xylan
contained in the obtained liquid phase to conditions sufficient to
hydrolyze xylan to xylose, and recovering the xylose, where the
extracting step includes at least one treatment of an aqueous
suspension of pulp or an alkali-soluble material a xylanase enzyme,
as described in, e.g., U.S. Pat. No. 6,512,110. Enzymes of the
invention can be used in processes for dissolving pulp from
cellulosic fibers such as recycled paper products made from
hardwood fiber, a mixture of hardwood fiber and softwood fiber,
waste paper, e.g., from unprinted envelopes, de-inked envelopes,
unprinted ledger paper, de-inked ledger paper, and the like, as
described in, e.g., U.S. Pat. No. 6,254,722.
[0623] In another aspect of the invention, the xylanases of the
invention can also be used in any wood, wood product, paper, paper
product, paper or wood pulp, Kraft pulp, or wood or paper recycling
treatment or industrial process, e.g., any wood, wood pulp, paper
waste, paper or pulp treatment or wood or paper deinking process as
an antimicrobial or microbial repellent. Alternatively, the
xylanases of the invention can be part of a wood, wood product,
paper, paper product, paper or wood pulp, Kraft pulp, or recycled
paper composition, and/or a composition comprising one or more
wood, wood product, paper, paper product, paper or wood pulp, Kraft
pulp, or recycled paper compositions, wherein the xylanases of the
invention act as an antimicrobial or microbial repellent in the
composition.
Treating Fibers and Textiles
[0624] The invention provides methods of treating fibers and
fabrics using one or more xylanases of the invention. The xylanases
can be used in any fiber- or fabric-treating method, which are well
known in the art, see, e.g., U.S. Pat. Nos. 6,261,828; 6,077,316;
6,024,766; 6,021,536; 6,017,751; 5,980,581; US Patent Publication
No. 20020142438 A1. For example, xylanases of the invention can be
used in fiber and/or fabric desizing. In one aspect, the feel and
appearance of a fabric is improved by a method comprising
contacting the fabric with a xylanase of the invention in a
solution. In one aspect, the fabric is treated with the solution
under pressure. For example, xylanases of the invention can be used
in the removal of stains.
[0625] The xylanases of the invention can be used to treat any
cellulosic material, including fibers (e.g., fibers from cotton,
hemp, flax or linen), sewn and unsewn fabrics, e.g., knits, wovens,
denims, yarns, and toweling, made from cotton, cotton blends or
natural or manmade cellulosics (e.g., originating from
xylan-containing cellulose fibers such as from wood pulp) or blends
thereof. Examples of blends are blends of cotton or rayon/viscose
with one or more companion material such as wool, synthetic fibers
(e.g., polyamide fibers, acrylic fibers, polyester fibers,
polyvinyl alcohol fibers, polyvinyl chloride fibers, polyvinylidene
chloride fibers, polyurethane fibers, polyurea fibers, aramid
fibers), and cellulose-containing fibers (e.g., rayon/viscose,
ramie, hemp, flax/linen, jute, cellulose acetate fibers,
lyocell).
[0626] The textile treating processes of the invention (using
xylanases of the invention) can be used in conjunction with other
textile treatments, e.g., scouring and bleaching. Scouring is the
removal of non-cellulosic material from the cotton fiber, e.g., the
cuticle (mainly consisting of waxes) and primary cell wall (mainly
consisting of pectin, protein and xyloglucan). A proper wax removal
is necessary for obtaining a high wettability. This is needed for
dyeing. Removal of the primary cell walls by the processes of the
invention improves wax removal and ensures a more even dyeing.
Treating textiles with the processes of the invention can improve
whiteness in the bleaching process. The main chemical used in
scouring is sodium, hydroxide in high concentrations and at high
temperatures. Bleaching comprises oxidizing the textile. Bleaching
typically involves use of hydrogen peroxide as the oxidizing agent
in order to obtain either a fully bleached (white) fabric or to
ensure a clean shade of the dye.
[0627] The invention also provides alkaline xylanases (xylanases
active under alkaline conditions). These have wide-ranging
applications in textile processing, degumming of plant fibers
(e.g., plant bast fibers), treatment of pectic wastewaters,
paper-making, and coffee and tea fermentations. See, e.g., Hoondal
(2002) Applied Microbiology and Biotechnology 59:409-418.
[0628] In another aspect of the invention, the xylanases of the
invention can also be used in any fiber- and/or fabric-treating
process as an antimicrobial or microbial repellent. Alternatively,
the xylanases of the invention can be part of a fiber- and/or
fabric-composition, where the xylanases of the invention act as an
antimicrobial or microbial repellent in the fiber and/or
fabric.
Detergent, Disinfectant and Cleaning Compositions
[0629] The invention provides detergent, disinfectant or cleanser
(cleaning or cleansing) compositions comprising one or more
polypeptides (e.g., xylanases) of the invention, and methods of
making and using these compositions. The invention incorporates all
methods of making and using detergent, disinfectant or cleanser
compositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561;
6,365,561; 6,380,147. The detergent, disinfectant or cleanser
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 xylanases of the invention can also be used as a
detergent, disinfectant or cleanser additive product in a solid or
a liquid form. Such additive products are intended to supplement or
boost the performance of conventional detergent compositions and
can be added at any stage of the cleaning process.
[0630] The actual active enzyme content depends upon the method of
manufacture of a detergent, disinfectant or cleanser composition
and is not critical, assuming the detergent solution has the
desired enzymatic activity. In one aspect, the amount of xylanase
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 xylanases 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.
[0631] Xylanases of the invention can be formulated into powdered
and liquid detergents, disinfectants or cleansers 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, disinfectant or cleanser
compositions can also include other enzymes such as xylanases,
cellulases, lipases, esterases, proteases, or endoglycosidases,
endo-beta.-1,4-glucanases, beta-glucanases,
endo-beta-1,3(4)-glucanases, cutinases, peroxidases, catalases,
laccases, amylases, glucoamylases, pectinases, reductases,
oxidases, phenoloxidases, ligninases, pullulanases, arabinanases,
hemicellulases, mannanases, xyloglucanases, xylanases, pectin
acetyl esterases, rhamnogalacturonan acetyl esterases,
polygalacturonases, rhamnogalacturonases, galactanases, pectin
lyases, pectin methyl esterases, cellobiohydrolases and/or
transglutaminases. These detergent, disinfectant or cleanser
compositions can also include dyes, colorants, odorants, bleaches,
buffers, builders, enzyme "enhancing agents" (see, e.g., U.S.
Patent application no. 20030096394) and stabilizers.
[0632] The addition of xylanases 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 compositions of the invention as long as
the enzyme is active at or tolerant of the pH and/or temperature of
the intended use. In addition, the xylanases of the invention can
be used in a cleaning composition without detergents, again either
alone or in combination with builders and stabilizers.
[0633] 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.
[0634] 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 xylanase
of the invention may be included as a detergent, disinfectant or
cleanser additive. The detergent, disinfectant or cleanser
composition of the invention may, for example, be formulated as a
hand or machine laundry detergent, disinfectant or cleanser
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 xylanase of the invention. Alternatively, a xylanase of
the invention can be formulated as a detergent, disinfectant or
cleanser composition for use in general household hard surface
cleaning operations. In alternative aspects, detergent,
disinfectant or cleanser additives and detergent, disinfectant or
cleanser compositions of the invention may comprise one or more
other enzymes such as a xylanase, a lipase, a protease, a cutinase,
an esterase, another xylanase, a carbohydrase, a cellulase, a
pectinase, a mannanase, an arabinase, a galactanase, a xylanase, an
oxidase, e.g., a lactase, and/or a peroxidase (see also, above).
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, xylanase 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.
[0635] When formulated as compositions suitable for use in a
laundry machine washing method, the xylanases of the invention can
comprise both a surfactant and a builder compound. They can
additionally comprise one or more detergent components, e.g.,
organic polymeric compounds, bleaching agents, additional enzymes,
suds suppressors, dispersants, lime-soap dispersants, soil
suspension and anti-redeposition agents and corrosion inhibitors.
Laundry compositions of the invention can also contain softening
agents, as additional detergent components. Such compositions
containing carbohydrase can provide fabric cleaning, stain removal,
whiteness maintenance, softening, color appearance, dye transfer
inhibition and sanitization when formulated as laundry detergent
compositions.
[0636] The density of the laundry detergent, disinfectant or
cleanser compositions of the invention can range from about 200 to
1500 g/liter, or, about 400 to 1200 g/liter, or, about 500 to 950
g/liter, or, 600 to 800 g/liter, of composition; this can be
measured at about 20.degree. C.
[0637] In one aspect, the "compact" form of laundry detergent,
disinfectant or cleanser compositions of the invention is best
reflected by density and, in terms of composition, by the amount of
inorganic filler salt. Inorganic filler salts are conventional
ingredients of detergent compositions in powder form. In
conventional detergent compositions, the filler salts are present
in substantial amounts, typically 17% to 35% by weight of the total
composition. In one aspect of the compact compositions, the filler
salt is present in amounts not exceeding 15% of the total
composition, or, not exceeding 10%, or, not exceeding 5% by weight
of the composition. The inorganic filler salts can be selected from
the alkali and alkaline-earth-metal salts of sulphates and
chlorides, e.g., sodium sulphate.
[0638] Liquid detergent compositions of the invention can also be
in a "concentrated form." In one aspect, the liquid detergent,
disinfectant or cleanser compositions can contain a lower amount of
water, compared to conventional liquid detergents, disinfectants or
cleansers. In alternative aspects, the water content of the
concentrated liquid detergent is less than 40%, or, less than 30%,
or, less than 20% by weight of the detergent, disinfectant or
cleanser composition. Detergent, disinfectant or cleanser compounds
of the invention can comprise formulations as described in WO
97/01629.
[0639] Xylanases of the invention can be useful in formulating
various detergent, cleaning, disinfectant or cleanser compositions.
A number of known compounds are suitable surfactants including
nonionic, anionic, cationic, or zwitterionic detergents, can be
used, e.g., as disclosed in U.S. Pat. Nos. 4,404,128; 4,261,868;
5,204,015. In addition, xylanases can be used, for example, in bar
or liquid soap applications, dish care formulations, contact lens
cleaning solutions or products, peptide hydrolysis, waste
treatment, textile applications, as fusion-cleavage enzymes in
protein production, and the like. Xylanases may provide enhanced
performance in a detergent composition as compared to another
detergent xylanase, that is, the enzyme group may increase cleaning
of certain enzyme sensitive stains such as grass or blood, as
determined by usual evaluation after a standard wash cycle.
Xylanases can be formulated into known powdered and liquid
detergents having pH between 6.5 and 12.0 at levels of about 0.01
to about 5% (for example, about 0.1% to 0.5%) by weight. These
detergent cleaning compositions can also include other enzymes such
as known xylanases, xylanases, proteases, amylases, cellulases,
mannanases, lipases or endoglycosidases, redox enzymes such as
catalases and laccases, as well as builders, stabilizers,
fragrances and pigments.
[0640] In one aspect, the invention provides detergent,
disinfectant or cleanser compositions having xylanase activity (a
xylanase of the invention) for use with fruit, vegetables and/or
mud and clay compounds (see, for example, U.S. Pat. No.
5,786,316).
[0641] In another aspect of the invention, the xylanases of the
invention can also be used in any detergent, disinfectant or
cleanser (cleaning solution) manufacturing process, wherein the
xylanase is used as an antimicrobial or microbial repellent.
Alternatively, the xylanases of the invention can be used in any
cleansing or washing process, wherein the xylanase is used as an
antimicrobial or microbial repellent. In another aspect of the
invention, the xylanase of the invention can be included in any
detergent or cleanser composition, wherein the xylanases of the
invention act as an antimicrobial or microbial repellent in the
composition.
Treating Foods and Food Processing
[0642] The xylanases of the invention have numerous applications in
food processing industry. For example, in one aspect, the xylanases
of the invention are used to improve the extraction of oil from
oil-rich plant material, e.g., oil-rich seeds, for example, soybean
oil from soybeans, olive oil from olives, rapeseed oil from
rapeseed and/or sunflower oil from sunflower seeds.
[0643] The xylanases of the invention can be used for separation of
components of plant cell materials. For example, xylanases of the
invention can be used in the separation of xylan-rich material
(e.g., plant cells) into components. In one aspect, xylanases of
the invention can be used to separate xylan-rich or oil-rich crops
into valuable protein and oil and hull fractions. The separation
process may be performed by use of methods known in the art.
[0644] The xylanases of the invention can be used in the
preparation of fruit or vegetable juices, syrups, extracts and the
like to increase yield. The xylanases of the invention can be used
in the enzymatic treatment (e.g., hydrolysis of xylan-comprising
plant materials) of various plant cell wall-derived materials or
waste materials, e.g., from cereals, grains, wine or juice
production, or agricultural residues such as vegetable hulls, bean
hulls, sugar beet pulp, olive pulp, potato pulp, and the like. The
xylanases of the invention can be used to modify the consistency
and appearance of processed fruit or vegetables. The xylanases of
the invention can be used to treat plant material to facilitate
processing of plant material, including foods, facilitate
purification or extraction of plant components. The xylanases of
the invention can be used to improve feed value, decrease the water
binding capacity, improve the degradability in waste water plants
and/or improve the conversion of plant material to ensilage, and
the like.
[0645] In one aspect, xylanases of the invention are used in baking
applications, e.g., cookies and crackers, to hydrolyze xylans such
as arabinoxylans. In one aspect, xylanases of the invention are
used to create non-sticky doughs that are not difficult to machine
and to reduce biscuit size. Xylanases of the invention can be used
to hydrolyze arabinoxylans to prevent rapid rehydration of the
baked product resulting in loss of crispiness and reduced
shelf-life. In one aspect, xylanases of the invention are used as
additives in dough processing. In one aspect, xylanases of the
invention are used in dough conditioning, wherein in one aspect the
xylanases possess high activity over a temperature range of about
25-35.degree. C. and at near neutral pH (7.0-7.5). In one aspect,
dough conditioning enzymes can be inactivated at the extreme
temperatures of baking (>500.degree. F.). The enzymes of the
invention can be used in conjunction with any dough processing
protocol, e.g., as in U.S. Patent App. No. 20050281916.
[0646] In one aspect, xylanases of the invention are used as
additives in dough processing to perform optimally under dough pH
and temperature conditions. In one aspect, an enzyme of the
invention is used for dough conditioning. In one aspect, a xylanase
of the invention possesses high activity over a temperature range
of 25-35.degree. C. and at near neutral pH (7.0-7.5). In one
aspect, the enzyme is inactivated at the extreme temperatures of
baking, for example, >500.degree. F.
[0647] In another aspect of the invention, the xylanases of the
invention can also be used in any food or beverage treatment or
food or beverage processing process, wherein the xylanase is used
as an antimicrobial or microbial repellent. In another aspect of
the invention, the xylanase of the invention can be included in any
food or beverage composition, wherein the xylanases of the
invention act as an antimicrobial or microbial repellent in the
composition.
Animal Feeds and Food or Feed or Food Additives (Supplements)
[0648] The invention provides methods for treating animal feeds and
foods and food or feed additives (supplements) using xylanases of
the invention, animals including mammals (e.g., humans), birds,
fish and the like. The invention provides animal feeds, foods, and
additives (supplements) comprising xylanases of the invention. In
one aspect, treating animal feeds, foods and additives using
xylanases of the invention can help in the availability of
nutrients, e.g., starch, protein, and the like, in the animal feed
or additive (supplements). By breaking down difficult to digest
proteins or indirectly or directly unmasking starch (or other
nutrients), the xylanase makes nutrients more accessible to other
endogenous or exogenous enzymes. The xylanase can also simply cause
the release of readily digestible and easily absorbed nutrients and
sugars.
[0649] When added to animal feed, xylanases of the invention
improve the in vivo break-down of plant cell wall material partly
due to a reduction of the intestinal viscosity (see, e.g., Bedford
et al., Proceedings of the 1st Symposium on Enzymes in Animal
Nutrition, 1993, pp. 73-77), whereby a better utilization of the
plant nutrients by the animal is achieved. Thus, by using xylanases
of the invention in feeds the growth rate and/or feed conversion
ratio (i.e., the weight of ingested feed relative to weight gain)
of the animal is improved.
[0650] The animal feed additive of the invention may be a
granulated enzyme product which 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.
[0651] Xylanases 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. Xylanases
can be added to animal feed or food compositions containing high
amounts of xylans, e.g., feed or food containing plant material
from cereals, grains and the like. When added to the feed or food
the xylanase significantly improves the in vivo break-down of
xylan-containing material, e.g., plant cell walls, whereby a better
utilization of the plant nutrients by the animal (e.g., human) is
achieved. In one aspect, the growth rate and/or feed conversion
ratio (i.e., the weight of ingested feed relative to weight gain)
of the animal is improved. For example a partially or indigestible
xylan-comprising protein is fully or partially degraded by a
xylanase of the invention, e.g., in combination with another
enzyme, e.g., beta-galactosidase, to peptides and galactose and/or
galactooligomers. These enzyme digestion products are more
digestible by the animal. Thus, xylanases of the invention can
contribute to the available energy of the feed or food. Also, by
contributing to the degradation of xylan-comprising proteins, a
xylanase of the invention can improve the digestibility and uptake
of carbohydrate and non-carbohydrate feed or food constituents such
as protein, fat and minerals.
[0652] In another aspect, xylanase 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 xylanase of the invention is produced in
recoverable quantities. The xylanase 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, and rheological properties, or to
destroy an antinutritive factor.
[0653] In one aspect, the invention provides methods for removing
oligosaccharides from feed prior to consumption by an animal
subject using a xylanase of the invention. In this process a feed
is formed having an increased metabolizable energy value. In
addition to xylanases of the invention, galactosidases, cellulases
and combinations thereof can be used. In one aspect, the enzyme is
added in an amount equal to between about 0.1% and 1% by weight of
the feed material. In one aspect, the feed is a cereal, a wheat, a
grain, a soybean (e.g., a ground soybean) material. See, e.g., U.S.
Pat. No. 6,399,123.
[0654] In another aspect, the invention provides methods for
utilizing xylanase as a nutritional supplement in the diets of
animals by preparing a nutritional supplement containing a
recombinant xylanase enzyme comprising at least thirty contiguous
amino acids of a sequence of the invention, and administering the
nutritional supplement to an animal to increase the utilization of
xylan contained in food ingested by the animal.
[0655] In yet another aspect, the invention provides an edible
pelletized enzyme delivery matrix and method of use for delivery of
xylanase to an animal, for example as a nutritional supplement. The
enzyme delivery matrix readily releases a xylanase enzyme, such as
one having an amino acid sequence of the invention, or at least 30
contiguous amino acids thereof, in aqueous media, such as, for
example, the digestive fluid of an animal. The invention enzyme
delivery matrix is prepared from a granulate edible carrier
selected from such components as grain germ that is spent of oil,
hay, alfalfa, timothy, soy hull, sunflower seed meal, corn meal,
soy meal, wheat midd, and the like, that readily disperse the
recombinant enzyme contained therein into aqueous media. In use,
the edible pelletized enzyme delivery matrix is administered to an
animal to delivery of xylanase to the animal. Suitable grain-based
substrates may comprise or be derived from any suitable edible
grain, such as wheat, corn, soy, sorghum, alfalfa, barley, and the
like. An exemplary grain-based substrate is a corn-based substrate.
The substrate may be derived from any suitable part of the grain,
but is preferably a grain germ approved for animal feed use, such
as corn germ that is obtained in a wet or dry milling process. The
grain germ preferably comprises spent germ, which is grain germ
from which oil has been expelled, such as by pressing or hexane or
other solvent extraction. Alternatively, the grain germ is expeller
extracted, that is, the oil has been removed by pressing.
[0656] 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.
[0657] The enzyme delivery matrix can be in the form of granules
having a granule size ranging from about 4 to about 400 mesh (USS);
more preferably, about 8 to about 80 mesh; and most preferably
about 14 to about 20 mesh. If the grain germ is spent via solvent
extraction, use of a lubricity agent such as corn oil may be
necessary in the pelletizer, but such a lubricity agent ordinarily
is not necessary if the germ is expeller extracted. In other
aspects of the invention, the matrix is prepared by other
compacting or compressing processes such as, for example, by
extrusion of the grain-based substrate through a die and grinding
of the extrudate to a suitable granule size.
[0658] The enzyme delivery matrix may further include a
polysaccharide component as a cohesiveness agent to enhance the
cohesiveness of the matrix granules. The cohesiveness agent is
believed to provide additional hydroxyl groups, which enhance the
bonding between grain proteins within the matrix granule. It is
further believed that the additional hydroxyl groups so function by
enhancing the hydrogen bonding of proteins to starch and to other
proteins. The cohesiveness agent may be present in any amount
suitable to enhance the cohesiveness of the granules of the enzyme
delivery matrix. Suitable cohesiveness agents include one or more
of dextrins, maltodextrins, starches, such as corn starch, flours,
cellulosics, hemicellulosics, and the like. For example, the
percentage of grain germ and cohesiveness agent in the matrix (not
including the enzyme) is 78% corn germ meal and 20% by weight of
corn starch.
[0659] Because the enzyme-releasing matrix of the invention is made
from biodegradable materials and contains moisture, the matrix may
be subject to spoilage, such as by molding. To prevent or inhibit
such molding, the matrix may include a mold inhibitor, such as a
propionate salt, which may be present in any amount sufficient to
inhibit the molding of the enzyme-releasing matrix, thus providing
a delivery matrix in a stable formulation that does not require
refrigeration.
[0660] The xylanase enzyme contained in the invention enzyme
delivery matrix and methods is preferably a thermostable xylanase,
as described herein, so as to resist inactivation of the xylanase
during manufacture where elevated temperatures and/or steam may be
employed to prepare the pelletized 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.
[0661] A coating can be applied to the invention 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.
Or, the coating may be 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.
[0662] 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 xylanase enzyme encoded by an amino acid sequence of the
invention or at least 30 consecutive amino acids thereof.
Preferably, the process includes compacting or compressing the
particles of enzyme-releasing matrix into granules, which can be
accomplished by pelletizing. The mold inhibitor and cohesiveness
agent, when used, can be added at any suitable time, and can be
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 can be in the ranges set forth above with
respect to the moisture content in the finished product, and can be
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 can be 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.
[0663] In one aspect, the pellet mill is operated with a 1/8 in. by
2 inch die at 100 lb./min. pressure at 82.degree. C. to provide
pellets, which then are crumbled in a pellet mill crumbler to
provide discrete plural particles having a particle size capable of
passing through an 8 mesh screen but being retained on a 20 mesh
screen.
[0664] The thermostable xylanases of the invention can be used in
the pellets of the invention. They can have high optimum
temperatures and high heat resistance such that an enzyme reaction
at a temperature not hitherto carried out can be achieved. The gene
encoding the xylanase according to the present invention (e.g., as
set forth in any of the sequences in the invention) can be used in
preparation of xylanases (e.g., using GSSM as described herein)
having characteristics different from those of the xylanases of the
invention (in terms of optimum pH, optimum temperature, heat
resistance, stability to solvents, specific activity, affinity to
substrate, secretion ability, translation rate, transcription
control and the like). Furthermore, a polynucleotide of the
invention may be employed for screening of variant xylanases
prepared by the methods described herein to determine those having
a desired activity, such as improved or modified thermostability or
thermotolerance. For example, U.S. Pat. No. 5,830,732, describes a
screening assay for determining thermotolerance of a xylanase.
[0665] In another aspect of the invention, the xylanases of the
invention can also be used in any animal feed, animal food or feed
additive production process, wherein the xylanase is used as an
antimicrobial or microbial repellent. In another aspect of the
invention, the xylanase of the invention can be included in any
animal feed, animal food or feed additive composition, wherein the
xylanases of the invention act as an antimicrobial or microbial
repellent in the composition.
Waste Treatment
[0666] The xylanases of the invention can be used in a variety of
other industrial applications, e.g., in waste treatment. For
example, in one aspect, the invention provides a solid waste
digestion process using xylanases 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
xylanases 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.
[0667] In another aspect of the invention, the xylanases of the
invention can also be used in any waste treatment process, wherein
the xylanase is used as an antimicrobial or microbial repellent. In
another aspect of the invention, the xylanase of the invention can
be included in any waste treatment composition, wherein the
xylanases of the invention act as an antimicrobial or microbial
repellent in the composition.
Oral Care Products
[0668] The invention provides oral care product comprising
xylanases of the invention, including the enzyme mixtures or
"cocktails" of the invention. Exemplary oral care products include
toothpastes, dental creams, gels or tooth powders, odontics, mouth
washes, pre- or post brushing rinse formulations, chewing gums,
lozenges, or candy. See, e.g., U.S. Pat. No. 6,264,925.
[0669] In another aspect of the invention, the xylanases of the
invention, including the enzyme mixtures or "cocktails" of the
invention, can also be used in any oral care manufacturing process,
wherein the xylanase is used as an antimicrobial or microbial
repellent. In another aspect of the invention, the xylanase of the
invention, including the enzyme mixtures or "cocktails" of the
invention, can be included in any oral care composition, wherein
the xylanases of the invention act as an antimicrobial or microbial
repellent in the composition.
Brewing and Fermenting
[0670] The invention provides methods of brewing (e.g., fermenting)
beer comprising xylanases of the invention, including the enzyme
mixtures or "cocktails" of the invention. In one exemplary process,
starch-containing raw materials are disintegrated and processed to
form a malt. A xylanase of the invention is used at any point in
the fermentation process. For example, xylanases 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 to 25.degree. C. for 3 to 6 days
when enzyme synthesis is stimulated under the control of
gibberellins. In one aspect, xylanases of the invention are added
at this (or any other) stage of the process. Xylanases of the
invention can be used in any beer or alcoholic beverage 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.
[0671] In one aspect, an enzyme of the invention is used to improve
filterability and wort viscosity and to obtain a more complete
hydrolysis of endosperm components. Use of an enzyme of the
invention would also increase extract yield. The process of brewing
involves germination of the barley grain (malting) followed by the
extraction and the breakdown of the stored carbohydrates to yield
simple sugars that are used by yeast for alcoholic fermentation.
Efficient breakdown of the carbohydrate reserves present in the
barley endosperm and brewing adjuncts requires the activity of
several different enzymes.
[0672] In one aspect, an enzyme of the invention has activity in
slightly acidic pH (e.g., 5.5-6.0) in, e.g., the 40.degree. C. to
70.degree. C. temperature range; and, in one aspect, with
inactivation at 95.degree. C. Activity under such conditions would
be optimal, but are not an essential requirement for efficacy. In
one aspect, an enzyme of the invention has activity between
40-75.degree. C., and pH 5.5-6.0; stable at 70.degree. C. for at
least 50 minutes, and, in one aspect, is inactivated at
96-100.degree. C. Enzymes of the invention can be used with other
enzymes, e.g., beta-1,4-endoglucanases and amylases.
[0673] In another aspect of the invention, the xylanases of the
invention, including the enzyme mixtures or "cocktails" of the
invention, can also be used in any brewing or fermentation process,
wherein the xylanase is used as an antimicrobial or microbial
repellent. In another aspect of the invention, the xylanase of the
invention can be included in any brewed or fermented composition,
wherein the xylanases of the invention act as an antimicrobial or
microbial repellent in the composition.
Biomass Conversion and Biofuel Production
[0674] The invention provides methods and processes for biomass
conversion, e.g., to a biofuel, such as bioethanol, biomethanol,
biopropanol and/or biobutanol and the like, using enzymes of the
invention, including the enzyme mixtures or "cocktails" of the
invention. Thus, the invention provides fuels, e.g., biofuels, such
as bioethanols, comprising a polypeptide of the invention,
including the enzyme mixtures or "cocktails" of the invention, or a
polypeptide encoded by a nucleic acid of the invention. In
alternative aspects, the fuel is derived from a plant material,
which optionally comprises potatoes, soybean (rapeseed), barley,
rye, corn, oats, wheat, beets or sugar cane, and optionally the
fuel comprises a bioethanol or a gasoline-ethanol mix.
[0675] The invention provides methods for making a fuel comprising
contacting a composition comprising a xylan, hemicellulose,
cellulose or a fermentable sugar with a polypeptide of the
invention, or a polypeptide encoded by a nucleic acid of the
invention, or any one of the mixtures or "cocktails" or products of
manufacture of the invention. In alternative embodiments, the
composition comprising a xylan, hemicellulose, a cellulose or a
fermentable sugar comprises a plant, plant product or plant
derivative, and the plant or plant product can comprise cane sugar
plants or plant products, beets or sugarbeets, wheat, corn,
soybeans, potato, rice or barley. In alternative embodiments, the
polypeptide has activity comprising catalyzing hydrolysis of
internal .beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase
linkages; and/or degrading a linear polysaccharide beta-1,4-xylan
into xylose. In one aspect, the fuel comprises a bioethanol or a
gasoline-ethanol mix, or a biopropanol or a gasoline-propanol mix,
or a biobutanol or a gasoline-butanol mix, or a biomethanol or a
gasoline-methanol mix, or any combination thereof.
[0676] The invention provides methods for making bioethanol,
biobutanol, biomethanol and/or a biopropanol comprising contacting
a composition comprising a xylan, hemi-cellulose, cellulose or a
fermentable sugar with a polypeptide of the invention, or a
polypeptide encoded by a nucleic acid of the invention, or any one
of the mixtures or "cocktails" or products of manufacture of the
invention. In alternative embodiments, the composition comprising a
cellulose or a fermentable sugar comprises a plant, plant product
or plant derivative, and the plant or plant product can comprise
cane sugar plants or plant products, beets or sugarbeets, wheat,
corn, soybeans, potato, rice or barley, and the polypeptide can
have activity comprising cellulase, glucanase, cellobiohydrolase,
beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, and/or
arabinofuranosidase activity.
[0677] The invention provides enzyme ensembles, or "cocktails", for
depolymerization of cellulosic and hemicellulosic polymers, xylans,
and polysaccharides to metabolizeable carbon moieties comprising a
polypeptide of the invention, or a polypeptide encoded by a nucleic
acid of the invention. In alternative embodiments, the polypeptide
has activity comprising catalyzing hydrolysis of internal
.beta.-1,4-xylosidic linkages or endo-.beta.-1,4-glucanase
linkages; and/or degrading a linear polysaccharide beta-1,4-xylan
into xylose. The enzyme ensembles, or "cocktails", of the invention
can be in the form of a composition (e.g., a formulation, liquid or
solid), e.g., as a product of manufacture. The invention further
enzymes, enzyme ensembles, or "cocktails" for depolymerization of
cellulosic and hemicellulosic polymers, xylans and polysaccharides,
to simpler moieties, such as sugars, which are then microbially
fermented to generate products such as succinic acid, lactic acid,
or acetic acid.
[0678] The invention provides compositions (including products of
manufacture, enzyme ensembles, or "cocktails") comprising a mixture
(or "cocktail") of hemicellulose- and cellulose-hydrolyzing
enzymes, wherein the xylan-hydrolyzing enzymes comprise at least
one of each of a xylanase of the invention and at least one,
several or all of a cellulase, glucanase, a cellobiohydrolase
and/or a .beta.-glucosidase. In alternative embodiments, the
xylan-hydrolyzing and/or hemicellulose-hydrolyzing mixtures of the
invention comprise at least one of each of a xylanase of the
invention and at least one or both of a .beta.-xylosidase and/or an
arabinofuranosidase.
[0679] The invention provides compositions (including products of
manufacture, enzyme ensembles, or "cocktails") comprising a mixture
(or "cocktail") of xylan-hydrolyzing, hemicellulose- and/or
cellulose-hydrolyzing enzymes comprising at least one, several or
all of a cellulase, a glucanase, a cellobiohydrolase and/or an
arabinofuranosidase, and a xylanase of this invention.
[0680] The invention provides compositions (including products of
manufacture, enzyme ensembles, or "cocktails") comprising mixture
(or "cocktail") of xylan-hydrolyzing, hemicellulose- and/or
cellulose-hydrolyzing enzymes comprising at least one, several or
all of a cellulase, a glucanase; a cellobiohydrolase; an
arabinofuranosidase; a xylanase; a .beta.-glucosidase; a
.beta.-xylosidase; and at least one enzyme of the invention.
[0681] The invention provides compositions (including products of
manufacture, enzyme ensembles, or "cocktails") comprising mixture
(or "cocktail") of enzymes comprising, in addition to at least one
enzyme of the invention: (1) a glucanase which cleaves internal
.beta.-1,4 linkages resulting in shorter glucooligosaccharides, (2)
a cellobiohydrolase which acts in an "exo" manner processively
releasing cellobiose units (.beta.-1,4 glucose--glucose
disaccharide), and/or (3) a .beta.-glucosidase for releasing
glucose monomer from short cellooligosaccharides (e.g.,
cellobiose).
Biomass Conversion and Production of Clean Biofuels
[0682] The invention provides compositions and processes using
enzymes of this invention, including mixtures, or "cocktails" of
enzymes of the invention, for the conversion of a biomass, or any
organic material, e.g., any xylan-comprising or lignocellulosic
material (e.g., any composition comprising a xylan, cellulose,
hemicellulose and/or lignin), to a fuel, such as a biofuel (e.g.,
bioethanol, biobutanol, biomethanol and/or a biopropanol),
including biodiesels, in addition to feeds, foods, food or feed
supplements (additives), pharmaceuticals and chemicals. Thus, the
compositions and methods of the invention provide effective and
sustainable alternatives or adjuncts to use of petroleum-based
products, e.g., as a mixture of a biofuel (e.g., bioethanol,
biobutanol, biomethanol and/or a biopropanol) and gasoline and/or
diesel fuel.
[0683] The invention provides cells and/or organisms expressing
enzymes of the invention (e.g., wherein the cells or organisms
comprise as heterologous nucleic acids a sequence of this
invention) for participation in chemical cycles involving natural
biomass (e.g., plant) conversion. In one aspect, enzymes and
methods for the conversion are used in enzyme ensembles (or
"cocktails") for the efficient depolymerization of xylan-comprising
compositions, or xylan, cellulosic and hemicellulosic polymers, to
metabolizeable carbon moieties. 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.
[0684] The invention provides methods, enzymes and mixtures of
enzymes or "cocktails" of the invention, for processing a material,
e.g., a biomass material, comprising a cellooligsaccharide, an
arabinoxylan oligomer, a lignin, a lignocellulose, a xylan, a
glucan, a cellulose and/or a fermentable sugar comprising
contacting the composition with a polypeptide of the invention, or
a polypeptide encoded by a nucleic acid of the invention, wherein
optionally the material is derived from an agricultural crop (e.g.,
wheat, barley, potatoes, switchgrass, poplar wood), is a byproduct
of a food or a feed production, is a lignocellulosic waste product,
or is a plant residue or a waste paper or waste paper product, and
optionally the plant residue comprise stems, leaves, hulls, husks,
corn or corn cobs, corn stover, corn fiber, hay, straw (e.g., rice
straw or wheat straw), sugarcane bagasse, sugar beet pulp, citrus
pulp, and citrus peels, wood, wood thinnings, wood chips, wood
pulp, pulp waste, wood waste, wood shavings and sawdust,
construction and/or demolition wastes and debris (e.g., wood, wood
shavings and sawdust), and optionally the paper waste comprises
discarded or used photocopy paper, computer printer paper, notebook
paper, notepad paper, typewriter paper, newspapers, magazines,
cardboard and paper-based packaging materials, and recycled paper
materials. In addition, urban wastes, e.g., the paper fraction of
municipal solid waste, municipal wood waste, and municipal green
waste, along with other materials containing sugar, starch, and/or
cellulose can be used. Optionally the processing of the material,
e.g., the biomass material, generates a bioalcohol, e.g., a
bioethanol, biomethanol, biobutanol or biopropanol.
[0685] Alternatively, the polypeptide of the invention may be
expressed in the biomass plant material or feedstock itself.
[0686] The methods of the invention also include taking the
converted biomass (e.g., lignocellulosic) material (processed by
enzymes of the invention) and making it into a fuel (e.g., a
biofuel such as a bioethanol, biobutanol, biomethanol, a
biopropanol, or a biodiesel) by fermentation and/or by chemical
synthesis. In one aspect, the produced sugars are fermented and/or
the non-fermentable products are gasified.
[0687] The methods of the invention also include converting algae,
virgin vegetable oils, waste vegetable oils, animal fats and
greases (e.g., tallow, lard, and yellow grease), or sewage, using
enzymes of the invention, and making it into a fuel (e.g., a
bioalcohol, e.g., a bioethanol, biomethanol, biobutanol or
biopropanol, or biodiesel) by fermentation and/or by chemical
synthesis or conversion.
[0688] The enzymes of the invention (including, for example,
organisms, such as microorganisms, e.g., fungi, yeast or bacteria,
and plants and plant cells and plant parts, e.g., seeds, making and
in some aspects secreting recombinant enzymes of the invention) can
be used in or included/integrated at any stage of any organic
matter/biomass conversion process, e.g., at any one step, several
steps, or included in all of the steps, or all of the following
methods of biomass conversion processes, or all of these biofuel
alternatives: [0689] Direct combustion: the burning of material by
direct heat and is the simplest biomass technology; can be very
economical if a biomass source is nearby. [0690] 1. Pyrolysis: is
the thermal degradation of biomass by heat in the absence of
oxygen. In one aspect, biomass is heated to a temperature between
about 800 and 1400 degrees Fahrenheit, but no oxygen is introduced
to support combustion resulting in the creation of gas, fuel oil
and charcoal. [0691] 2. Gasification: Biomass can be used to
produce methane through heating or anaerobic digestion. Syngas, a
mixture of carbon monoxide and hydrogen, can be derived from
biomass. [0692] Landfill Gas: is generated by the decay (anaerobic
digestion) of buried garbage in landfills. When the organic waste
decomposes, it generates gas consisting of approximately 50%
methane, the major component of natural gas. [0693] Anaerobic
Digestion: converts organic matter to a mixture of methane, the
major component of natural gas, and carbon dioxide. In one aspect,
biomass such as waterwaste (sewage), manure, or food processing
waste, is mixed with water and fed into a digester tank without
air. [0694] Fermentation [0695] Alcohol Fermentation: Fuel alcohol
is produced by converting cellulosic mass and/or starch to sugar,
fermenting the sugar to alcohol, then separating the alcohol water
mixture by distillation. Feedstocks such as dedicated crops (e.g.,
wheat, barley, potatoes, switchgrass, poplar wood), agricultural
residues and wastes (e.g., rice straw, corn stover, wheat straw,
sugarcane bagasse, rice hulls, corn fiber, sugar beet pulp, citrus
pulp, and citrus peels), forestry wastes (e.g., hardwood and
softwood thinnings, hardwood and softwood residues from timber
operations, wood shavings, and sawdust), urban wastes (e.g., paper
fraction of municipal solid waste, municipal wood waste, municipal
green waste), wood wastes (e.g., saw mill waste, pulp mill waste,
construction waste, demolition waste, wood shavings, and sawdust),
and waste paper or other materials containing sugar, starch, and/or
cellulose can be converted to sugars and then to alcohol by
fermentation with yeast. Alternatively, materials containing sugars
can be converted directly to alcohol by fermentation. [0696]
Transesterification: An exemplary reaction for converting oil to
biodiesel is called transesterification. The transesterification
process reacts an alcohol (like methanol) with the triglyceride
oils contained in vegetable oils, animal fats, or recycled greases,
forming fatty acid alkyl esters (biodiesel) and glycerin. The
reaction requires heat and a strong base catalyst, such as sodium
hydroxide or potassium hydroxide. [0697] Biodiesel: Biodiesel is a
mixture of fatty acid alkyl esters made from vegetable oils, animal
fats or recycled greases. Biodiesel can be used as a fuel for
vehicles in its pure form, but it is usually used as a petroleum
diesel additive to reduce levels of particulates, carbon monoxide,
hydrocarbons and air toxics from diesel-powered vehicles. [0698]
Hydrolysis: includes hydrolysis of a compound, e.g., a biomass,
such as a lignocellulosic material, catalyzed using an enzyme of
the instant invention. [0699] Cogeneration: is the simultaneous
production of more than one form of energy using a single fuel and
facility. In one aspect, biomass cogeneration has more potential
growth than biomass generation alone because cogeneration produces
both heat and electricity.
[0700] In one aspect, the polypeptides of the invention have
sufficient enzymatic activity, e.g., a xylanase, a mannanase and/or
a glucanase activity, for, or can be used with other enzymes in a
process for, generating a biodiesel or a fuel, (e.g., a bioalcohol,
e.g., a bioethanol, biomethanol, biobutanol or biopropanol, or
biodiesel) from an organic material, e.g., a biomass, such as
compositions derived from plants and animals, including any
agricultural crop or other renewable feedstock, an agricultural
residue or an animal waste, the organic components of municipal and
industrial wastes, or construction or demolition wastes or debris,
or microorganisms such as algae or yeast.
[0701] In one aspect, polypeptides of the invention are used in
processes for converting an organic material, e.g., a biomass, such
as a lignocellulosic biomass, to a biofuel, such as a bioethanol,
biobutanol, biomethanol, a biopropanol, or otherwise are used in
processes for hydrolyzing or digesting biomaterials such that they
can be used as a biofuel (including biodiesel or bioethanol,
biobutanol, biomethanol or biopropanol), or for making it easier
for the biomass to be processed into a fuel. In an alternative
aspect, polypeptides of the invention are used in processes for a
transesterification process reacting an alcohol (like methanol,
butanol, propanol, ethanol) with a triglyceride oil contained in a
vegetable oil, animal fat or recycled greases, forming fatty acid
alkyl esters (biodiesel) and glycerin. In one aspect, biodiesel is
made from soybean oil or recycled cooking oils. Animal's fats,
other vegetable oils, and other recycled oils can also be used to
produce biodiesel, depending on their costs and availability. In
another aspect, blends of all kinds of fats and oils are used to
produce a biodiesel fuel of the invention.
[0702] Enzymes of the invention can also be used in glycerin
refining. The glycerin by-product contains unreacted catalyst and
soaps that are neutralized with an acid. Water and alcohol are
removed to produce 50% to 80% crude glycerin. The remaining
contaminants include unreacted fats and oils, which can be
processes using the polypeptides of the invention. In a large
biodiesel plants of the invention, the glycerin can be further
purified, e.g., to 99% or higher purity, for the pharmaceutical and
cosmetic industries.
[0703] Fuels (including bioalcohols such as bioethanols,
biomethanols, biobutanols or biopropanols, or biodiesels) made
using the polypeptides of the invention, including the mixture of
enzymes or "cocktails" of the invention, can be used with fuel
oxygenates to improve combustion characteristics. Adding oxygen
results in more complete combustion, which reduces carbon monoxide
emissions. This is another environmental benefit of replacing
petroleum fuels with biofuels (e.g., a fuel of the invention). A
biofuel made using the compositions and/or methods of this
invention can be blended with gasoline to form an E10 blend (about
5% to 10% ethanol and about 90% to 95% gasoline), but it can be
used in higher concentrations such as E85 or in its pure form. A
biofuel made using the compositions and/or methods of this
invention can be blended with petroleum diesel to form a B20 blend
(20% biodiesel and 80% petroleum diesel), although other blend
levels can be used up to B100 (pure biodiesel).
[0704] In one aspect, the polypeptides of this invention are used
in processes for converting organic material, e.g., a biomass, such
as a lignocellulosic biomass, to methanol, butanol, propanol and/or
ethanol. The invention also provides processes for making ethanol
("bioethanol") methanol, butanol and/or propanol from compositions
comprising organic material, e.g., a biomass, such as a
lignocellulosic biomass. The organic material, e.g., a biomass,
such as a lignocellulose biomass material, can be obtained from
agricultural crops, as a byproduct of food or feed production, or
as biomass waste products, such as plant residues and waste paper
or construction and/or demolition wastes or debris. Examples of
suitable plant residues for treatment with polypeptides of the
invention include grains, seeds, stems, leaves, hulls, husks, corn
cobs, corn stover, straw, grasses (e.g., Indian grass, such as
Sorghastrum nutans; or, switch grass, e.g., Panicum species, such
as Panicum virgatum), 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. Examples
of construction and demolition wastes and debris include wood, wood
scraps, wood shavings and sawdust.
[0705] In one aspect, the enzymes and methods of the invention can
be used in conjunction with more "traditional" means of making
methanol, butanol, propanol and/or ethanol from biomass, e.g., as
methods comprising hydrolyzing biomass (e.g., lignocellulosic
materials) by subjecting dried biomass 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.
[0706] Another exemplary method that incorporated use of enzymes of
the invention comprises hydrolyzing biomass (e.g., lignocellulosic
materials) containing xylan, hemicellulose, cellulose and/or 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.
[0707] Another exemplary method that incorporated use of enzymes of
the invention comprises processing a 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.
[0708] Another exemplary method that incorporated use of enzymes of
the invention comprises prehydrolyzing biomass (e.g.,
lignocellulosic materials) 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.
[0709] 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.
[0710] In one aspect, methods of the invention for the enzymatic
degradation of biomass (e.g., lignocellulosic materials), e.g., for
production of a biofuel, e.g., an ethanol, from a biomass or any
organic material, can also comprise use of ultrasonic treatment of
a biomass material; see, e.g., U.S. Pat. No. 6,333,181.
[0711] In another aspect, methods of the invention for producing a
biofuel, e.g., an ethanol (a bioethanol) from a biomass (e.g., a
cellulosic) substrate comprise providing a reaction mixture in the
form of a slurry comprising biomass (e.g., a cellulosic) substrate,
an enzyme of this invention and a fermentation agent (e.g., within
a reaction vessel, such as a semi-continuously solids-fed
bioreactor), and the reaction mixture is reacted under conditions
sufficient to initiate and maintain a fermentation reaction (as
described, e.g., in U.S. Pat. App. No. 20060014260). In one aspect,
experiment or theoretical calculations can determine an optimum
feeding frequency. In one aspect, additional quantities of the
biomass (e.g., a cellulosic) substrate and the enzyme are provided
into the reaction vessel at an interval(s) according to the
optimized feeding frequency.
[0712] One exemplary process for making a biofuels and biodiesels
of the invention is described in U.S. Pat. App. Pub. Nos.
20050069998; 20020164730; and in one aspect comprises stages of
grinding the biomass (e.g., lignocellulosic material) (e.g., to a
size of 15-30 mm), subjecting the product obtained to steam
explosion pre-treatment (e.g., at a temperature of 190-230.degree.
C.) for between 1 and 10 minutes in a reactor; collecting the
pretreated material in a cyclone or related product of manufacture;
and separating the liquid and solid fractions by filtration in a
filter press, introducing the solid fraction in a fermentation
deposit and adding one or more enzymes of the invention, and in one
aspect, another enzyme is also added, e.g., a cellulase and/or
beta-glucosidase enzyme (e.g., dissolved in citrate buffer pH
4.8).
[0713] Another exemplary process for making a biofuels and
biodiesels of the invention comprising methanol, butanol, propanol
and/or ethanol using enzymes of the invention comprises pretreating
a starting material comprising a biomass (e.g., a lignocellulosic)
feedstock comprising at least a xylan, a hemicellulose and/or a
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 biomass (e.g., hemicellulose and/or 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.
[0714] Exemplary conditions for hydrolysis of biomass (e.g., a
lignocellulosic material) by an enzyme of this invention 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.
Biofuels and Biologically Produced Alcohols
[0715] The invention provides biofuels and synthetic fuels,
including liquids and gases (e.g., syngas) and biologically
produced alcohols, and methods for making them, using the
compositions (e.g., enzyme and nucleic acids, and transgenic
plants, animal, seeds and microorganisms) and methods of the
invention. The invention provides biofuels and biologically
produced alcohols comprising enzymes, nucleic acids, transgenic
plants, animals (e.g., microorganisms, such as bacteria or yeast)
and/or seeds of the invention. In one aspect, these biofuels and
biologically produced alcohols are produced from a biomass.
[0716] The invention provides biologically produced alcohols, such
as ethanol, methanol, propanol and butanol produced by methods of
the invention, which include the action of microbes and enzymes of
the invention through fermentation (hydrolysis) to result in an
alcohol fuel.
Biofuels as a Liquid or a Gas Gasoline
[0717] The invention provides biofuels and synthetic fuels in the
form of a gas, or gasoline, e.g., a syngas. In one aspect, methods
of the invention comprising use of enzymes of the invention for
chemical cycles for natural biomass conversion, e.g., for the
hydrolysis of a biomass to make a biofuel, e.g., a bioethanol,
biopropanol, bio-butanol or a biomethanol, or a synthetic fuel, in
the form of a liquid or as a gas, such as a "syngas".
[0718] For example, invention provides methods for making biofuel
gases and synthetic gas fuels ("syngas") comprising a bioethanol,
biopropanol, bio-butanol and/or a biomethanol made using a
polypeptide of the invention, or made using a method of the
invention; and in one aspect this biofuel gas of the invention is
mixed with a natural gas (can also be produced from biomass), e.g.,
a hydrogen or a hydrocarbon-based gas fuel.
[0719] In one aspect, the invention provides methods for processing
biomass to a synthetic fuel, e.g., a syngas, such as a syngas
produced from a biomass by gasification. In one aspect, the
invention provides methods for making an ethanol, propanol, butanol
and/or methanol gas from a sugar cane, e.g., a bagasse. In one
aspect, this fuel, or gas, is used as motor fuel, e.g., an
automotive, truck, airplane, boat, small engine, etc. fuel. In one
aspect, the invention provides methods for making an ethanol,
propanol, butanol and/or methanol from a plant, e.g., corn, or a
plant product, e.g., hay or straw (e.g., a rice straw or a wheat
straw, or any the dry stalk of any cereal plant), or an
agricultural waste product. Cellulosic ethanol, propanol, butanol
and/or methanol can be manufactured from a plant, e.g., corn, or
plant product, e.g., hay or straw, or an agricultural waste product
(e.g., as processed by Iogen Corporation of Ontario, Canada).
[0720] In one aspect, the ethanol, propanol, butanol and/or
methanol made using a method of composition of the invention can be
used as a fuel (e.g., a gasoline) additive (e.g., an oxygenator) or
in a direct use as a fuel. For example, a ethanol, propanol,
butanol and/or methanol, including a fuel, made by a method of the
invention can be mixed with ethyl tertiary butyl ether (ETBE), or
an ETBE mixture such as ETBE containing 47% ethanol as a biofuel,
or with MTBE (methyl tertiary-butyl ether). In another aspect, a
ethanol, propanol, butanol and/or methanol, including a fuel, made
by a method of the invention can be mixed with:
TABLE-US-00005 IUPAC name Common name but-1-ene .alpha.-butylene
cis-but-2-ene cis-.beta.-butylene trans-but-2-ene
trans-.beta.-butylene 2-methylpropene isobutylene
[0721] A butanol and/or ethanol made by a method of the invention
(e.g., using an enzyme of the invention) can be further processed
using "A.B.E." (Acetone, Butanol, Ethanol) fermentation; in one
aspect, butanol being the only liquid product. In one aspect, this
butanol and/or ethanol is burned "straight" in existing gasoline
engines (without modification to the engine or car), produces more
energy and is less corrosive and less water soluble than ethanol,
and can be distributed via existing infrastructures.
[0722] The invention also provides mixed alcohols wherein one,
several or all of the alcohols are made by processes comprising at
least one method of the invention (e.g., using an enzyme of the
invention), e.g., comprising a mixture of ethanol, propanol,
butanol, pentanol, hexanol, and heptanol, such as ECALENE.TM.
(Power Energy Fuels, Inc., Lakewood, Colo.), e.g.:
TABLE-US-00006 Exemplary Fuel of the Invention Component Weight %
Methanol 0% Ethanol 75% Propanol 9% Butanol 7% Pentanol 5% Hexanol
& Higher 4%
[0723] In one aspect, one, several or all of these alcohols are
made by a process of the invention using an enzyme of the
invention, and the process can further comprise a biomass-to-liquid
technology, e.g., a gasification process to produce syngas followed
by catalytic synthesis, or by a bioconversion of biomass to a mixed
alcohol fuel.
[0724] The invention also provides processes comprising use of an
enzyme of the invention incorporating (or, incorporated into) "gas
to liquid", or GTL; or "coal to liquid", or CTL; or "biomass to
liquid" or BTL; or "oilsands to liquid", or OTL, processes; and in
one aspect these processes of the invention are used to make
synthetic fuels. In one aspect, one of these processes of the
invention comprises making a biofuel (e.g., a synfuel) out of a
biomass using, e.g., the so-called "Fischer Tropsch" process (a
catalyzed chemical reaction in which carbon monoxide and hydrogen
are converted into liquid hydrocarbons of various forms; typical
catalysts used are based on iron and cobalt; the principal purpose
of this process is to produce a synthetic petroleum substitute for
use as synthetic lubrication oil or as synthetic fuel). In one
aspect, this synthetic biofuel of the invention can contain oxygen
and can be used as additive in high quality diesel and petrol.
[0725] In alternative aspects, the processes of the invention use
various pretreatments, which can be grouped into three categories:
physical, chemical, and multiple (physical+chemical). Any chemicals
can be used as a pretreatment agent, e.g., acids, alkalis, gases,
cellulose solvents, alcohols, oxidizing agents and reducing agents.
Among these chemicals, alkali is the most popular pretreatment
agent because it is relatively inexpensive and results in less
cellulose degradation. The common alkalis sodium hydroxide and lime
also can be used as pretreatment agents. Although sodium hydroxide
increases biomass digestibility significantly, it is difficult to
recycle, is relatively expensive, and is dangerous to handle. In
contrast, lime has many advantages: it is safe and very
inexpensive, and can be recovered by carbonating wash water with
carbon dioxide.
[0726] In one aspect, the invention provides a multi-enzyme system
(including at least one enzyme of this invention) that can
hydrolyze polysaccharides in a biomass, e.g., sugarcane, e.g.,
bagasse, a component of sugarcane processed in sugar mills. In one
aspect, the biomass is processed by an enzyme of the invention made
by an organism (e.g., transgenic animal, plants, transformed
microorganism) and/or byproduct (e.g., harvested plant, fruit,
seed) expressing an enzyme of the invention. In one aspect, the
enzyme is a recombinant enzyme made by the plant or biomass which
is to be processed to a fuel, e.g., the invention provides a
transgenic sugarcane bagasse comprising an enzyme of the invention.
In one aspect, these compositions and products used in methods of
the invention comprising chemical cycles for natural biomass
conversion, e.g., for the hydrolysis of a biomass to make a
biofuel, e.g., bioethanol, biopropanol, bio-butanol, biomethanol, a
synthetic fuel in the form of a liquid or a gas, such as a
"syngas".
[0727] In one aspect, the invention provides a biofuel, e.g., a
biogas, produced by the process of anaerobic digestion of organic
material by anaerobes, wherein the process comprises use of an
enzyme of the invention or a method of the invention. This biofuel,
e.g., a biogas, can be produced either from biodegradable waste
materials or by the use of energy crops fed into anaerobic
digesters to supplement gas yields. The solid output, digestate,
can also be used as a biofuel.
[0728] In one aspect, the invention provides a biofuel, e.g., a
biogas, comprising a methane, wherein the process comprises use of
an enzyme of the invention or a method of the invention. This
biofuel, e.g., a biogas, can be recovered in industrial anaerobic
digesters and mechanical biological treatment systems. Landfill gas
can be further processed using an enzyme of this invention or a
process of this invention; before processing landfill gas can be a
less clean form of biogas produced in landfills through naturally
occurring anaerobic digestion. Paradoxically if landfill gas is
allowed to escape into the atmosphere it is a potent greenhouse
gas.
[0729] The invention provides methods for making biologically
produced oils and gases from various wastes, wherein the process
comprises use of an enzyme of the invention or a method of the
invention. In one aspect, these methods comprise thermal
depolymerization of waste to extract methane and other oils similar
to petroleum; or, e.g., a bioreactor system that utilizes nontoxic
photosynthetic algae to take in smokestacks flue gases and produce
biofuels such as biodiesel, biogas and a dry fuel comparable to
coal, e.g., as designed by GreenFuel Technologies Corporation, of
Cambridge, Mass.
[0730] The invention provides methods for making biologically
produced oils, including crude oils, and gases that can be used in
diesel engines, wherein the process comprises use of an enzyme of
the invention or a method of the invention. In one aspect, these
methods can refine petroleum, e.g., crude oils, into kerosene,
petroleum, diesel and other fractions.
[0731] The invention provides methods (using an enzyme of the
invention or a method of the invention) for making biologically
produced oils from: [0732] Straight vegetable oil (SVO). [0733]
Waste vegetable oil (WVO)--waste cooking oils and greases produced
in quantity mostly by commercial kitchens. [0734] Biodiesel
obtained from transesterification of animal fats and vegetable oil,
directly usable in petroleum diesel engines. [0735] Biologically
derived crude oil, together with biogas and carbon solids via the
thermal depolymerization of complex organic materials including non
oil based materials; for example, waste products such as old tires,
offal, wood and plastic. [0736] Pyrolysis oil; which may be
produced out of biomass, wood waste etc. using heat only in the
flash pyrolysis process (the oil may have to be treated before
using in conventional fuel systems or internal combustion engines).
[0737] Wood, charcoal, and dried dung.
Medical and Research Applications
[0738] Xylanases of the invention, including the enzyme mixtures or
"cocktails" of the invention, can be used as antimicrobial agents
due to their bacteriolytic properties. Xylanases of the invention
can be used to eliminating or protecting animals from salmonellae,
as described in e.g., PCT Application Nos. WO0049890 and WO9903497.
In another aspect of the invention, the xylanases of the invention
can also be used an antimicrobial surface cleanser or microbial
repellent.
Other Industrial and Medical Applications
[0739] As discussed above, xylanases of the invention, including
the enzyme mixtures or "cocktails" of the invention, can be used
can be used, e.g., in a wide variety of industrial processes,
medical and research (laboratory) applications, and food, animal
feed and beverage applications. New xylanases are discovered by
screening existing libraries and DNA libraries constructed from
diverse mesophilic and moderately thermophilic locations as well as
from targeted sources including digestive flora, microorganisms in
animal waste, soil bacteria and highly alkaline habitats. Biotrap
and primary enrichment strategies using arabinoxylan substrates
and/or non-soluble polysaccharide fractions of animal feed material
are also useful.
[0740] Two screening formats (activity-based and sequence-based)
are used in the discovery of novel xylanases. The activity-based
approach is direct screening for xylanase activity in agar plates
using a substrate such as azo-xylan (Megazyme). Alternatively a
sequence-based approach may be used, which relies on bioinformatics
and molecular biology to design probes for hybridization and
biopanning. See, for example, U.S. Pat. Nos. 6,054,267, 6,030,779,
6,368,798, 6,344,328. Hits from the screening are purified,
sequenced, characterized (for example, determination of
specificity, temperature and pH optima), analyzed using
bioinformatics, subcloned and expressed for basic biochemical
characterization. These methods may be used in screening for
xylanases useful in a myriad of applications, including dough
conditioning and as animal feed additive enzymes.
[0741] In characterizing enzymes obtained from screening, the
exemplary utility in dough processing and baking applications may
be assessed. Characterization may include, for example, measurement
of substrate specificity (xylan, arabinoxylan, CMC, BBG),
temperature and pH stability and specific activity. A commercial
enzyme may be used as a benchmark. In one aspect, the enzymes of
the invention have significant activity at pH.gtoreq.7 and
25-35.degree. C., are inactive on insoluble xylan, are stable and
active in 50-67% sucrose.
[0742] In another aspect, utility as feed additives may be assessed
from characterization of candidate enzymes. Characterization may
include, for example, measurement of substrate specificity (xylan,
arabinoxylan, CMC, B.beta.G), temperature and pH stability,
specific activity and gastric stability. In one aspect the feed is
designed for a monogastric animal and in another aspect the feed is
designed for a ruminant animal. In one aspect, the enzymes of the
invention have significant activity at pH 2-4 and 35-40.degree. C.,
a half-life greater than 30 minutes in gastric fluid, formulation
(in buffer or cells) half-life greater than 5 minutes at 85.degree.
C. and are used as a monogastric animal feed additive. In another
aspect, the enzymes of the invention have one or more of the
following characteristics: significant activity at pH 6.5-7.0 and
35-40.degree. C., a half-life greater than 30 minutes in rumen
fluid, formulation stability as stable as dry powder and are used
as a ruminant animal feed additive.
[0743] Enzymes are reactive toward a wide range of natural and
unnatural substrates, thus enabling the modification of virtually
any organic lead compound. Moreover, unlike traditional chemical
catalysts, enzymes are highly enantio- and regio-selective. The
high degree of functional group specificity exhibited by enzymes
enables one to keep track of each reaction in a synthetic sequence
leading to a new active compound. Enzymes are also capable of
catalyzing many diverse reactions unrelated to their physiological
function in nature. For example, peroxidases catalyze the oxidation
of phenols by hydrogen peroxide. Peroxidases can also catalyze
hydroxylation reactions that are not related to the native function
of the enzyme. Other examples are xylanases which catalyze the
breakdown of polypeptides. In organic solution some xylanases can
also acylate sugars, a function unrelated to the native function of
these enzymes.
[0744] 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. Each biocatalyst is
specific for one functional group, or several related functional
groups and can react with many starting compounds containing this
functional group. 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 compound can be produced with each
iteration of biocatalytic derivatization.
[0745] 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.
[0746] Many of the procedural steps are performed using robotic
automation enabling the execution of many thousands of biocatalytic
reactions and screening assays per day as well as ensuring a high
level of accuracy and reproducibility. As a result, a library of
derivative compounds can be produced in a matter of weeks which
would take years to produce using current chemical methods. (For
further teachings on modification of molecules, including small
molecules, see PCT/US94/09174).
[0747] In one aspect, the invention provides a composition
comprising at least one mucoadhesive polymer that is capable of
forming a hydrogel and at one least water soluble polymer, and one
or more enzymes of the invention. This formulation can be used in
any industrial, food or feed processing or medical or research
application of the invention, i.e., any application using an enzyme
or nucleic acid of the invention. In one aspect, the formulation
forms a hydrogel in aqueous solution that has mucoadhesive
properties; this can be capable of releasing enzymes,
microorganisms capable of generating enzymes of the invention, or
antibodies of the invention, over an extended period of time.
Alternatively, the hydrogel can entrap enzymes, microorganisms
capable of generating enzymes of the invention, or antibodies of
the invention and release them over a defined (e.g., an extended)
period of time.
[0748] The invention will be further described with reference to
the following examples; however, it is to be understood that the
invention is not limited to such examples.
EXAMPLES
Example 1
Xylanase Assay with Wheat Arabinoxylan as Substrate
[0749] The following example describes an exemplary xylanase assay
that can be used, for example, to determine if an enzyme is within
the scope of the invention. Enzymes of the invention, e.g., SEQ ID
NO:2 having one or more amino acid residue changes (mutations) as
set forth in Table 1 and as described herein, also include a genus
of polypeptides having various sequence identities based on 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 or SEQ ID NO:24, can be subjected to an
assay at pH 8 (Na-phosphate buffer) and 70.degree. C. using wheat
arabinoxylan as a substrate.
Example 2
Determination of Melting Temperature and Xylanase Activity
Differential Scanning Calorimetry (DSC)
[0750] The melting temperature transition midpoint (T.sub.m) for
each enzyme of the invention, e.g., SEQ ID NO:2 having one or more
amino acid residue changes (mutations) as set forth in Table 1 and
as described herein, can be determined by Differential Scanning
calorimetry (DSC). Baseline subtracted DSC data can be normalized
for protein concentration.
[0751] In one assay, calorimetry can be performed using a Model
6100 NANO II DSC.TM. apparatus (calorimetry Sciences Corporation,
American Fork, UT) using the DSCRUN.TM. (DSCRun) software package
for data acquisition, CPCALC.TM. (CpCalc) for analysis,
CPCONVERT.TM. (CpConvert) for conversion into molar heat capacity
from microwatts and CPDECONVOLUTE.TM. (CpDeconvolute) for
deconvolution. Analysis can be carried out with 1 mg/ml recombinant
protein in 20 mM potassium phosphate (pH 7.0) and 100 mM KCl at a
scan rate of 1.degree. C./min. A constant pressure of 5 atm can be
maintained during all DSC experiments to prevent possible degassing
of the solution on heating. The instrumental baseline can be
recorded routinely before the experiments with both cells filled
with buffer. Reversibility of the thermally induced transitions can
be tested by reheating the solution in the calorimeter cell
immediately after cooling the first run.
[0752] Alternatively, DSC measurements can be made using a VP-DSC
microcalorimeter (Micro-Cal) in duplicate. In one aspect, a
required sample volume is 540 .mu.L. The concentrations of the
protein can be between 0.1 to 0.5 mg/mL in 50 mM HEPES, pH 7.2; a
sample of the dialysis buffer can be retained for base line
controls. Each sample can be heated from 40.degree. C. to
110.degree. C. Samples and/or buffer can be heated and cooled at a
scan rate of 90.degree. C./h. Buffer baselines were recorded
multiple times until the system reached a stable state. The T.sub.m
value was the temperature where maximum heat was released.
Xylanase Activity Assays
[0753] Enzymatic activities can be determined using 400 .mu.L of 2%
Azo-xylan as substrate in 550 .mu.L of CP (citrate-phosphate)
buffer, pH 6.0 at the indicated temperatures. Activity measurements
as a function of pH can be determined using 50 mM Britton and
Robinson buffer solutions (pH 3.0, 5.0, 6.0, 7.0, 8.0 and 9.0)
prepared by mixing solutions of 0.1 M phosphoric acid solution, 0.1
M boric acid and 0.1 M acetic acid followed by pH adjustment with 1
M sodium hydroxide. Reactions can be initiated by adding 50 .mu.L
of 0.1 mg/ml of purified enzyme. Time points can be taken from 0 to
15 minutes where 50 .mu.L of reaction mixture are added to 200
.mu.L of precipitation solution (100% ethanol). When all time
points have been taken, samples are mixed, incubated for 10 minutes
and centrifuged at 3000 g for 10 minutes at 4.degree. C.
Supernatant (150 .mu.L) can be aliquoted into a fresh 96 well plate
and absorbance is measured at 590 nm. A.sub.590 values can be
plotted against time and the initial rate is determined from the
slope of the line.
Polysaccharide Fingerprinting
[0754] Polysaccharide fingerprints can be determined by
polysaccharide analysis using carbohydrate gel electrophoresis
(PACE). Beechwood xylan (0.1 mg/mL, 100 .mu.L, Sigma, Poole,
Dorset, UK) or xylooligosaccharides (1 mM, 20 .mu.L, Megazyme,
Wicklow, Ireland) can be treated with enzyme (1-3 .mu.g) in a total
volume of 250 .mu.L for 16 hours. The reaction is buffered in 0.1 M
ammonium acetate pH 5.5. Controls without substrates or enzymes are
performed under the same conditions to identify any unspecific
compounds in the enzymes, polysaccharides/oligosaccharides or
labeling reagents. The reactions are stopped by boiling for 20 min.
Assays can be independently performed at least 2 times for each
condition. Derivatization using ANTS
(8-aminonaphthalene-1,3,6-trisulfonic acid, Molecular Probes,
Leiden, The Netherlands), electrophoresis and imaging are carried
out as described (Goubet, F., Jackson, P., Deery, M. and Dupree, P.
(2002) Anal. Biochem. 300, 53-68).
Fitness Calculation
[0755] The fitness (F.sub.n), for a given enzyme variant, n, can be
calculated by equally weighting increase in denaturation
temperature transition midpoint (T.sub.m) and increase (or
decrease) in enzymatic activity relative to the largest difference
in each parameter across all variants: F.sub.n=F.sub.Tn+F.sub.Vn,
where F.sub.Tn=T.sub.m fitness factor of the variant and
F.sub.Vn=activity fitness factor of the variant. The fitness
factors for each (T.sub.m and activity) are relative to the largest
difference in T.sub.m or rate across all of the variants.
F.sub.Tn=(T.sub.m-T.sub.mL)/(T.sub.mH-T.sub.mL) where T.sub.mn is
the T.sub.m for the given variant, n, and T.sub.mL is the lowest
T.sub.m across all variants and T.sub.mH the highest T.sub.m across
all variants and F.sub.Vn=(V.sub.n-V.sub.L)/(V.sub.H-V.sub.L) where
V.sub.n is the relative rate for the given variant, n, and V.sub.L
is the lowest rate across all variants and V.sub.H the highest rate
across all variants.
Example 3
Pre-Treating Paper Pulp with Xylanases of the Invention
[0756] In one aspect, xylanases of the invention are used to
treat/pretreat paper pulp, or recycled paper or paper pulp, waste
wood or wood chips, and the like. In one aspect, enzyme(s) of the
invention are used to increase the "brightness" of the paper via
their use in treating/pretreating paper pulp, or recycled paper or
paper pulp, and the like.
[0757] In one aspect, xylanases of the invention are used to
treat/pretreat paper pulp, or recycled paper or paper pulp, and the
like to reduce the Kappa number. Kappa number is defined as a
numerical value indicating a paper's relative lignin content--the
higher the Kappa number, the higher the lignin content. In some
aspects, reduction in Kappa # has benefits when treating unbleached
pulp (kappa #70-90), when then is used for, e.g., processing, such
as in board manufacture. In some aspects, a reduction in Kappa
across the X stage allows lower alkali use in cooking or cooking to
a higher target Kappa #. In some aspects, this results in higher
pulp strength, less machine refining and higher machine speeds. In
some aspects, such results are seen using digester additives
(surfactants) in linerboard mills; this can allow for better liquor
penetration, and allow lower effective alkali charge leading to
higher pulp strength, lower refining and a 200 fpm (feet per
minute) increase in machine speed.
[0758] This example describes an exemplary routine screening
protocol to determine whether a xylanase is useful in pretreating
paper pulp; e.g., in reducing the use of bleaching chemicals (e.g.,
chlorine dioxide, ClO.sub.2) when used to pretreat Kraft paper
pulp.
[0759] The screening protocol has two alternative test parameters:
Impact of xylanase treatment after an oxygen delignification step
(post-O.sub.2 pulp); and, impact of xylanase in a process that does
not include oxygen delignification (pre-O.sub.2 brownstock).
[0760] The invention provides pulp or paper treatment conditions
that simulate process conditions in industrial situations, e.g.,
factories: for example, at about pH 8.0; 70.degree. C.; 60 min
duration. For example, an exemplary process of the invention is
schematically depicted in the Flow Diagram of FIG. 5; see also FIG.
6. However, the conditions of a process of method of the invention
can be adjusted to any temperature, time duration and/or pH,
depending on the exemplary enzyme(s) of the invention used and the
objective of the process; for example, there are a variety of ways
to adjust pH in the various pulp and paper processes of the
invention: [0761] adding acid and/or base: [0762] Hydrochloric acid
(HCl) [0763] Sodium hydroxide (NaOH) [0764] H.sub.2SO.sub.4
(sulfuric acid) [0765] NaHSO.sub.4 (sodium hydrogen sulfate) [0766]
H.sub.2SO.sub.3 (sulfurous acid) [0767] H3PO.sub.4 (phosphoric
acid) [0768] HF (hydrofluoric acid) [0769] CH3CO.sub.2H (acetic
acid) [0770] H.sub.2CO.sub.3 (carbonic acid) [0771] H.sub.2S
(hydrogen sulfide) [0772] NaH.sub.2PO.sub.4 (sodium dihydrogen
phosphate) [0773] NH.sub.4Cl (ammonium chloride) [0774] HCN
(hydrocyanic acid) [0775] Na.sub.2SO.sub.4 (sodium sulfate) [0776]
NaCl (sodium chloride) [0777] NaCH.sub.3CO.sub.2 (sodium acetate)
[0778] NaHCO.sub.3 (sodium bicarbonate) [0779] Na.sub.2HPO.sub.4
(sodium hydrogen phosphate) [0780] Na.sub.2SO.sub.3 (sodium
sulfite) [0781] NaCN (sodium cyanide) [0782] NH.sub.3 (aqueous
ammonia) [0783] Na.sub.2CO.sub.3 (sodium carbonate) [0784]
Na3PO.sub.4 (sodium phosphate) [0785] bubbling in gas, e.g.,
CO.sub.2 (which forms an acid with water when dissolved) Dose
Response Determination for Xylanases on Pre-O2 Brownstock
Conditions for xylanase stage (X-stage) as follows: [0786] pH 8
[0787] Temperature 70.degree. C. [0788] Time 60 min [0789] Kappa
factor 0.24 [0790] For no-enzyme control, kappa factor was 0.30
[0791] Pretreatment of Intercontinental Pre-O.sub.2 Brownstock
Xylanase [0792] Determination of ClO.sub.2 Dose Response in D.sub.o
[0793] Experimental outline [0794] Pre-O.sub.2 Brownstock [0795]
Initial kappa 31.5 [0796] X stage conditions [0797] Xylanase charge
0.7 U/gm [0798] Temperature 70.degree. C. [0799] pH 8 [0800]
Treatment time 1 hr [0801] Pulp consistency 10% [0802] Bleach
sequence XDE.sub.p [0803] Kappa factor 0.22, 0.26 and 0.30 (% D on
pulp: 2.63, 3.12 and 3.60)
[0804] Determination of ClO.sub.2 Dose Response in D.sub.0: [0805]
Xylanase 0.7 U/g, pH 8.0, 70.degree. C., 1 hr [0806] Pulp:
Pre-O.sub.2 Brownstock, initial kappa 31.5
[0807] Percentage saving of ClO.sub.2 is of little significance to
the industry. Their primary concern is lbs of ClO.sub.2 required
per ton OD pulp. This makes sense when one considers that a lower
percentage saving seen with a high initial kappa brownstock can be
more valuable in terms of lbs of ClO.sub.2 saved than a higher
percentage reduction for a low initial kappa pulp which will
require a lower total charge of ClO.sub.2 to reach target
brightness.
[0808] Relationship Between Brightness, Yield and Kappa Factor for
Bleached Control Pulp:
[0809] Bleaching with increasing doses of ClO.sub.2 to achieve
higher target brightness results in increased loss of pulp yield.
This is an issue because pulp at this stage of the process has a
value of almost $400 per ton and loss of cellulose costs money.
[0810] A benefit of xylanase (e.g., a xylanase of the invention) is
that use of a lower ClO.sub.2 dose can reduce yield losses as long
as the action of the xylanase itself doesn't cancel out the
gain.
[0811] Dose Response Data for Pretreatment of Pre-O.sub.2
Brownstock with Xylanase
[0812] Experimental Outline [0813] Northwood Pre-O.sub.2 Brownstock
[0814] Initial kappa 28.0 [0815] Initial consistency 32.46% [0816]
Initial brightness 28.37 [0817] X stage conditions [0818] Xylanase
charge 0 to 2.70 U/gm [0819] Temperature 58.degree. C. to
61.degree. C. [0820] pH 8.2 to 8.5 [0821] Treatment time 1 hr
[0822] Bleach sequence XDE.sub.p [0823] Kappa factor 0.24 [0824]
ClO.sub.2 saving calculated for Kappa factors between 0.24 and
0.30
[0825] The purpose of this experiment is to evaluate xylanases on
unwashed SPF brownstock. Results can show dose-dependent increases
in final brightness for pulp treated with XYLB (E.c), with
brightness achieved in presence of xylanase at lower Kf of 0.24,
approaching brightness achieved at higher Kf of 0.30
asymptotically.
Example 4
Novel Biobleaching Assay for Assessing Xylanase Performance in
Enhancing the Brightness of Pulp
[0826] This example describes an exemplary protocol, a
"biobleaching assay," that can be used to determine if a
polypeptide has xylanase activity and is within the scope of the
invention. This assay can be used to assess the performance of an
exemplary enzyme of the invention, for example SEQ ID NO:2 having
one or more amino acid residue changes (mutations) as set forth in
Table 1 and as described herein, or a sequence having a sequence
identity (as described herein) to an 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 or
SEQ ID NO:24; in enhancing the brightness of a pulp, e.g., a Kraft
Pulp.
[0827] The invention provides biobleaching procedures, e.g., a
three-stage biobleaching procedure that closely simulates the
conditions of an actual pulp mill bleach plant, as illustrated in
FIG. 5; including a process as illustrated in FIG. 6. This bleach
sequence is designated by (X)DoEp, in which X represents the
xylanase treatment stage (using, e.g., an enzyme of the invention),
D for chlorine dioxide bleaching stage, and Ep for alkaline
peroxide extraction stage. Many different feedstocks may be used,
for example, Southern Softwood Kraft Brownstock (without oxygen
delignification) and hardwood Kraft pulp (e.g., maple and aspen).
Upon completion of each biobleaching round, ensuing pulp can be
used to produce TAPPI (Technical Association of Pulp and Paper
Industries, the technical association for the worldwide pulp, paper
and converting industry)--standard handsheets. The GE % brightness
of each handsheet can be measured, and the brightness values can be
used as the indication of how well each enzyme performs on the pulp
during the enzymatic pretreatment stage (X).
[0828] Pulp Biobleaching:
[0829] Pulp was bleached in 10-g batches in sealed plastic bags
using a 3-stage (X)DoEp sequence, as illustrated in FIG. 5. The
treatment conditions at the three stages can be summarized as
follows: [0830] X stage: 10% (w/v) consistency at 65.degree. C. and
pH=8 for 60 min [0831] Do stage: 4% (w/v) consistency at 60.degree.
C. for 30 min; Kappa Factor=0.18 for enzyme treated samples, and
0.18 and 0.21 for no-enzyme controls. [0832] Ep stage: 10% (w/v)
consistency at 75.degree. C. for 90 min; caustic charge: 1.7% (w
NaOH/w OD pulp) and H.sub.2O.sub.2 charge: 0.5% (w/w)
[0833] As noted in FIG. 5, in one aspect, raw pulp is washed to
reduce pH to pH 8.5; pulp is filter pressed and divided into bags.
At each stage, bags can be incubated in a water bath at the desired
temperature and each bag is taken out and kneaded thoroughly every
10 min to ensure uniform mass and heat transfer within the pulp
mass. After each treatment, pulp can be filtered, washed with 2 L
of DI water and filtered again before receiving the next treatment.
The moisture content of the pulp can be measured using a
Mettler-Toledo moisture analyzer (Fisher Scientific, USA).
[0834] As noted in FIG. 5, in one aspect, after the pulp is filter
pressed and divided into bags, in the X stage, the pulp can be
resuspended, filter pressed, the pH adjusted; and then, incubated
with enzyme at 10% solids, 65.degree. C., 1 hour; then kneaded for
10 minutes. At the Do stage the pulp can be resuspended, washed, pH
set to 4.0, and filter pressed; then, impregnated with ClO.sub.2 at
4% solids (i.e., 4% (w/v) consistency) at 60.degree. C. for 30 min;
then kneaded for 10 minutes. At the Do stage the Kappa Factor=0.18
for enzyme treated samples, and 0.18 and 0.21 for no-enzyme
controls. At the Ep stage the pulp can be resuspended, washed, and
filter pressed; then, incubated with NaOH and H.sub.2 O.sub.2 at
10% solids (i.e., 10% (w/v) consistency) at 75.degree. C. for 90
min; then kneaded for 10 minutes. The caustic charge: 1.7% (w
NaOH/w OD pulp) and H.sub.2O.sub.2 charge: 0.5% (w/w). After
kneading, handsheets were formed.
[0835] Handsheets: As noted in FIG. 5, in one aspect, handsheets
can be formed (4 m pulp, pH about 6.5); handsheets can be made from
unbleached and bleached pulp using TAPPI standard equipment
(Kalamazoo Paper Chemicals, Richland, Mich.) according to TAPPI
method T-272 sp-97. The GE % brightness of each handsheet can be
measured using a BRIGHTMETER MICRO S-5/BC.TM. (Technidyne Corp.,
New Albany, Ind.) according to TAPPI method T-452 om-98 (reference
at 457 nm).
Example 5
Novel Biobleaching Process
[0836] This example describes a novel biobleaching process of the
invention, as illustrated in FIG. 6. This process can be practiced
using any xylanase enzyme, including a polypeptide of the
invention, which includes a polypeptide having at 50% to 99% or
more sequence identity to an exemplary enzyme 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, and also includes any
polypeptide having the sequence of SEQ ID NO:2 having one or more
amino acid residue changes (mutations) as set forth in Table 1 and
as described herein.
[0837] This exemplary process of the invention can have a starting
material comprising "brownstock," which can be described as: 1)
feedstock preparation--logs coming into the paper mill are
debarked, chipped and screened to remove overthick chips, fines,
knots and foreign matter, 2) pulping--wood chips are cooked at
160.degree. C. to 190.degree. C. under pressure for several hours
in a concentrated liquor of sodium hydroxide and sodium sulfide to
separate cellulose fibers and increase cellulose content by
extracting the majority of unwanted lignin. The output of this step
is referred to as "brownstock".
[0838] This process of the invention comprises a "Bleaching
Step"--a multistage process by which residual lignin and other
chromophores are removed to whiten the pulp to target brightness in
preparation for making paper or other products. Pulp is treated
with oxidizing chemicals, for example chlorine and chlorine
dioxide, that attack lignin preferentially. In one aspect the
process comprises a bleaching sequence where pulp is reacted with
chlorine dioxide, the "D.sub.0" stage (see also FIG. 5); extracted
with alkali in the presence of hydrogen peroxide, the "Ep" stage
(see also FIG. 5, the "Ep" stage); reacted with chlorine dioxide a
second time, a "D1" stage; extracted with alkali and hydrogen
peroxide, an Ep stage; and, reacted with chlorine dioxide a third
time, a D2 stage. In practicing this process, bleaching can be
subject to many variations with respect to type and quantity of
oxidizing chemicals used and the number of process steps (however,
chlorine dioxide is currently the most widely used chemical
oxidant). In one aspect, this process comprises pretreatment of
cooked pulp with oxygen under pressure; the oxygen reactor can be
at high pressure--at about 200 to 230.degree. F. and pH 12 to 14
(this is a common first step in bleaching, known as "oxygen
delignification").
[0839] In one aspect, this process comprises refining. For example,
prior to papermaking bleached pulp is mechanically fined to
collapse the cellulose fibers into flat ribbons, fibrilate their
surfaces and improve their physical characteristics for
papermaking. At any stage of the process following pulping, the
pulp may be dewatered, washed and adjusted to a predetermined
consistency by the addition of clean water or recycled streams.
[0840] Xylanase (e.g., an enzyme of the invention) can be just
added after pulping, in the oxygen reactor or in the storage
container just before the oxygen reactor. Xylanase (e.g., an enzyme
of the invention) can be added at multiple points (one or more or
all points) in the bleaching process. In one aspect, a laccase is
added to catalyze break-down of lignin. The laccase may be added at
any stage of the process, including in the oxygen reactor. Pulp may
release various components that self-mediate the laccase.
Alternatively, in one aspect, organic or inorganic mediators can be
added (see, e.g., DE 19723890 describing an oxidation system
comprising an organic mediator and a laccase; alternative exemplary
mediators include
2,2'-azinobis(3-ethylbenzth-iazoline-5-sulphonate) (ABTS) as an
exemplary organic mediator and potassium octacyanomolybdate
[K.sub.4Mo(CN).sub.8] as an exemplary inorganic mediator).
Mediators as described in U.S. patent application no. 20030096394,
can also be used in the processes of the invention, including any
compound capable of enhancing the activities of laccase and
laccase-related enzymes.
[0841] In one aspect, an esterase, e.g., lipase, or oxidoreductase,
e.g., peroxidase is added. In addition, pH and/or temperature can
be modified in the reactor. In monitoring reactions of the
invention, any lignin content-measuring technique can be used,
e.g., see U.S. Patent Application No. 20020144795, describing a
method to measure kappa number or lignin content of kraft pulps
based on the voltammetric measurement of catalytic reactions
involving lignin and redox mediators.
[0842] Enzymes of the invention can also be used in with
alkali-oxygen bleaching (oxygen delignification) processes as
described, e.g., in U.S. Pat. No. 6,824,646, the process comprising
bleaching lignocellulose pulp in aqueous alkali solution with
oxygen and treating the pulp with a hemicellulase, while a liquid
fraction delivered from the enzyme treatment step is separated from
the hemicellulase treated reaction mixture, and subjected to a
penetration treatment through a separation membrane, for example,
reverse osmosis membrane, to separate a permeated fraction from a
non-permeated fraction; and then the permeated fraction is fed to
the alkali-oxygen bleaching (oxygen delignification) step
comprising use of an enzyme of the invention.
[0843] In alternative aspects of this or any other process (method)
of the invention xylanases (e.g., enzymes of the invention) are
used to reduce bleaching chemicals, e.g., chlorine, chlorine
dioxide, caustic, peroxide, or any combination thereof; and in
alternative aspects, a reduction of up to about 1%, 5%, 10%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95% or more, or 100%, of chemicals can be seen in practicing
the methods and using the enzymes of the invention. In one aspect,
a 100% reduction in chemicals can be achieved when the xylanase is
used in combination with a laccase or other enzyme, e.g., by use of
enzyme cocktails; noting the invention provides enzyme mixtures, or
"cocktails" comprising at least one enzyme of the invention and one
or more other enzyme(s), which can be another xylanase, or any
other enzyme.
[0844] In one aspect xylanases of the invention are used to reduce
chlorine dioxide to allow recycling of water in the process; thus,
there is less water used and less water dumped into the sewer. In
one aspect xylanases of the invention are used to allow more
lignin-rich pulp to enter the bleaching plant, allowing for better
pulp yield and better quality pulp (i.e., less destruction during
the cooking process). In one aspect, xylanases of the invention are
used to increase the overall brightness of the paper. In one
aspect, xylanases of the invention are used to lower the kappa
number of the pulp.
[0845] Xylanases of the invention can be used, and the processes of
the invention can be practiced, on all wood types, including, for
example, on hard wood with, e.g., oxygen delignification, hard wood
without oxygen delignification, soft wood with oxygen
delignification and soft wood without oxygen delignification, and
the like. Xylanases of the invention can be used, and the processes
of the invention can be practiced for processing of recycled paper
and/or pulp.
[0846] Oxygen delignification typically requires the addition of a
reaction tower between a brownstock washer and a bleach plant.
Typically, oxygen and sodium hydroxide are added to brownstock.
Reduction of bleaching chemistry by 50% can be achieved in the
bleaching process if preceded by oxygen delignification. Washing
follows oxygen delignification; effluent can be recovered or
discharged. Ozone delignification can be used in place of oxygen
delignification.
Example 6
Novel Biobleaching Assay
[0847] This example describes assays that can demonstrate xylanase
activity in polypeptides of the invention, e.g., the exemplary
polypeptides of the invention, or enzymes of the invention, e.g.,
SEQ ID NO:2 having one or more amino acid residue changes
(mutations) as set forth in Table 1 and as described herein, also
include a genus of polypeptides having various sequence identities
based on 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 or SEQ ID NO:24.
[0848] These xylanase activity studies can be based on those
described by Nelson (1944) J. Biol. Chem. 153:375-380, "Reducing
Sugar Assay for Xylanase"; and, Somogyi (1952) J. Biol. Chem.
195:19-23. This "Nelson-Somogyi" assay can be used to determine
units of activity; data from "Nelson-Somogyi" assays demonstrating
xylanase activity in polypeptides of the invention by determining
units of activity is set forth, below.
[0849] Enzyme unit determinations also can be determined using the
Nelson-Somogyi assay. Biobleaching assays can be based on methods
from TAPPI ((Technical Association of Pulp and Paper Industries,
see above). Below a description along with references to the TAPPI
protocols.
[0850] Pulp:
[0851] In one aspect, two batches of southern softwood Kraft
brownstock are obtained, e.g., from the Department of Wood and
Fiber Science at North Carolina State University (Raleigh, N.C.).
The pulp Kappa Numbers can be determined, e.g., typically are or
are between 21.4 or 29.7, as analyzed using TAPPI method T-236
om-99; see e.g., TAPPI Test Methods (2000-2001, 2003 173).
[0852] Pulp Biobleaching:
[0853] Pulp can be pretreated with xylanase and bleached in 10 g
batches in sealed plastic bags using a 3-stage xylanase/chlorine
dioxide/alkaline peroxide sequence: (X)DoEp (see explanation
above). The treatment conditions at the three stages can be: [0854]
X stage: 10% (w/v) consistency at 65.degree. C. and pH 8 for 60
min. [0855] Do stage: 4% (w/v) consistency at 60.degree. C. for 30
min; a Kappa Factor of 0.18 was used for enzyme treated samples,
and 0.18 and 0.21 for no-enzyme control samples. The concentration
of chlorine dioxide used during the Do stage was calculated using
equation (1):
[0855] ClO 2 % = KF .times. K # 2.63 ( 1 ) ##EQU00001## [0856]
Where ClO.sub.2% is equal to g pure chlorine dioxide per 100 g
oven-dried (OD) pulp
[0857] KF is the Kappa Factor and K# is the Kappa Number of the
pulp as determined by TAPPI method T-236 om-99, TAPPI Test Methods
(2000-2001, 2003 173), Ep stage: 10% (w/v) consistency at
75.degree. C. for 90 min; caustic charge is 1.7% on pulp (w/w) and
H.sub.2O.sub.2 charge is 0.5% on pulp (w/w).
[0858] At each stage, replicate bags can be incubated in a water
bath at the desired temperature and then removed and kneaded
thoroughly every 10 min to ensure uniform mass and heat transfer
within the pulp mass. After each stage, pulp can be filtered, e.g.,
through a Buchner funnel lined with a hard polypropylene filter
(297-micron mesh, Spectrum Labs, Ft. Lauderdale, Fla.). The
filtrate can be recycled once to catch the fines, and the pulp cake
can be washed, e.g., with 2 L of DI water. The pulp cake can then
be resuspended, e.g., in 1.5 L of DI water and pH can be adjusted,
e.g., to pH 8 and pH 4 prior to X and Do stages, respectively. The
moisture content of the pulp can be measured using a Mettler-Toledo
moisture analyzer (Fisher Scientific, USA).
[0859] Handsheets can be made from the bleached pulp using TAPPI
standard equipment (Kalamazoo Paper Chemicals, Richland, Mich.)
according to TAPPI method T-272 sp-97, TAPPI Test Methods
(2000-2001, 2003 173). The GE % brightness of each handsheet TAPPI
Test Methods (2000-2001, 2003 173) can be measured, e.g., using a
Technidyne BRIGHTMETER MICRO S-5BC.TM. (Technidyne Corp., New
Albany, Ind.) according to TAPPI method T-452 om-98.
TABLE-US-00007 COMPONENTS used in assay (1) 1M NaOH Solution 1: 12
g K.sup.+/Na.sup.+ tartrate; 24 g 0.5M Sodium phosphate buffer pH 8
Na.sub.2CO.sub.3; 16 g NaHCO.sub.3; 144 g Na.sub.2SO.sub.4 1%
Arabinoxylan-(Megazyme #P- in 800 mL H.sub.2O WAXYM) prepared
according to the Solution 2: 4 g CuSO.sub.4*5H.sub.2O; 36 g
manufacturer's instructions Na.sub.2SO.sub.4 in 200 mL H.sub.2O
Xylose-prepare standards 0.15 mM- Reagent A: Mix 4 volumes of
solution 1 2 mM using D-xylose dissolved in H.sub.2O with 1 volume
of solution 2. Note- 96 well PCR plate (Fisher 05 500-48) make
fresh daily PCR plate seals Reagent B: 25 g
(NH.sub.4).sub.2MoO.sub.4 in 450 mL Standard 96 well clear plates
H.sub.2O; add 21 mL conc. H.sub.2SO.sub.4, mix. 1 mL tubes (E&K
671511-RC) for the 96 Dissolve 3 g Na.sub.2HAsO.sub.4*7H.sub.2O in
25 well block mL dH.sub.2O; mix with ammonium molybdate solution
and incubate reagent at 37.degree. C. for 24-48 h. Store solution
in a dark bottle i.e. away from light at room temperature.
[0860] Procedure [0861] 1. Prepare reagent A [0862] 2. Pipet 5 uL
of 1 M NaOH into each well of a 96 well PCR plate. Keep plate on
ice. [0863] 3. Prepare reaction mixture. Alternatively, you can
make a master mix for multiple samples. Here is the 1.times. mix.
Add to the 1 mL tubes and place into the 96 well block. [0864] a.
50 uL pH8 Na-phosphate buffer [0865] b. 250 uL of 1% substrate (to
make a final concentration of 0.5%) [0866] c. 150 uL H.sub.2O
[0867] 4. Preheat reaction mixture to desired temperature for 3
minutes. [0868] 5. Dilute the 0.5 M phosphate buffer to 5 mM pH 8
and make enzyme dilutions using this buffer. [0869] 6. Pipet 75 uL
of diluted enzyme into a well of a 96 well microtiter plate [0870]
7. Pipet 50 uL of diluted enzyme into the 1 mL tube containing the
reaction mix. [0871] 8. At the desired timepoint, pipet 50 uL from
each reaction mixture into tubes containing the NaOH (the NaOH will
raise the pH to 12, quenching the reaction). [0872] 9. Add 50 uL of
each standard to separate tubes also containing NaOH. Standards are
linear within the range of 0.25 mM xylose to 2.0 mM. Use at least 4
standards to generate the standard curve. [0873] 10. Add 50 uL of
Reagent A to each well. Seal plate using the Microseal.TM. `A`
Film. [0874] 11. Heat the plate for 20 min. at 100.degree. C. in a
PCR machine. Set the machine to cool down to 4.degree. C. after
heating the samples. [0875] 12. Add 50 uL of reagent B to each
tube, mix. [0876] 13.--note a significant amount of CO.sub.2 is
formed after addition of reagent B. Care should be taken so sample
does not contaminate adjacent wells. [0877] 14. Pipet 100 uL of
each sample or standard into separate wells of a 96 well microtiter
plate. [0878] 15. Read plate at 560 nm. [0879] 16. Plot standard
curve data and express standards as umoles of xylose i.e., 50 uL of
2.5 mM xylose is 0.125 .mu.moles of xylose. [0880] 17. Subtract
buffer control from sample data for each timepoint and plot the
data [0881] 18. Divide timepoint curve slope value by the xylose
standard curve slope value [0882] 19. Multiply by 10 (accounts for
the 50 uL samples ( 1/10 of the total assay volume) [0883] 20.
Divide by the volume used in the assay (0.05) to get .mu.moles of
xylose released per min per mL of enzyme or U/mL of enzyme. [0884]
21. Divide this number by the protein concentration to get
U/mg.
[0885] "Units of Activity" data from the "Nelson-Somogyi" assays
can be used to determine dosing in biobleaching assays (based on
TAPPI methods).
[0886] As noted above, the enzymes and processes of the invention
can also be used in conjunction with a second approach to enzymatic
bleaching using oxidative enzymes such as laccase and/or manganese
peroxidase (MnP) to delignify pulp. In one aspect of this second
approach, of these enzymes, laccase is preferred, because MnP
requires hydrogen peroxide, manganese (II) ions and a chelator.
Laccase can cause delignification of pulp under slight oxygen
pressure, but is considerably more effective when mediators are
added, as discussed above.
[0887] Catalyst improved delignification methods can also be used
in conjunction with the methods of the invention, for example,
polysulfide or anthraquinone. Anthraquinone is a pulping reaction
catalyst which can increase the speed of pulping, increase yield,
and reduce pulping chemical usage by up to 10%. It is possible to
use both anthraquinone and polysulfide together.
[0888] In one aspect, laccase is used in conjunction with the
methods of the invention, as discussed above. For example, laccase
is used in an oxygen reactor in a process of the invention, where
the laccase breaks down the lignin in the oxygen reactor. While
pulp may release various components that self-mediate the laccase,
in one aspect organic or inorganic mediators are added (see
discussion above, e.g., alternative exemplary mediators include
2,2'-azinobis(3-ethylbenzth-iazoline-5-sulphonate) (ABTS) as an
exemplary organic mediator and potassium octacyanomolybdate
[K.sub.4Mo(CN).sub.8] as an exemplary inorganic mediator, or
mediators as described in U.S. patent application no. 20030096394).
In one aspect, another hydrolase, such as an esterase (e.g., a
lipase) and/or an oxidoreductase (e.g., a peroxidase) is also
added. In alternative aspects, pH and/or temperature are modified
in the reactor.
Example 7
Studies Demonstrating the Enzymatic Activity of Enzymes of the
Invention
[0889] This example describes studies demonstrating the enzymatic
activity of the exemplary xylanase enzymes of the invention, which
demonstrates that polypeptides of this invention, which includes a
polypeptide having at 50% to 99% or more sequence identity to an
exemplary enzyme 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 and/or SEQ
ID NO:24, and also includes any polypeptide having the sequence of
SEQ ID NO:2 having one or more amino acid residue changes
(mutations) as set forth in Table 1 and as described herein, have
xylanase activity.
[0890] An exemplary assay for evaluating these xylanases: [0891] 1.
Initial Screen--using an azo-xylan (solution-based) substrate
[0892] a. Enzymatic activity of enzymes can be determined by an
azo-xylan assay using MEGAZYME.RTM. substrate Birchwood Azo-xylan
in 100 mM sodium phosphate, pH 8, according to manufacturer's
recommended assay protocol. The concentrations of enzyme samples
can be adjusted such that they had equal amounts of xylanase
activity at pH8. [0893] b. The azo-xylan assay are then repeated
with normalized samples in 100 mM sodium borate buffer at pH 10.4.
[0894] 2. Initial Screen--ENZ-CHEK ULTRA XYLANASE ASSAY KIT.TM.
(Invitrogen) [0895] a. Xylanase enzyme samples can be prepared in
the same manner as for the azo-xylan assay (section 1, above).
[0896] b. The level of enzymatic activity of enzymes can be
measured by employing commercially available assay kit, e.g., sold
by Invitrogen under the name ENZ-CHEK ULTRA XYLANASE ASSAY KIT.TM.
(Product number E33650). The ENZ-CHEK.TM. kit substrate produces
fluorescent signal in the presence of xylanases, which can be used
to quantify xylanase activities using kit-supplied standards. The
protocol used for testing xylanase enzymes can be slightly modified
from any manufacturer-recommended protocol. The modifications can
primarily involve, e.g., testing xylanases at different pH and
temperature that what is recommended by the manufacturer. [0897]
Secondary Screen--Exemplary Pulp Assays [0898] a. The enzymes from
azo-xylan assay can be tested for activity on wheat arabinoxylan
using, e.g., a Nelson-Somogyi assay as already described herein.
They can be then tested in a laboratory scale bleaching assays to
determine the amount of chemical savings each can achieved for a
given pulp type and chlorine dioxide loading. The ones that meet
desired performance characteristics can be tested in TAPPI bag
biobleaching assay (e.g., in triplicate) at a range of loadings and
pH levels. [0899] Exemplary enzyme characterization
screen--Temperature profile [0900] a. Thermotolerance of xylanases
can be assayed using azo-xylan assay at pH 8 and pH 10.4 at
progressively more elevated temperatures; and enzymes of the
invention were tested using this assay. The initial rates of
reaction at each temperature can be recorded and plotted to
determine optimal performance temperature of xylanases. [0901] b.
Residual activity--Another exemplary assay that can be employed for
testing thermostability of enzymes is the residual activity method,
whereby a sample of enzyme is treated at an elevated temperature at
a particular pH for a specific period of time, and then assayed
under standard conditions under permissive temperature (typically
37.degree. C.). A half-life at a particular temperature is then
determined and provides a measure of a given enzyme fitness under
those temperature conditions.
Example 8
Studies Demonstrating the Enzymatic Activity of Enzymes of the
Invention
[0902] This example describes studies demonstrating the enzymatic
activity of the exemplary xylanase enzymes of the invention,
including the enzymatic activity of any polypeptide of this
invention, which includes a polypeptide having at 50% to 99% or
more sequence identity to an exemplary enzyme 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 and/or SEQ ID NO:24, and also includes any
polypeptide having the sequence of SEQ ID NO:2 having one or more
amino acid residue changes (mutations) as set forth in Table 1 and
as described herein, have xylanase activity.
[0903] The evolution of endoxylanase SEQ ID NO:2 (Xyl 11) utilizing
GSSM technology and xylanase screening identified point mutations
(Xyl 11 mutants) having increased xylanase activity, as well
increased sugar release from alkaline pretreated corn stover, when
used in combination with 7 other cellulosic enzymes (Table 2,
below) after 36 hrs in saccharification cocktail assays at
50.degree. C. These assays contain alkaline pretreated dry corn
cobs at 5% (w/v) with a total enzyme loading of 10.2 mg/g cellulose
in the solids.
TABLE-US-00008 TABLE 2 Composition of Enzyme Cocktail Conc. mg/g
Enzyme SEQ ID NOs: Cellulose Endoglucanase{circumflex over ( )} SEQ
ID NO: 4 (encoded by SEQ ID NO: 3) 1.7 Oligomerase I SEQ ID NO: 6
(encoded by SEQ ID NO: 5) 0.5 (beta-glucosidase){circumflex over (
)} CBH1 (GH family 7){circumflex over ( )} SEQ ID NO: 8 (encoded by
SEQ ID NO: 7) 5 CBH2 (GH family 6){circumflex over ( )} SEQ ID NO:
10 (encoded by SEQ ID NO: 9) 1 Xylanase (GH family 11) VARIES
(control*, Xyl 11 or Xyl 11 mutants) 0.6
Arabinofuranosidase{circumflex over ( )} SEQ ID NO: 14 (encoded by
SEQ ID NO: 13) 0.25 Xylanase (GH family10){circumflex over ( )} SEQ
ID NO: 16 (encoded by SEQ ID NO: 15) 0.15 Oligomerase II SEQ ID NO:
18 (encoded by SEQ ID NO: 17) 1 (beta-xylosidase){circumflex over (
)} *control xylanase is SEQ ID NO: 12 (encoded by SEQ ID NO: 11)
{circumflex over ( )}previously described in PCT Publication No. WO
07/094852
[0904] The new xylanase mutants improved xylose release over the
wild type at 0.6 mg/g cellulose as well as 0.2 mg/g cellulose
loading (FIG. 2). At the standard loading of 0.6 mg/g cellulose
these new variants achieved conversion rates of up to 90% monomeric
xylose released vs. 63% with the wild type. Some of the
polypeptides of the invention (the mutants of SEQ ID NO:2), in
particular, Xyl 11 mutant 11 and Xyl 11 mutant 14, also achieved
greater than 90% xylose release even at the reduced loading of 0.2
mg cellulose. These novel polypeptides of the invention (the
mutants of SEQ ID NO:2) therefore not only improve the rate of
xylose release but also can do so at a reduced enzyme loading.
Similar positive effects on xylose release and enzyme loading could
be envisioned for comparable saccharification reactions using
different feed stocks (switch grass, hard and soft woods, energy
cane, bagasse etc.) applied to alkaline or acidic pretreatments and
with different initial enzyme loadings (1 mg-100 mg/g cellulose)
and different ratios of cocktail components.
[0905] The enzymes of this invention can be used to process/treat
cellulosic material for, e.g., biological alcohol (e.g., EtOH, or
ethanol) fermentation; cellulosic material that is processed using
compositions and methods of the invention can be mainly composed of
cellulose (containing glucose), and hemicellulose--which is mostly
containing xylose. In one aspect, glucose as well as xylose can be
used as a sugar source for EtOH fermentation. In one aspect,
xylanases of the invention are active in the enzymatic breakdown of
the hemicellulose portion of cellulosic material, releasing a
monomeric xylose. In one aspect, the improved xylanase activity of
polypeptides of the invention increases the amount of xylose
available for fermentation.
[0906] In one aspect, by removing the hemicellulose the cellulose
becomes more accessible to cellulases, which can also increase the
conversion of cellulose to glucose. Using xylanases of the
invention, e.g., the sequence variations of the exemplary
endoxylanase Xyl 11 (SEQ ID NO:2), including the exemplary 18 amino
acid substitutions described herein, an increased specific activity
can be achieved over the "wild type" xylanase, as described in
Table 1, above. Note: in Table 1 tertiary assay activity is
indicated as the Absorbance at 560 nm measured in the BCA assay
reached after 9.5 h of hydrolysis. Referencing Table 1, when each
of these clones (xylanases of the invention) was evaluated in
cocktail saccharification assays with the xylanase as the variable,
fourteen of these clones (xylanases of the invention) improved
xylose conversion rates when compared to assays with the wild type
at the same loading, as noted in Table 3 (see Table 1 for the
sequence referenced in Table 3, e.g., Table 1 sets for the sequence
of Xyl 11 mutant 5, Xyl 11 mutant 5, etc., based on the exemplary
SEQ ID NO:2; note also, "Xyl 11 (WT)" refers to the "wild type"
exemplary SEQ ID NO:2):
TABLE-US-00009 TABLE 3 Xylose Conversion by the Cocktail Shown in
Table 2 (above). Note the xylanase component (Xyl 11 WT or Xyl 11
mutant) varies in each cocktail. Xylose Conversion @ 0.2 mg Xylose
Conversion @ 0.6 mg Xyl 11 or Xyl 11 Xyl 11 or Xyl 11 mutant used
in mutant used cocktail 36 hr STDEV in cocktail 36 hr STDEV Xyl 11
(WT) 51.57% 0.01 Xyl 11 WT) 62.84% 0.02 Xyl 11 mutant 5 55.98% 0.00
Xyl 11 mutant 16 68.10% 0.01 Xyl 11 mutant 16 57.43% 0.01 Xyl 11
mutant 5 69.69% 0.00 Xyl 11 mutant 12 59.03% 0.02 Xyl 11 mutant 12
71.75% 0.01 Xyl 11 mutant 4 59.46% 0.00 Xyl 11 mutant 7 74.25% 0.00
Xyl 11 mutant 9 60.34% 0.01 Xyl 11 mutant 9 74.45% 0.00 Xyl 11
mutant 17 60.45% 0.01 Xyl 11 mutant 17 74.76% 0.01 Xyl 11 mutant 7
61.23% 0.02 Xyl 11 mutant 4 74.94% 0.01 Xyl 11 mutant 2 61.73% 0.02
Xyl 11 mutant 13 75.30% 0.01 Xyl 11 mutant 6 62.31% 0.00 Xyl 11
mutant 2 78.24% 0.05 Xyl 11 mutant 13 63.58% 0.01 Xyl 11 mutant 15
80.03% 0.00 Xyl 11 mutant 15 65.95% 0.00 Xyl 11 mutant 6 80.36%
0.09 Xyl 11 mutant 10 66.34% 0.00 Xyl 11 mutant 10 80.61% 0.01 Xyl
11 mutant 11 71.76% 0.01 Xyl 11 mutant 14 84.74% 0.01 Xyl 11 mutant
14 73.69% 0.03 Xyl 11 mutant 11 90.36% 0.06
[0907] Accordingly, the invention provides an enzyme cocktail
comprising, or consisting of, the enzymes: Endoglucanase,
Oligomerase I (beta-glucosidase), CBH1 (GH family 7), CBH2 (GH
family 6), Xylanase (GH family 11), Arabinofuranosidase, Xylanase
(GH family 10) and an Oligomerase II (beta-xylosidase); wherein
one, two, three, four, five, six, seven and/or all eight of these
enzyme are a polypeptide of this invention, and methods for
treating polysaccharide compositions using these cocktails, or any
cocktail of this invention, for, e.g., treating/processing wood,
pulp, paper, waste(s) and the like, or making biofuels or foods or
feeds, or any other industrial process or method, e.g., as
described herein.
Screens and Assays for Identifying Enzymes of the Invention
[0908] The following screens and assays were used in identifying
exemplary enzymes of the invention, and in one aspect, these
screens and assays can be applied to determine if any polypeptide
has sufficient xylanase activity to fall with the scope of this
invention--assuming of course it also has the requisite sequence
identity, as described herein:
[0909] Xylanase Evolution Screen:
[0910] Utilizing the GSSM technology (Verenium Corporation, U.S.
Pat. No. 6,171,820) an evolution library for endoxylanase Xyl 11
(SEQ ID NO:2) representing all possible amino acid exchanges for
each of the 194 residues of this enzyme was created. Point
mutations were introduced using degenerate oligonucleotides, one
amino acid position at a time, so that each original codon could be
substituted with each of the 20 naturally encoded amino acids. The
mutated variants were transformed into XL1-Blue (recA-strain,
Stratagene) and then into Pseudomonas fluorescens MB214 (Dow Global
Technologies Inc., US Patent Publication No. 20050130160), using
vector pWZ82T (SEQ ID NO:25). All variants were grown and expressed
(from Pseudomonas fluorescens MB214) and lysed in 96 well plates.
Hydrolysis reactions with the lysates were carried out in 96 well
plates (200 ul of 200 mM citrate buffer, pH 5.5, 0.5% dried and
milled alkaline pretreated corn stover--CP-15, 50 C). Aliquots were
removed from the reaction at 1, 3, 5 and 10 hrs and added to 800 mM
carbonate buffer pH 10 to stop the reaction. The extent of
hydrolysis at each time point was evaluated via a reducing ends
assay (BCA), as described by Johnston et al. 1998 (see below),
recording absorption at 560 nm (A560). In addition a quantitative
ELISA utilizing Xyl 11 (SEQ ID NO:2) specific antibodies was used
to normalize activity to protein expression. Both functional and
quantitative assays were automated for high through put. In the
primary screen, clones exhibiting normalized activity exceeding Xyl
11 (SEQ ID NO:2) controls on the plate by at least 2 standard
deviations (>1.0+2 STDV wt) were moved on to a secondary screen.
In the secondary screen, all primary hits were re-screened in
duplicate applying the same assay and hit criteria as in the
primary screen. Clones that confirmed for both duplicates were then
moved on to a tertiary screen. In tertiary screens, these clones
again were assayed in duplicate using the BCA assay, but this time
with different defined concentrations of protein (0.1, 0.05 and
0.025 mg/ml). Total protein of lysates was determined via Bradford
assays (e.g., as described in Bradford 1976, see below) the
relative content of xylanase then was determined via densitometry
of SDS PAGE gels after running defined amounts of total protein.
All clones exceeding wt activity, recorded as absorption at 560 nm
(A560), for at least one enzyme concentration in the tertiary
screen were then assayed in saccharification assays.
[0911] Saccharification/Cocktail Assay:
[0912] Cocktail reactions were set up in capped 10 ml glass vials
containing two metal ball bearings. The reaction volume was 5 ml
(200 mM Sodium Citrate-1 mM Sodium Azide pH 5.5) with 5% solids
(size 40 grit milled alkaline pretreated corn stover). Enzyme
composition and loadings were according to Table 2, above, only
varying the family 11 endoxylanase. Reaction vials were incubated
for 36 h at 50 C under shaking at 300 rpm. The concentration of
xylose monomers released was determined by HPLC (RI detector,
Shodex SP-0810 column, flow rate of 0.5 ml/min) using a set of
standards and calibration curves. [0913] Johnston, D. B.;
Shoemaker, S. P.; Smith, G. M. and Whitaker, J. R.: Kinetic
Measurement of Cellulase Activity on Insoluble Substrates Using
Disodium 2,2' Bicinchoninate. Journal of Food Biochemistry (22)
Issue 4 pp. 301-319, 1998 [0914] Bradford, M. M. (1976) A Rapid and
Sensitive Method for the Quantitation of Microgram Quantities of
Protein Utilizing the Principle of Protein-Dye Binding. Anal.
Biochem. 72:248-25
[0915] While the invention has been described in detail with
reference to certain preferred aspects thereof, it will be
understood that modifications and variations are within the spirit
and scope of that which is described and claimed.
Sequence CWU 1
1
241585DNAArtificial SequenceSynthetically engineered 1atggcccaga
cctgcctcac gtcgccccaa accggctttc acaatggctt cttctattcc 60ttctggaagg
acagtccggg cacggtgaat ttttgcctgt tggagggcgg ccgttacaca
120tcgaactgga gcggcatcaa caactgggtg ggcggcaagg gatggcagac
cggttcacgc 180cggaacatca cgtactcggg cagcttcaat acaccgggca
acggctacct ggcgctttac 240ggatggacca ccaatccact cgtcgagtac
tacgtcgtcg atagctgggg gagctggcgt 300ccgccgggtt cggacggaac
gttcctgggg acggtcaaca gcgatggcgg aacgtatgac 360atctatcgcg
cgcagcgggt caacgcgccg tccatcatcg gcaacgccac gttctatcaa
420tactggagcg ttcggcagtc gaagcgggta ggtgggacga tcaccaccgg
aaaccacttc 480gacgcgtggg ccagcgtggg cctgaacctg ggcactcaca
actaccagat catggcgacc 540gagggctacc aaagcagcgg cagctccgac
atcacggtga gttga 5852194PRTArtificial SequenceSynthetically
engineered 2Met Ala Gln Thr Cys Leu Thr Ser Pro Gln Thr Gly Phe His
Asn Gly 1 5 10 15 Phe Phe Tyr Ser Phe Trp Lys Asp Ser Pro Gly Thr
Val Asn Phe Cys 20 25 30 Leu Leu Glu Gly Gly Arg Tyr Thr Ser Asn
Trp Ser Gly Ile Asn Asn 35 40 45 Trp Val Gly Gly Lys Gly Trp Gln
Thr Gly Ser Arg Arg Asn Ile Thr 50 55 60 Tyr Ser Gly Ser Phe Asn
Thr Pro Gly Asn Gly Tyr Leu Ala Leu Tyr65 70 75 80 Gly Trp Thr Thr
Asn Pro Leu Val Glu Tyr Tyr Val Val Asp Ser Trp 85 90 95 Gly Ser
Trp Arg Pro Pro Gly Ser Asp Gly Thr Phe Leu Gly Thr Val 100 105 110
Asn Ser Asp Gly Gly Thr Tyr Asp Ile Tyr Arg Ala Gln Arg Val Asn 115
120 125 Ala Pro Ser Ile Ile Gly Asn Ala Thr Phe Tyr Gln Tyr Trp Ser
Val 130 135 140 Arg Gln Ser Lys Arg Val Gly Gly Thr Ile Thr Thr Gly
Asn His Phe145 150 155 160 Asp Ala Trp Ala Ser Val Gly Leu Asn Leu
Gly Thr His Asn Tyr Gln 165 170 175 Ile Met Ala Thr Glu Gly Tyr Gln
Ser Ser Gly Ser Ser Asp Ile Thr 180 185 190 Val
Ser31365DNAClostridium thermocellum 3gtgaaaaaaa ttgtttcttt
ggtttgtgtg cttgtgatgc tggtaagcat cttaggctcg 60ttttcagtcg tagcggcatc
accggtaaaa ggctttcagg tatcgggaac aaagcttttg 120gatgcaagcg
gaaacgagct tgtaatgagg ggcatgcgtg atatttcagc aatagatttg
180gttaaagaaa taaaaatcgg atggaatttg ggaaatactt tggatgctcc
tacagagact 240gcctggggaa atccaaggac aaccaaggca atgatagaaa
aggtaaggga aatgggcttt 300aatgccgtca gagtgcctgt tacctgggat
acgcacatcg gacctgctcc ggactataaa 360attgacgaag catggctgaa
cagagttgag gaagtggtaa actatgttct tgactgcggt 420atgtacgcga
tcataaatgt tcaccatgac aatacatgga ttatacctac atatgccaat
480gagcaaagga gtaaagaaaa acttgtaaaa gtttgggaac aaatagcaac
ccgttttaaa 540gattatgacg accatttgtt gtttgagaca atgaacgaac
cgagagaagt aggttcacct 600atggaatgga tgggcggaac gtatgaaaac
cgagatgtga taaacagatt taatttggcg 660gttgttaata ccatcagagc
aagcggcgga aataacgata aaagattcat actggttccg 720accaatgcgg
caaccggcct ggatgttgca ttaaacgacc ttgtcattcc gaacaatgac
780agcagagtca tagtatccat acatgcttat tcaccgtatt tctttgctat
ggatgtcaac 840ggaacttcat attggggaag tgactatgac aaggcttctc
ttacaagtga acttgatgct 900atttacaaca gatttgtgaa aaacggaagg
gctgtaatta tcggagaatt cggaaccatt 960gacaagaaca acctgtcttc
aagggtggct catgccgagc actatgcaag agaagcagtt 1020tcaagaggaa
ttgctgtttt ctggtgggat aacggctatt acaatccggg tgatgcagag
1080acttatgcat tgctgaacag aaaaactctc tcatggtatt atcctgaaat
tgtccaggct 1140cttatgagag gtgccggcgt tgaaccttta gtttcaccga
ctcctacacc tacattaatg 1200ccgaccccct cgcccacggt gacagcaaat
attttgtacg gtgacgtaaa cggggacgga 1260aaaataaatt ctacagactg
tacaatgcta aagagatata ttttgcgtgg catagaagaa 1320ttcccaagtc
ctagcggaat tatagccgct gacgtaaatg cggat 13654455PRTClostridium
thermocellumSIGNAL(1)...(25)DOMAIN(71)...(359)Cellulase (glycosyl
hydrolase family 5)DOMAIN(415)...(435)Dockerin type I
repeatSITE(186)...(195)Glycosyl hydrolases family 5 signature.
Prosite id = PS00659SITE(280)...(283)N-glycosylation site. Prosite
id = PS00001SITE(415)...(434)Clostridium cellulosome enzymes
repeated domain signature. Prosite id =
PS00448SITE(423)...(426)N-glycosylation site. Prosite id = PS00001
4Met Lys Lys Ile Val Ser Leu Val Cys Val Leu Val Met Leu Val Ser 1
5 10 15 Ile Leu Gly Ser Phe Ser Val Val Ala Ala Ser Pro Val Lys Gly
Phe 20 25 30 Gln Val Ser Gly Thr Lys Leu Leu Asp Ala Ser Gly Asn
Glu Leu Val 35 40 45 Met Arg Gly Met Arg Asp Ile Ser Ala Ile Asp
Leu Val Lys Glu Ile 50 55 60 Lys Ile Gly Trp Asn Leu Gly Asn Thr
Leu Asp Ala Pro Thr Glu Thr 65 70 75 80 Ala Trp Gly Asn Pro Arg Thr
Thr Lys Ala Met Ile Glu Lys Val Arg 85 90 95 Glu Met Gly Phe Asn
Ala Val Arg Val Pro Val Thr Trp Asp Thr His 100 105 110 Ile Gly Pro
Ala Pro Asp Tyr Lys Ile Asp Glu Ala Trp Leu Asn Arg 115 120 125 Val
Glu Glu Val Val Asn Tyr Val Leu Asp Cys Gly Met Tyr Ala Ile 130 135
140 Ile Asn Val His His Asp Asn Thr Trp Ile Ile Pro Thr Tyr Ala Asn
145 150 155 160 Glu Gln Arg Ser Lys Glu Lys Leu Val Lys Val Trp Glu
Gln Ile Ala 165 170 175 Thr Arg Phe Lys Asp Tyr Asp Asp His Leu Leu
Phe Glu Thr Met Asn 180 185 190 Glu Pro Arg Glu Val Gly Ser Pro Met
Glu Trp Met Gly Gly Thr Tyr 195 200 205 Glu Asn Arg Asp Val Ile Asn
Arg Phe Asn Leu Ala Val Val Asn Thr 210 215 220 Ile Arg Ala Ser Gly
Gly Asn Asn Asp Lys Arg Phe Ile Leu Val Pro 225 230 235 240 Thr Asn
Ala Ala Thr Gly Leu Asp Val Ala Leu Asn Asp Leu Val Ile 245 250 255
Pro Asn Asn Asp Ser Arg Val Ile Val Ser Ile His Ala Tyr Ser Pro 260
265 270 Tyr Phe Phe Ala Met Asp Val Asn Gly Thr Ser Tyr Trp Gly Ser
Asp 275 280 285 Tyr Asp Lys Ala Ser Leu Thr Ser Glu Leu Asp Ala Ile
Tyr Asn Arg 290 295 300 Phe Val Lys Asn Gly Arg Ala Val Ile Ile Gly
Glu Phe Gly Thr Ile 305 310 315 320 Asp Lys Asn Asn Leu Ser Ser Arg
Val Ala His Ala Glu His Tyr Ala 325 330 335 Arg Glu Ala Val Ser Arg
Gly Ile Ala Val Phe Trp Trp Asp Asn Gly 340 345 350 Tyr Tyr Asn Pro
Gly Asp Ala Glu Thr Tyr Ala Leu Leu Asn Arg Lys 355 360 365 Thr Leu
Ser Trp Tyr Tyr Pro Glu Ile Val Gln Ala Leu Met Arg Gly 370 375 380
Ala Gly Val Glu Pro Leu Val Ser Pro Thr Pro Thr Pro Thr Leu Met 385
390 395 400 Pro Thr Pro Ser Pro Thr Val Thr Ala Asn Ile Leu Tyr Gly
Asp Val 405 410 415 Asn Gly Asp Gly Lys Ile Asn Ser Thr Asp Cys Thr
Met Leu Lys Arg 420 425 430 Tyr Ile Leu Arg Gly Ile Glu Glu Phe Pro
Ser Pro Ser Gly Ile Ile 435 440 445 Ala Ala Asp Val Asn Ala Asp 450
455 52610DNACochliobolus heterostrophus ATCC 48331 5atgctgtggc
ttgcacaagc attgttggtc ggccttgccc aggcatcgcc caggttccct 60cgtgctacca
acgacaccgg cagtgattct ttgaacaatg cccagagccc gccattctac
120ccaagtcctt gggtagatcc caccaccaag gactgggcgg ctgcctatga
aaaagcaaag 180gcttttgtta gccaattgac tcttattgag aaggtcaacc
tcaccaccgg cactggatgg 240cagagcgacc actgcgttgg taacgtgggc
gctattcctc gccttggctt tgatcccctc 300tgcctccagg acagccctct
cggcatccgt ttcgcagact acgtttctgc tttcccagca 360ggtggcacca
ttgctgcatc atgggaccgc tatgagtttt acacccgcgg taacgagatg
420ggtaaggagc accgaaggaa gggagtcgac gttcagcttg gtcctgccat
tggacctctt 480ggtcgccacc ccaagggcgg tcgtaactgg gaaggcttca
gtcctgatcc tgtactttcc 540ggtgtggccg tgagcgaaac agtccgcggt
atccaggatg ctggtgtcat tgcctgcact 600aagcacttcc ttctgaacga
gcaagaacat ttccgtcagc ccggcagttt cggagatatc 660ccctttgtcg
atgccatcag ctccaatacc gatgacacga ctctacacga gctctacctg
720tggccctttg ccgacgccgt ccgcgctggt actggtgcca tcatgtgctc
ttacaacaag 780gccaacaact cgcaactctg ccaaaactcg caccttcaaa
actatattct caagggcgag 840cttggcttcc agggtttcat tgtatctgac
tgggatgcac agcactcggg cgttgcgtcg 900gcttatgctg gattggacat
gactatgcct ggtgatactg gattcaacac tggactgtcc 960ttctggggcg
ctaacatgac cgtctccatt ctcaacggca ccattcccca gtggcgtctc
1020gacgatgcgg ccatccgtat catgaccgca tactactttg tcggccttga
tgagtctatc 1080cctgtcaact ttgacagctg gcaaactagc acgtacggat
tcgagcattt tttcggaaag 1140aagggcttcg gtctgatcaa caagcacatt
gacgttcgcg aggagcactt ccgctccatc 1200cgccgctctg ctgccaagtc
aaccgttctc ctcaagaact ctggcgtcct tcccctctct 1260ggaaaggaga
agtggactgc tgtatttgga gaagatgctg gcgaaaaccc gctgggcccc
1320aacggatgcg ctgaccgcgg ctgcgactct ggcaccttgg ccatgggctg
gggttcggga 1380actgcagact tcccttacct cgtcactcct ctcgaagcca
tcaagcgtga ggttggcgag 1440aatggcggcg tgatcacttc ggtcacagac
aactacgcca cttcgcagat ccagaccatg 1500gccagcaggg ccagccactc
gattgtcttc gtcaatgccg actctggtga aggttacatc 1560actgttgata
acaacatggg tgaccgcaac aacatgactg tgtggggcaa tggtgatgtg
1620cttgtcaaga atatctctgc tctgtgcaac aacacgattg tggttatcca
ctctgtcggc 1680ccagtcatta ttgacgcctg gaaggccaac gacaacgtga
ctgccattct ctgggctggt 1740cttcctggcc aggagtctgg taactcgatt
gctgacattc tatacggaca ccacaaccct 1800ggtggcaagc tccccttcac
cattggcagc tcttcagagg agtatggccc tgatgtcatc 1860tacgagccca
cgaacggcat cctcagccct caggccaact ttgaagaggg cgtcttcatt
1920gactaccgcg cgtttgacaa ggcgggcatt gagcccacgt acgaatttgg
ctttggtctt 1980tcgtacacga cttttgaata ctcggacctc aaggtcactg
cgcagtctgc cgaggcttac 2040aagcctttca ccggccagac ttcggctgcc
cctacattcg gaaacttcag caagaacccc 2100gaggactacc agtaccctcc
cggccttgtt taccccgaca cgttcatcta cccctacctc 2160aactcgactg
acctcaagac ggcatctcag gatcccgagt acggcctcaa cgttacctgg
2220cccaagggct ctaccgatgg ctcgcctcag acccgcattg cggctggtgg
tgcgcccggc 2280ggtaaccccc agctctggga cgttttgttc aaggtcgagg
ccacgatcac caacactggt 2340cacgttgctg gtgacgaggt ggcccaggcg
tacatctcgc ttggtggccc caacgacccc 2400aaggtgctac tccgtgactt
tgaccgcttg accatcaagc ctggtgagag cgctgttttc 2460acagccaaca
tcacccgccg tgatgtcagc aactgggaca ctgtcagcca gaactgggtc
2520attaccgagt accccaagac gatccacgtt ggtgccagtt cgaggaacct
tcctctttct 2580gccccactgg acactagcag ctttagataa
26106869PRTCochliobolus heterostrophus ATCC
48331DOMAIN(89)...(310)Glycosyl hydrolase family 3 N terminal
domainDOMAIN(408)...(642)Glycosyl hydrolase family 3 C terminal
domainSITE(24)...(27)N-glycosylation site. Prosite id =
PS00001SITE(73)...(76)N-glycosylation site. Prosite id =
PS00001SITE(262)...(265)N-glycosylation site. Prosite id =
PS00001SITE(276)...(293)Glycosyl hydrolases family 3 active site.
Prosite id = PS00775SITE(332)...(335)N-glycosylation site. Prosite
id = PS00001SITE(539)...(542)N-glycosylation site. Prosite id =
PS00001SITE(531)...(534)N-glycosylation site. Prosite id =
PS00001SITE(550)...(553)N-glycosylation site. Prosite id =
PS00001SITE(572)...(575)N-glycosylation site. Prosite id =
PS00001SITE(695)...(698)N-glycosylation site. Prosite id =
PS00001SITE(721)...(724)N-glycosylation site. Prosite id =
PS00001SITE(737)...(740)N-glycosylation site. Prosite id =
PS00001SITE(823)...(826)N-glycosylation site. Prosite id = PS00001
6Met Leu Trp Leu Ala Gln Ala Leu Leu Val Gly Leu Ala Gln Ala Ser 1
5 10 15 Pro Arg Phe Pro Arg Ala Thr Asn Asp Thr Gly Ser Asp Ser Leu
Asn 20 25 30 Asn Ala Gln Ser Pro Pro Phe Tyr Pro Ser Pro Trp Val
Asp Pro Thr 35 40 45 Thr Lys Asp Trp Ala Ala Ala Tyr Glu Lys Ala
Lys Ala Phe Val Ser 50 55 60 Gln Leu Thr Leu Ile Glu Lys Val Asn
Leu Thr Thr Gly Thr Gly Trp 65 70 75 80 Gln Ser Asp His Cys Val Gly
Asn Val Gly Ala Ile Pro Arg Leu Gly 85 90 95 Phe Asp Pro Leu Cys
Leu Gln Asp Ser Pro Leu Gly Ile Arg Phe Ala 100 105 110 Asp Tyr Val
Ser Ala Phe Pro Ala Gly Gly Thr Ile Ala Ala Ser Trp 115 120 125 Asp
Arg Tyr Glu Phe Tyr Thr Arg Gly Asn Glu Met Gly Lys Glu His 130 135
140 Arg Arg Lys Gly Val Asp Val Gln Leu Gly Pro Ala Ile Gly Pro Leu
145 150 155 160 Gly Arg His Pro Lys Gly Gly Arg Asn Trp Glu Gly Phe
Ser Pro Asp 165 170 175 Pro Val Leu Ser Gly Val Ala Val Ser Glu Thr
Val Arg Gly Ile Gln 180 185 190 Asp Ala Gly Val Ile Ala Cys Thr Lys
His Phe Leu Leu Asn Glu Gln 195 200 205 Glu His Phe Arg Gln Pro Gly
Ser Phe Gly Asp Ile Pro Phe Val Asp 210 215 220 Ala Ile Ser Ser Asn
Thr Asp Asp Thr Thr Leu His Glu Leu Tyr Leu 225 230 235 240 Trp Pro
Phe Ala Asp Ala Val Arg Ala Gly Thr Gly Ala Ile Met Cys 245 250 255
Ser Tyr Asn Lys Ala Asn Asn Ser Gln Leu Cys Gln Asn Ser His Leu 260
265 270 Gln Asn Tyr Ile Leu Lys Gly Glu Leu Gly Phe Gln Gly Phe Ile
Val 275 280 285 Ser Asp Trp Asp Ala Gln His Ser Gly Val Ala Ser Ala
Tyr Ala Gly 290 295 300 Leu Asp Met Thr Met Pro Gly Asp Thr Gly Phe
Asn Thr Gly Leu Ser 305 310 315 320 Phe Trp Gly Ala Asn Met Thr Val
Ser Ile Leu Asn Gly Thr Ile Pro 325 330 335 Gln Trp Arg Leu Asp Asp
Ala Ala Ile Arg Ile Met Thr Ala Tyr Tyr 340 345 350 Phe Val Gly Leu
Asp Glu Ser Ile Pro Val Asn Phe Asp Ser Trp Gln 355 360 365 Thr Ser
Thr Tyr Gly Phe Glu His Phe Phe Gly Lys Lys Gly Phe Gly 370 375 380
Leu Ile Asn Lys His Ile Asp Val Arg Glu Glu His Phe Arg Ser Ile 385
390 395 400 Arg Arg Ser Ala Ala Lys Ser Thr Val Leu Leu Lys Asn Ser
Gly Val 405 410 415 Leu Pro Leu Ser Gly Lys Glu Lys Trp Thr Ala Val
Phe Gly Glu Asp 420 425 430 Ala Gly Glu Asn Pro Leu Gly Pro Asn Gly
Cys Ala Asp Arg Gly Cys 435 440 445 Asp Ser Gly Thr Leu Ala Met Gly
Trp Gly Ser Gly Thr Ala Asp Phe 450 455 460 Pro Tyr Leu Val Thr Pro
Leu Glu Ala Ile Lys Arg Glu Val Gly Glu 465 470 475 480 Asn Gly Gly
Val Ile Thr Ser Val Thr Asp Asn Tyr Ala Thr Ser Gln 485 490 495 Ile
Gln Thr Met Ala Ser Arg Ala Ser His Ser Ile Val Phe Val Asn 500 505
510 Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Asp Asn Asn Met Gly Asp
515 520 525 Arg Asn Asn Met Thr Val Trp Gly Asn Gly Asp Val Leu Val
Lys Asn 530 535 540 Ile Ser Ala Leu Cys Asn Asn Thr Ile Val Val Ile
His Ser Val Gly 545 550 555 560 Pro Val Ile Ile Asp Ala Trp Lys Ala
Asn Asp Asn Val Thr Ala Ile 565 570 575 Leu Trp Ala Gly Leu Pro Gly
Gln Glu Ser Gly Asn Ser Ile Ala Asp 580 585 590 Ile Leu Tyr Gly His
His Asn Pro Gly Gly Lys Leu Pro Phe Thr Ile 595 600 605 Gly Ser Ser
Ser Glu Glu Tyr Gly Pro Asp Val Ile Tyr Glu Pro Thr 610 615 620 Asn
Gly Ile Leu Ser Pro Gln Ala Asn Phe Glu Glu Gly Val Phe Ile 625 630
635 640 Asp Tyr Arg Ala Phe Asp Lys Ala Gly Ile Glu Pro Thr Tyr Glu
Phe 645 650 655 Gly Phe Gly Leu Ser Tyr Thr Thr Phe Glu Tyr Ser Asp
Leu Lys Val 660 665 670 Thr Ala Gln Ser Ala Glu Ala Tyr Lys Pro Phe
Thr Gly Gln Thr Ser 675 680 685 Ala Ala Pro Thr Phe Gly Asn Phe Ser
Lys Asn Pro Glu Asp Tyr Gln 690 695 700 Tyr Pro Pro Gly Leu Val Tyr
Pro Asp Thr Phe Ile Tyr Pro Tyr Leu 705 710
715 720 Asn Ser Thr Asp Leu Lys Thr Ala Ser Gln Asp Pro Glu Tyr Gly
Leu 725 730 735 Asn Val Thr Trp Pro Lys Gly Ser Thr Asp Gly Ser Pro
Gln Thr Arg 740 745 750 Ile Ala Ala Gly Gly Ala Pro Gly Gly Asn Pro
Gln Leu Trp Asp Val 755 760 765 Leu Phe Lys Val Glu Ala Thr Ile Thr
Asn Thr Gly His Val Ala Gly 770 775 780 Asp Glu Val Ala Gln Ala Tyr
Ile Ser Leu Gly Gly Pro Asn Asp Pro 785 790 795 800 Lys Val Leu Leu
Arg Asp Phe Asp Arg Leu Thr Ile Lys Pro Gly Glu 805 810 815 Ser Ala
Val Phe Thr Ala Asn Ile Thr Arg Arg Asp Val Ser Asn Trp 820 825 830
Asp Thr Val Ser Gln Asn Trp Val Ile Thr Glu Tyr Pro Lys Thr Ile 835
840 845 His Val Gly Ala Ser Ser Arg Asn Leu Pro Leu Ser Ala Pro Leu
Asp 850 855 860 Thr Ser Ser Phe Arg 865 71527DNAUnknownObtained
from environmental sample 7atgtaccgca ttctcgccac cgcctcggct
ctgctggcaa ccgcccgtgc ccagcaagcc 60tgcaccctca acgccgaaag caagcctgcc
ttgacctggt ccaagtgcac atccagcggc 120tgcagcaacg tccgcggatc
tgtcgtggtt gacgccaact ggcgatggac ccatagcacc 180tccagcagca
ccaactgcta caccggcaac acctgggaca agactctctg ccccgatgga
240aagacctgcg ctgacaagtg ctgtcttgat ggtgccgact actctggcac
ctacggagtc 300acctcgagcg gcaaccagct caacctcaag tttgtgactg
ttggaccata cagcaccaat 360gttggcagcc gtctctacct catggaggat
gagaacaact accagatgtt cgacctcctg 420ggcaacgaat tcacctttga
tgtcgatgtc aacaacatcg gatgcggcct gaacggcgcc 480ctctacttcg
tctccatgga caaggatggt ggcaagagcc gcttcagcac caacaaggct
540ggtgccaagt acggaactgg ctactgcgat gcccagtgcc ctcgcgatgt
caagttcatc 600aacggagttg ccaactccga cgactggcag ccctccgcca
gcgacaagaa cgccggtgtt 660ggcaagtacg gcacctgctg ccctgagatg
gatatctggg aggccaacaa gatctccacg 720gcttacactc cccatccctg
caagagcctc acccagcagt cctgcgaggg cgatgcctgc 780ggtggcacct
actcttctac tcgctatgct ggaacttgcg atcccgatgg ttgcgatttc
840aacccttacc gccagggcaa ccacaccttc tacggtcccg gctccggctt
caacgttgat 900accaccaaga aggtgactgt cgtgacccag ttcatcaagg
gcagcgacgg caagctctct 960gagatcaagc gtctctatgt tcagaacggc
aaggtcattg gcaaccccca gtccgagatt 1020gccaacaacc ccggcagctc
cgtcaccgac agcttctgca aggcccagaa ggttgcattc 1080aacgaccccg
atgacttcaa caagaagggt ggctggagcg gcatgaacga cgccctcgcc
1140aagcccatgg ttctcgtcat gagcctgtgg cacgaccact acgccaacat
gctctggctc 1200gactctacct accccaaggg ctccaagact cccggctctg
ctcgtggctc ttgccctgag 1260gactctggtg tccccgccac tctcgagaag
gaggtcccca actccagcgt cagcttctcc 1320aacatcaagt tcggtcccat
cggcagcacc tactccggca ccggcggcaa caaccccgac 1380cccgaggagc
ctgaggagcc cgaggagcct gtcggcaccg tcccccagtg gggccagtgc
1440ggcggcatca actacagcgg ccccaccgcc tgcgtgtctc cctacaagtg
caacaagatc 1500aacgactact actcccagtg ctactag
15278508PRTUnknownObtained from environmental
sampleSIGNAL(1)...(20)DOMAIN(19)...(453)Glycosyl hydrolase family
7DOMAIN(476)...(504)Fungal cellulose binding domain 8Met Tyr Arg
Ile Leu Ala Thr Ala Ser Ala Leu Leu Ala Thr Ala Arg1 5 10 15 Ala
Gln Gln Ala Cys Thr Leu Asn Ala Glu Ser Lys Pro Ala Leu Thr 20 25
30 Trp Ser Lys Cys Thr Ser Ser Gly Cys Ser Asn Val Arg Gly Ser Val
35 40 45 Val Val Asp Ala Asn Trp Arg Trp Thr His Ser Thr Ser Ser
Ser Thr 50 55 60 Asn Cys Tyr Thr Gly Asn Thr Trp Asp Lys Thr Leu
Cys Pro Asp Gly65 70 75 80 Lys Thr Cys Ala Asp Lys Cys Cys Leu Asp
Gly Ala Asp Tyr Ser Gly 85 90 95 Thr Tyr Gly Val Thr Ser Ser Gly
Asn Gln Leu Asn Leu Lys Phe Val 100 105 110 Thr Val Gly Pro Tyr Ser
Thr Asn Val Gly Ser Arg Leu Tyr Leu Met 115 120 125 Glu Asp Glu Asn
Asn Tyr Gln Met Phe Asp Leu Leu Gly Asn Glu Phe 130 135 140 Thr Phe
Asp Val Asp Val Asn Asn Ile Gly Cys Gly Leu Asn Gly Ala145 150 155
160 Leu Tyr Phe Val Ser Met Asp Lys Asp Gly Gly Lys Ser Arg Phe Ser
165 170 175 Thr Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp
Ala Gln 180 185 190 Cys Pro Arg Asp Val Lys Phe Ile Asn Gly Val Ala
Asn Ser Asp Asp 195 200 205 Trp Gln Pro Ser Ala Ser Asp Lys Asn Ala
Gly Val Gly Lys Tyr Gly 210 215 220 Thr Cys Cys Pro Glu Met Asp Ile
Trp Glu Ala Asn Lys Ile Ser Thr225 230 235 240 Ala Tyr Thr Pro His
Pro Cys Lys Ser Leu Thr Gln Gln Ser Cys Glu 245 250 255 Gly Asp Ala
Cys Gly Gly Thr Tyr Ser Ser Thr Arg Tyr Ala Gly Thr 260 265 270 Cys
Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Gln Gly Asn His 275 280
285 Thr Phe Tyr Gly Pro Gly Ser Gly Phe Asn Val Asp Thr Thr Lys Lys
290 295 300 Val Thr Val Val Thr Gln Phe Ile Lys Gly Ser Asp Gly Lys
Leu Ser305 310 315 320 Glu Ile Lys Arg Leu Tyr Val Gln Asn Gly Lys
Val Ile Gly Asn Pro 325 330 335 Gln Ser Glu Ile Ala Asn Asn Pro Gly
Ser Ser Val Thr Asp Ser Phe 340 345 350 Cys Lys Ala Gln Lys Val Ala
Phe Asn Asp Pro Asp Asp Phe Asn Lys 355 360 365 Lys Gly Gly Trp Ser
Gly Met Asn Asp Ala Leu Ala Lys Pro Met Val 370 375 380 Leu Val Met
Ser Leu Trp His Asp His Tyr Ala Asn Met Leu Trp Leu385 390 395 400
Asp Ser Thr Tyr Pro Lys Gly Ser Lys Thr Pro Gly Ser Ala Arg Gly 405
410 415 Ser Cys Pro Glu Asp Ser Gly Val Pro Ala Thr Leu Glu Lys Glu
Val 420 425 430 Pro Asn Ser Ser Val Ser Phe Ser Asn Ile Lys Phe Gly
Pro Ile Gly 435 440 445 Ser Thr Tyr Ser Gly Thr Gly Gly Asn Asn Pro
Asp Pro Glu Glu Pro 450 455 460 Glu Glu Pro Glu Glu Pro Val Gly Thr
Val Pro Gln Trp Gly Gln Cys465 470 475 480 Gly Gly Ile Asn Tyr Ser
Gly Pro Thr Ala Cys Val Ser Pro Tyr Lys 485 490 495 Cys Asn Lys Ile
Asn Asp Tyr Tyr Ser Gln Cys Tyr 500 505 91413DNAUnknownObtained
from an environmental sample 9atgcgctata catggtcggt cgcggcggcg
ctgctgccat gcgcaatcca ggctcagcaa 60accctctatg gacaatgtgg tggtcagggc
tactccggac tcaccagctg cgtggcggga 120gcaacatgct ccaccgtaaa
tgaatactac gctcagtgta cgccagcagc aggcagcgcc 180acttccacca
ccttgaagac aactacgacc accgctgggg cgacgacgac gacgactagc
240aagacttctg cttcccagac gtctactact aaaacctcaa ccagtaccgc
ctcaacaacc 300acggctacaa ccacggccag cgcgagcggc aacccgttca
gtgggtacca gctctacgtg 360aacccctact actcctccga agtggcctcc
ctggctatcc catccctcac ggggacactt 420tcctcgctcc aggctgcagc
cacagccgca gccaaggtgc cctctttcgt ctggctggac 480gtggctgcca
aggtgccgac gatggccacc tacctggccg acatcaaagc ccagaatgca
540gcgggagcca acccccccgt cgccggccag tttgtggtct acgacctccc
tgaccgcgac 600tgcgccgcgc tggccagcaa cggcgagtac tccatcgcca
acaacggtgt ggccaactac 660aaggcctaca tcgactccat ccgcaaggtc
ctggtgcagt actcggatgt gcacaccatt 720ctggtgatcg agcccgacag
tctcgccaac ctggtgacca acctcaatgt ggccaaatgt 780gccaacgctc
agagcgccta cctcgaatgc accaactatg ccctggagca gctgaacctc
840cccaacgtgg ccatgtatct tgatgccgga cacgccggct ggctcggctg
gcccgcgaac 900cagcaaccgg ccgccaatct gtacgcgagc gtgtacaaga
acgccagctc gcccgccgca 960gtgcgcggcc tggccacgaa cgtcgccaac
tacaacgcct tcaccatcgc ctcgtgcccg 1020tcgtacaccc agggcaacag
cgtctgcgac gagcagcagt acatcaacgc gatcgccccg 1080ctcctgtcag
cgcagggctt caacgcccac ttcatcgtcg acaccggccg caacggcaaa
1140cagcccaccg gccaacaagc ctggggcgac tggtgcaacg tcatcaacac
ggggttcggc 1200gtgcgcccga ccaccaacac gggcgacgcg ctcgtcgacg
ccttcgtctg ggtcaagccc 1260ggcggcgaga gcgacggcac ctccgatagc
tcggcgaccc gctacgacgc ccactgcggg 1320tacagcgatg ccttgcagcc
ggcgccggag gcggggacct ggttccaggc ctacttcgta 1380caattgctct
cgaacgccaa tccggctttc tag 141310470PRTUnknownObtained from an
environmental sampleSIGNAL(1)...(18)DOMAIN(22)...(50)Fungal
cellulose binding domainDOMAIN(120)...(437)Glycosyl hydrolases
family 6SITE(26)...(53)Cellulose-binding domain, fungal type.
Prosite id = PS00562SITE(240)...(249)Glycosyl hydrolases family 6
signature 2. Prosite id = PS00656SITE(314)...(317)N-glycosylation
site. Prosite id = PS00001 10Met Arg Tyr Thr Trp Ser Val Ala Ala
Ala Leu Leu Pro Cys Ala Ile 1 5 10 15 Gln Ala Gln Gln Thr Leu Tyr
Gly Gln Cys Gly Gly Gln Gly Tyr Ser 20 25 30 Gly Leu Thr Ser Cys
Val Ala Gly Ala Thr Cys Ser Thr Val Asn Glu 35 40 45 Tyr Tyr Ala
Gln Cys Thr Pro Ala Ala Gly Ser Ala Thr Ser Thr Thr 50 55 60 Leu
Lys Thr Thr Thr Thr Thr Ala Gly Ala Thr Thr Thr Thr Thr Ser 65 70
75 80 Lys Thr Ser Ala Ser Gln Thr Ser Thr Thr Lys Thr Ser Thr Ser
Thr 85 90 95 Ala Ser Thr Thr Thr Ala Thr Thr Thr Ala Ser Ala Ser
Gly Asn Pro 100 105 110 Phe Ser Gly Tyr Gln Leu Tyr Val Asn Pro Tyr
Tyr Ser Ser Glu Val 115 120 125 Ala Ser Leu Ala Ile Pro Ser Leu Thr
Gly Thr Leu Ser Ser Leu Gln 130 135 140 Ala Ala Ala Thr Ala Ala Ala
Lys Val Pro Ser Phe Val Trp Leu Asp 145 150 155 160 Val Ala Ala Lys
Val Pro Thr Met Ala Thr Tyr Leu Ala Asp Ile Lys 165 170 175 Ala Gln
Asn Ala Ala Gly Ala Asn Pro Pro Val Ala Gly Gln Phe Val 180 185 190
Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly 195
200 205 Glu Tyr Ser Ile Ala Asn Asn Gly Val Ala Asn Tyr Lys Ala Tyr
Ile 210 215 220 Asp Ser Ile Arg Lys Val Leu Val Gln Tyr Ser Asp Val
His Thr Ile 225 230 235 240 Leu Val Ile Glu Pro Asp Ser Leu Ala Asn
Leu Val Thr Asn Leu Asn 245 250 255 Val Ala Lys Cys Ala Asn Ala Gln
Ser Ala Tyr Leu Glu Cys Thr Asn 260 265 270 Tyr Ala Leu Glu Gln Leu
Asn Leu Pro Asn Val Ala Met Tyr Leu Asp 275 280 285 Ala Gly His Ala
Gly Trp Leu Gly Trp Pro Ala Asn Gln Gln Pro Ala 290 295 300 Ala Asn
Leu Tyr Ala Ser Val Tyr Lys Asn Ala Ser Ser Pro Ala Ala 305 310 315
320 Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Ala Phe Thr Ile
325 330 335 Ala Ser Cys Pro Ser Tyr Thr Gln Gly Asn Ser Val Cys Asp
Glu Gln 340 345 350 Gln Tyr Ile Asn Ala Ile Ala Pro Leu Leu Ser Ala
Gln Gly Phe Asn 355 360 365 Ala His Phe Ile Val Asp Thr Gly Arg Asn
Gly Lys Gln Pro Thr Gly 370 375 380 Gln Gln Ala Trp Gly Asp Trp Cys
Asn Val Ile Asn Thr Gly Phe Gly 385 390 395 400 Val Arg Pro Thr Thr
Asn Thr Gly Asp Ala Leu Val Asp Ala Phe Val 405 410 415 Trp Val Lys
Pro Gly Gly Glu Ser Asp Gly Thr Ser Asp Ser Ser Ala 420 425 430 Thr
Arg Tyr Asp Ala His Cys Gly Tyr Ser Asp Ala Leu Gln Pro Ala 435 440
445 Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Ser
450 455 460 Asn Ala Asn Pro Ala Phe 465 470 11594DNAArtificial
SequenceSynthetically generated 11atggcccaga cctgcctcac gtcgagtcaa
accggcacta acaatggctt ctattattcc 60ttctggaagg acagtccggg cacggtgaat
ttttgcctgc agtccggcgg ccgttacaca 120tcgaactgga gcggcatcaa
caactgggtg ggcggcaagg gatggcagac cggttcacgc 180cggaacatca
cgtactcggg cagcttcaat tcaccgggca acggctacct ggcgctttac
240ggatggacca ccaatccact cgtcgagtac tacgtcgtcg atagctgggg
gagctggcgt 300ccgccgggtt cggacggaac gttcctgggg acggtcaaca
gcgatggcgg aacgtatgac 360atctatcgcg cgcagcgggt caacgcgccg
tccatcatcg gcaacgccac gttctatcaa 420tactggagcg ttcggcagtc
gaagcgggta ggtgggacga tcaccaccgg aaaccacttc 480gacgcgtggg
ccagcgtggg cctgaacctg ggcactcaca actaccagat catggcgacc
540gagggctacc aaagcagcgg cagctccgac atcacggtga gtgaaggcgg ttga
59412197PRTArtificial SequenceSynthetically generated 12Met Ala Gln
Thr Cys Leu Thr Ser Ser Gln Thr Gly Thr Asn Asn Gly 1 5 10 15Phe
Tyr Tyr Ser Phe Trp Lys Asp Ser Pro Gly Thr Val Asn Phe Cys 20 25
30Leu Gln Ser Gly Gly Arg Tyr Thr Ser Asn Trp Ser Gly Ile Asn Asn
35 40 45Trp Val Gly Gly Lys Gly Trp Gln Thr Gly Ser Arg Arg Asn Ile
Thr 50 55 60Tyr Ser Gly Ser Phe Asn Ser Pro Gly Asn Gly Tyr Leu Ala
Leu Tyr65 70 75 80Gly Trp Thr Thr Asn Pro Leu Val Glu Tyr Tyr Val
Val Asp Ser Trp 85 90 95Gly Ser Trp Arg Pro Pro Gly Ser Asp Gly Thr
Phe Leu Gly Thr Val 100 105 110Asn Ser Asp Gly Gly Thr Tyr Asp Ile
Tyr Arg Ala Gln Arg Val Asn 115 120 125Ala Pro Ser Ile Ile Gly Asn
Ala Thr Phe Tyr Gln Tyr Trp Ser Val 130 135 140Arg Gln Ser Lys Arg
Val Gly Gly Thr Ile Thr Thr Gly Asn His Phe145 150 155 160Asp Ala
Trp Ala Ser Val Gly Leu Asn Leu Gly Thr His Asn Tyr Gln 165 170
175Ile Met Ala Thr Glu Gly Tyr Gln Ser Ser Gly Ser Ser Asp Ile Thr
180 185 190Val Ser Glu Gly Gly 195132637DNAUnknownObtained from
environmental sample 13atgactagtg gacgaaacac atgtgtgtgt ctgttgttga
ttgtgctggc gatcggtctt 60ctgtcaaagc caccggcgag cgcgcaaaat gaggcgcctt
ataaagccac gctgacgatc 120cggttggacc aaccgggagc ggtgatcaat
cgcaacatct acggccagtt tgcggagcat 180ctcggacgtt tgatctacga
cgggctctgg gttggtgaag gatcgtcgat cccgaacacg 240cgcggattgc
gtaacgacgt cgttacggcg ttaaaagaat tgcatgtgcc tgtgctgcgt
300tggcccggcg gctgttttgc cgacgagtat cactggcgtg acggcattgg
accacgcgac 360aagcgtccgc ggcggccgaa cgcgagttgg ggcggcgtcg
attcgaatgc gtttggcacg 420catgagttca tggagctgtg cgagatgttg
ggcgcagacg cttatatcaa tggcaacgtc 480ggcagcggca cgccgcagga
gatgatggaa tggatcgagt acatgacttc cgattccgat 540tcggatctcg
ccaacctgcg ccgtcgcaat ggccgcgaca agccgtggaa ggtgccgtat
600ttcgccgtcg gcaatgagac gtggggctgt ggtggaaata tgcggccgga
gttttacgcc 660gacgtgtatc gccagtacgc cacgttcatc aagaaccatt
caggcaatcg cattcagaaa 720ctcgcgagcg gtggttacga caacaattac
aactggaccg aggtgctgat ggcgcaggcg 780gcgaagcaga tcgatggcct
gtcgttgcac tattacacgc tgcccaccgg caactgggac 840aagaaaggat
cggcgacgga attcggcgaa agcgagtggc acgcgacgct cgccaggacg
900ttgcgcatcg aggagttcat tcagaagcac agcgcgatca tggacaagca
cgatccgcag 960aagcgcgtcg gtttgatggt tgacgagtgg ggcacgtggt
acgaccgcga cgagggccgc 1020gacatgggcg cgctttatca gcagaacacg
ttgcgcgatg cggttgcggc cggtatcaat 1080ctcaatatct ttcacaagta
tgccgatcgc gtgcgcatgg cgaacatcgc gcagatggtg 1140aacgtgttgc
aggcgatggt gttgacggac aaagagaaaa tggtgctgac gccgacgtat
1200cacgtttttc ggatgtatcg cgtgcatcag ggagcgacgc tgatcccggt
cgaggttagt 1260gcgccgcagt acacgctggg tggtgcgtct gtgccgtcgt
tgagcgtgtc ggcttcgcgt 1320gacggtgaag gacgggtgca tctgtcgatc
gtgaatctcg atccagcgcg ggcggcggag 1380atcgatgcga acggaccgtt
cagcagtgtc aagggagaag tattgactgc gccggcggtg 1440aatgcgctga
atactttcga tcacccggat agtgtcaagc ccgtgtcttt taatggatat
1500aaattagaag gctctaaatt aatcctgaat attccggcga aatccgtggt
ggtgttggaa 1560cttggaccac agaaacaagc aacgctcaaa gatgcattca
aaaacgattt catgatcggc 1620gcggcgctca accggcgaca gttcttcgaa
gaagacgctc gcggcgcaga gatcgtgcgc 1680atgcatttca actcgatcac
gccggagaac gtgttgaagt gggggctggt ccatcccgaa 1740ccgaacaagt
acgacttcac cgctcccgat cgcttcgtcg aattcggcga gaagcacggc
1800atgttcgtcg tcggacacac gctcgtctgg cataaccaaa cgccgcgctg
ggtttttgaa 1860gacgaaaaga aacagccgct cgatcgcgag acgttgctga
aacgaatgcg cgatcacatc 1920ttcaccgtcg tcggccgtta caagggacgc
attaaaggct gggacgtagt caacgaggcg 1980ctgaatcagg atggcacgat
gcggcagtcg ccgtggttca agatcatcgg cgaggattat 2040ctcgtcaaag
cgtttgagtt tgcccacgag gccgatccag ccgccgagct ttattacaac
2100gactacgatc
tcgagctgcc ggcgaagcgc gcaggcgccg tcgaactgct gaagaaactg
2160aaagccgcgg gtgtgtcgct tgctggtgtg ggattgcaga accacagtct
catggagtgg 2220ccgtcagccg cagatgtgga tgcgacgatc gcggcgttcg
cgaatctggg tttgaaggtt 2280cacatcacgg aactcgacgt cgacgtgctg
ccgcgcacga cgaaacccgg tgcggattac 2340gcagtcgacg tgaaggtgac
gccgcagttg aacccgtatc tcgacggctt accggaggcg 2400cgacagtcgg
cgttggcgag gcgttatgcg gagctgtttc acgtgtttag aaaacatcgc
2460gacgcgatcg agcgtgtgac gttctgggga gttgcggacg gcgattcgtg
gttgaacaac 2520tggcccatcc gcggcaggac aaactatccg ctgctcttcg
atcgttccgg ccaaccgaaa 2580ccggcgttag cgtcggtgat cgaaaccgct
aattattcaa cggaacgtcg acggtga 263714878PRTUnknownObtained from
environmental
sampleSIGNAL(1)...(28)DOMAIN(328)...(515)Alpha-L-arabinofuranosidase
C-terminusDOMAIN(528)...(870)Glycosyl hydrolase family
10SITE(127)...(130)N-glycosylation site. Prosite id =
PS00001SITE(205)...(208)N-glycosylation site. Prosite id =
PS00001SITE(232)...(235)N-glycosylation site. Prosite id =
PS00001SITE(251)...(254)N-glycosylation site. Prosite id =
PS00001SITE(734)...(737)N-glycosylation site. Prosite id =
PS00001SITE(757)...(767)Glycosyl hydrolases family 10 active site.
Prosite id = PS00591SITE(871)...(874)N-glycosylation site. Prosite
id = PS00001 14Met Thr Ser Gly Arg Asn Thr Cys Val Cys Leu Leu Leu
Ile Val Leu 1 5 10 15 Ala Ile Gly Leu Leu Ser Lys Pro Pro Ala Ser
Ala Gln Asn Glu Ala 20 25 30 Pro Tyr Lys Ala Thr Leu Thr Ile Arg
Leu Asp Gln Pro Gly Ala Val 35 40 45 Ile Asn Arg Asn Ile Tyr Gly
Gln Phe Ala Glu His Leu Gly Arg Leu 50 55 60 Ile Tyr Asp Gly Leu
Trp Val Gly Glu Gly Ser Ser Ile Pro Asn Thr 65 70 75 80 Arg Gly Leu
Arg Asn Asp Val Val Thr Ala Leu Lys Glu Leu His Val 85 90 95 Pro
Val Leu Arg Trp Pro Gly Gly Cys Phe Ala Asp Glu Tyr His Trp 100 105
110 Arg Asp Gly Ile Gly Pro Arg Asp Lys Arg Pro Arg Arg Pro Asn Ala
115 120 125 Ser Trp Gly Gly Val Asp Ser Asn Ala Phe Gly Thr His Glu
Phe Met 130 135 140 Glu Leu Cys Glu Met Leu Gly Ala Asp Ala Tyr Ile
Asn Gly Asn Val 145 150 155 160 Gly Ser Gly Thr Pro Gln Glu Met Met
Glu Trp Ile Glu Tyr Met Thr 165 170 175 Ser Asp Ser Asp Ser Asp Leu
Ala Asn Leu Arg Arg Arg Asn Gly Arg 180 185 190 Asp Lys Pro Trp Lys
Val Pro Tyr Phe Ala Val Gly Asn Glu Thr Trp 195 200 205 Gly Cys Gly
Gly Asn Met Arg Pro Glu Phe Tyr Ala Asp Val Tyr Arg 210 215 220 Gln
Tyr Ala Thr Phe Ile Lys Asn His Ser Gly Asn Arg Ile Gln Lys 225 230
235 240 Leu Ala Ser Gly Gly Tyr Asp Asn Asn Tyr Asn Trp Thr Glu Val
Leu 245 250 255 Met Ala Gln Ala Ala Lys Gln Ile Asp Gly Leu Ser Leu
His Tyr Tyr 260 265 270 Thr Leu Pro Thr Gly Asn Trp Asp Lys Lys Gly
Ser Ala Thr Glu Phe 275 280 285 Gly Glu Ser Glu Trp His Ala Thr Leu
Ala Arg Thr Leu Arg Ile Glu 290 295 300 Glu Phe Ile Gln Lys His Ser
Ala Ile Met Asp Lys His Asp Pro Gln 305 310 315 320 Lys Arg Val Gly
Leu Met Val Asp Glu Trp Gly Thr Trp Tyr Asp Arg 325 330 335 Asp Glu
Gly Arg Asp Met Gly Ala Leu Tyr Gln Gln Asn Thr Leu Arg 340 345 350
Asp Ala Val Ala Ala Gly Ile Asn Leu Asn Ile Phe His Lys Tyr Ala 355
360 365 Asp Arg Val Arg Met Ala Asn Ile Ala Gln Met Val Asn Val Leu
Gln 370 375 380 Ala Met Val Leu Thr Asp Lys Glu Lys Met Val Leu Thr
Pro Thr Tyr 385 390 395 400 His Val Phe Arg Met Tyr Arg Val His Gln
Gly Ala Thr Leu Ile Pro 405 410 415 Val Glu Val Ser Ala Pro Gln Tyr
Thr Leu Gly Gly Ala Ser Val Pro 420 425 430 Ser Leu Ser Val Ser Ala
Ser Arg Asp Gly Glu Gly Arg Val His Leu 435 440 445 Ser Ile Val Asn
Leu Asp Pro Ala Arg Ala Ala Glu Ile Asp Ala Asn 450 455 460 Gly Pro
Phe Ser Ser Val Lys Gly Glu Val Leu Thr Ala Pro Ala Val 465 470 475
480 Asn Ala Leu Asn Thr Phe Asp His Pro Asp Ser Val Lys Pro Val Ser
485 490 495 Phe Asn Gly Tyr Lys Leu Glu Gly Ser Lys Leu Ile Leu Asn
Ile Pro 500 505 510 Ala Lys Ser Val Val Val Leu Glu Leu Gly Pro Gln
Lys Gln Ala Thr 515 520 525 Leu Lys Asp Ala Phe Lys Asn Asp Phe Met
Ile Gly Ala Ala Leu Asn 530 535 540 Arg Arg Gln Phe Phe Glu Glu Asp
Ala Arg Gly Ala Glu Ile Val Arg 545 550 555 560 Met His Phe Asn Ser
Ile Thr Pro Glu Asn Val Leu Lys Trp Gly Leu 565 570 575 Val His Pro
Glu Pro Asn Lys Tyr Asp Phe Thr Ala Pro Asp Arg Phe 580 585 590 Val
Glu Phe Gly Glu Lys His Gly Met Phe Val Val Gly His Thr Leu 595 600
605 Val Trp His Asn Gln Thr Pro Arg Trp Val Phe Glu Asp Glu Lys Lys
610 615 620 Gln Pro Leu Asp Arg Glu Thr Leu Leu Lys Arg Met Arg Asp
His Ile 625 630 635 640 Phe Thr Val Val Gly Arg Tyr Lys Gly Arg Ile
Lys Gly Trp Asp Val 645 650 655 Val Asn Glu Ala Leu Asn Gln Asp Gly
Thr Met Arg Gln Ser Pro Trp 660 665 670 Phe Lys Ile Ile Gly Glu Asp
Tyr Leu Val Lys Ala Phe Glu Phe Ala 675 680 685 His Glu Ala Asp Pro
Ala Ala Glu Leu Tyr Tyr Asn Asp Tyr Asp Leu 690 695 700 Glu Leu Pro
Ala Lys Arg Ala Gly Ala Val Glu Leu Leu Lys Lys Leu 705 710 715 720
Lys Ala Ala Gly Val Ser Leu Ala Gly Val Gly Leu Gln Asn His Ser 725
730 735 Leu Met Glu Trp Pro Ser Ala Ala Asp Val Asp Ala Thr Ile Ala
Ala 740 745 750 Phe Ala Asn Leu Gly Leu Lys Val His Ile Thr Glu Leu
Asp Val Asp 755 760 765 Val Leu Pro Arg Thr Thr Lys Pro Gly Ala Asp
Tyr Ala Val Asp Val 770 775 780 Lys Val Thr Pro Gln Leu Asn Pro Tyr
Leu Asp Gly Leu Pro Glu Ala 785 790 795 800 Arg Gln Ser Ala Leu Ala
Arg Arg Tyr Ala Glu Leu Phe His Val Phe 805 810 815 Arg Lys His Arg
Asp Ala Ile Glu Arg Val Thr Phe Trp Gly Val Ala 820 825 830 Asp Gly
Asp Ser Trp Leu Asn Asn Trp Pro Ile Arg Gly Arg Thr Asn 835 840 845
Tyr Pro Leu Leu Phe Asp Arg Ser Gly Gln Pro Lys Pro Ala Leu Ala 850
855 860 Ser Val Ile Glu Thr Ala Asn Tyr Ser Thr Glu Arg Arg Arg 865
870 875 151725DNAClostridium thermocellum 15gtgtggaagc ccggattgtg
gaatttcctt caaatggcag atgaagccgg attgacgagg 60gatggaaaca ctccggttcc
gacacccagt ccaaagccgg ctaacacacg tattgaagcg 120gaagattatg
acggtattaa ttcttcaagt attgagataa taggtgttcc acctgaagga
180ggcagaggaa taggttatat taccagtggt gattatctgg tatacaagag
tatagacttt 240ggaaacggag caacgtcgtt taaggccaag gttgcaaatg
caaatacttc caatattgaa 300cttagattaa acggtccgaa tggtactctc
ataggcacac tctcggtaaa atccacagga 360gattggaata catatgagga
gcaaacttgc agcattagca aagtcaccgg aataaatgat 420ttgtacttgg
tattcaaagg ccctgtaaac atagactggt tcacttttgg cgttgaaagc
480agttccacag gtctggggga tttaaatggt gacggaaata ttaactcgtc
ggaccttcag 540gcgttaaaga ggcatttgct cggtatatca ccgcttacgg
gagaggctct tttaagagcg 600gatgtaaata ggagcggcaa agtggattct
actgactatt cagtgctgaa aagatatata 660ctccgcatta ttacagagtt
ccccggacaa ggtgatgtac agacacccaa tccgtctgtt 720actccgacac
aaactcctat ccccacgatt tcgggaaatg ctcttaggga ttatgcggag
780gcaaggggaa taaaaatcgg aacatgtgtc aactatccgt tttacaacaa
ttcagatcca 840acctacaaca gcattttgca aagagaattt tcaatggttg
tatgtgaaaa tgaaatgaag 900tttgatgctt tgcagccgag acaaaacgtt
tttgattttt cgaaaggaga ccagttgctt 960gcttttgcag aaagaaacgg
tatgcagatg aggggacata cgttgatttg gcacaatcaa 1020aacccgtcat
ggcttacaaa cggtaactgg aaccgggatt cgctgcttgc ggtaatgaaa
1080aatcacatta ccactgttat gacccattac aaaggtaaaa ttgttgagtg
ggatgtggca 1140aacgaatgta tggatgattc cggcaacggc ttaagaagca
gcatatggag aaatgtaatc 1200ggtcaggact accttgacta tgctttcagg
tatgcaagag aagcagatcc cgatgcactt 1260cttttctaca atgattataa
tattgaagac ttgggtccaa agtccaatgc ggtatttaac 1320atgattaaaa
gtatgaagga aagaggtgtg ccgattgacg gagtaggatt ccaatgccac
1380tttatcaatg gaatgagccc cgagtacctt gccagcattg atcaaaatat
taagagatat 1440gcggaaatag gcgttatagt atcctttacc gaaatagata
tacgcatacc tcagtcggaa 1500aacccggcaa ctgcattcca ggtacaggca
aacaactata aggaacttat gaaaatttgt 1560ctggcaaacc ccaattgcaa
tacctttgta atgtggggat tcacagataa atacacatgg 1620attccgggaa
ctttcccagg atatggcaat ccattgattt atgacagcaa ttacaatccg
1680aaaccggcat acaatgcaat aaaggaagct cttatgggct attga
172516574PRTClostridium thermocellumDOMAIN(39)...(158)Carbohydrate
binding module (family 6)DOMAIN(167)...(187)Dockerin type I
repeatDOMAIN(201)...(221)Dockerin type I
repeatDOMAIN(254)...(571)Glycosyl hydrolase family
10SITE(47)...(50)N-glycosylation site. Prosite id =
PS00001SITE(95)...(98)N-glycosylation site. Prosite id =
PS00001SITE(107)...(110)N-glycosylation site. Prosite id =
PS00001SITE(167)...(179)EF-hand calcium-binding domain. Prosite id
= PS00018SITE(167)...(186)Clostridium cellulosome enzymes repeated
domain signature. Prosite id =
PS00448SITE(175)...(178)N-glycosylation site. Prosite id =
PS00001SITE(204)...(216)EF-hand calcium-binding domain. Prosite id
= PS00018SITE(201)...(213)Clostridium cellulosome enzymes repeated
domain signature. Prosite id =
PS00448SITE(203)...(206)N-glycosylation site. Prosite id =
PS00001SITE(276)...(279)N-glycosylation site. Prosite id =
PS00001SITE(484)...(594)Glycosyl hydrolases family 10 active site.
Prosite id = PS00591 16Met Trp Lys Pro Gly Leu Trp Asn Phe Leu Gln
Met Ala Asp Glu Ala 1 5 10 15 Gly Leu Thr Arg Asp Gly Asn Thr Pro
Val Pro Thr Pro Ser Pro Lys 20 25 30 Pro Ala Asn Thr Arg Ile Glu
Ala Glu Asp Tyr Asp Gly Ile Asn Ser 35 40 45 Ser Ser Ile Glu Ile
Ile Gly Val Pro Pro Glu Gly Gly Arg Gly Ile 50 55 60 Gly Tyr Ile
Thr Ser Gly Asp Tyr Leu Val Tyr Lys Ser Ile Asp Phe 65 70 75 80 Gly
Asn Gly Ala Thr Ser Phe Lys Ala Lys Val Ala Asn Ala Asn Thr 85 90
95 Ser Asn Ile Glu Leu Arg Leu Asn Gly Pro Asn Gly Thr Leu Ile Gly
100 105 110 Thr Leu Ser Val Lys Ser Thr Gly Asp Trp Asn Thr Tyr Glu
Glu Gln 115 120 125 Thr Cys Ser Ile Ser Lys Val Thr Gly Ile Asn Asp
Leu Tyr Leu Val 130 135 140 Phe Lys Gly Pro Val Asn Ile Asp Trp Phe
Thr Phe Gly Val Glu Ser 145 150 155 160 Ser Ser Thr Gly Leu Gly Asp
Leu Asn Gly Asp Gly Asn Ile Asn Ser 165 170 175 Ser Asp Leu Gln Ala
Leu Lys Arg His Leu Leu Gly Ile Ser Pro Leu 180 185 190 Thr Gly Glu
Ala Leu Leu Arg Ala Asp Val Asn Arg Ser Gly Lys Val 195 200 205 Asp
Ser Thr Asp Tyr Ser Val Leu Lys Arg Tyr Ile Leu Arg Ile Ile 210 215
220 Thr Glu Phe Pro Gly Gln Gly Asp Val Gln Thr Pro Asn Pro Ser Val
225 230 235 240 Thr Pro Thr Gln Thr Pro Ile Pro Thr Ile Ser Gly Asn
Ala Leu Arg 245 250 255 Asp Tyr Ala Glu Ala Arg Gly Ile Lys Ile Gly
Thr Cys Val Asn Tyr 260 265 270 Pro Phe Tyr Asn Asn Ser Asp Pro Thr
Tyr Asn Ser Ile Leu Gln Arg 275 280 285 Glu Phe Ser Met Val Val Cys
Glu Asn Glu Met Lys Phe Asp Ala Leu 290 295 300 Gln Pro Arg Gln Asn
Val Phe Asp Phe Ser Lys Gly Asp Gln Leu Leu 305 310 315 320 Ala Phe
Ala Glu Arg Asn Gly Met Gln Met Arg Gly His Thr Leu Ile 325 330 335
Trp His Asn Gln Asn Pro Ser Trp Leu Thr Asn Gly Asn Trp Asn Arg 340
345 350 Asp Ser Leu Leu Ala Val Met Lys Asn His Ile Thr Thr Val Met
Thr 355 360 365 His Tyr Lys Gly Lys Ile Val Glu Trp Asp Val Ala Asn
Glu Cys Met 370 375 380 Asp Asp Ser Gly Asn Gly Leu Arg Ser Ser Ile
Trp Arg Asn Val Ile 385 390 395 400 Gly Gln Asp Tyr Leu Asp Tyr Ala
Phe Arg Tyr Ala Arg Glu Ala Asp 405 410 415 Pro Asp Ala Leu Leu Phe
Tyr Asn Asp Tyr Asn Ile Glu Asp Leu Gly 420 425 430 Pro Lys Ser Asn
Ala Val Phe Asn Met Ile Lys Ser Met Lys Glu Arg 435 440 445 Gly Val
Pro Ile Asp Gly Val Gly Phe Gln Cys His Phe Ile Asn Gly 450 455 460
Met Ser Pro Glu Tyr Leu Ala Ser Ile Asp Gln Asn Ile Lys Arg Tyr 465
470 475 480 Ala Glu Ile Gly Val Ile Val Ser Phe Thr Glu Ile Asp Ile
Arg Ile 485 490 495 Pro Gln Ser Glu Asn Pro Ala Thr Ala Phe Gln Val
Gln Ala Asn Asn 500 505 510 Tyr Lys Glu Leu Met Lys Ile Cys Leu Ala
Asn Pro Asn Cys Asn Thr 515 520 525 Phe Val Met Trp Gly Phe Thr Asp
Lys Tyr Thr Trp Ile Pro Gly Thr 530 535 540 Phe Pro Gly Tyr Gly Asn
Pro Leu Ile Tyr Asp Ser Asn Tyr Asn Pro 545 550 555 560 Lys Pro Ala
Tyr Asn Ala Ile Lys Glu Ala Leu Met Gly Tyr 565 570
172232DNACochliobolus heterostrophus ATCC 48331 17gcaatcggtc
ctgattgtac caatggtccc ctgagtacca atgcaatttg cgatgtcaat 60gcgcctcctc
atgagagggc agcggctcta gtcgcagcta tggaaccgca agaaaagcta
120gataacctcg tcagtaaatc caaaggtgtg tcgagattag gtcttccagc
gtataactgg 180tggggcgaag ctctacacgg tgtagctgga gcgccaggaa
tcaaattcgt cgaaccttat 240aaaaacgcta cttcgtttcc tatgccaatc
cttatgtcgg cagcttttga tgatgatctc 300attttcaaaa ttgccaatat
tatcgggaac gaggcccgag ccttcggaaa tggtggagtc 360gctcctatgg
actattggac ccctgacatc aatcccgtcc gcgatatacg atggggccga
420gccagtgaat cacccggaga ggacattcga cgaataaaag ggtacaccaa
ggctctgctt 480gctggcctcg aaggtgacca agcccaaagg aagatcattg
caacatgcaa acactatgtg 540ggttacgaca tggaagcttg gggaggatac
gatcgacaca acttcagtgc aaagatcacc 600atgcaagacc tcgcagagta
ctacatgccg ccattccagc aatgtgcgcg tgactcgaag 660gtcgggtcat
tcatgtgcag ctacaatgca gtcaacggtg ttccaacatg cgctgacacc
720tacgttcttc aaacaatcct gagggaccac tggaactgga cagatagcaa
taactacatt 780actagcgatt gcgaagccgt tgcggatatc tctgagaacc
acaaatatgt cgaaaccctt 840gcgcaaggca ccgcacttgc ttttgccaag
ggtatggatc ttagctgtga atacagtgga 900tcgtcagata tcccaggagc
ttggtcacaa ggtcttctga atctttctgt tatcgacaaa 960gcattgactc
gacaatatga aggcttagtc catgccggct actttgatgg cgcgaaggcg
1020acttacgcaa acttgagtta taatgacatc aacacacccg aagcacgaca
gctatccttg 1080caagttacct ctgaaggttt ggtcatgcta aagaacgatc
acacacttcc attgcctctc 1140acgaagggat caaaggtggc tatgataggt
ttctgggcca acgactcttc caaactccag 1200ggcatctaca gcggtccacc
tccttaccgg cactctccag tattcgctgg tgaacaaatg 1260ggattagata
tggccatagc ctggggccca atgattcaga actcaagtgt gcccgacaac
1320tggactacca acgcgctcga cgcggccgag aagtccgact atattctcta
ctttggtggt 1380caagactgga cagtggcgca agaaggctac gatcgcacta
caatcagttt tcctcaagtg 1440caaatcgacc ttcttgccaa actggctaaa
cttggcaagc cgcttgttgt catcacgctt 1500ggtgatatga ctgatcactc
ccctctcttg tccatggaag gcatcaactc aattatctgg 1560gcgaattggc
ctggccaaga tggcggtcca gcgatactaa acgtgatttc cggtgtgcat
1620gctcctgcag gtcgtttgcc aataacggaa tacccggcag
attatgtcaa gctctctatg 1680cttgacatga acttgcgacc acatgccgag
agccctggcc gtacttatcg ctggttcaat 1740gagtctgttc agccatttgg
cttcggtcta cattacacta cttttgaggc tggttttgct 1800agcgaagaag
gtctaaccta cgatatccag gaaaccttgg atagctgtac acagcagtac
1860aaggatttgt gtgaggttgc accactggag gtcaccgtgg caaacaaggg
taaccgaaca 1920tcggatttcg tcgctctcgc tttcatcaag ggcgaggttg
gacctaagcc atacccacta 1980aagactctga ttacgtacgg gaggctcaga
gatatccatg ggggcgcgaa gaagtcggcg 2040tcacttccgc ttacacttgg
agaattggcc agagtggatc aatcaggcaa caccgttatc 2100tatcccggcg
aatacaccct gctccttgac gagcctactc aggctgagct gaaattgact
2160attacgggcg aggagacaat tctggacaaa tggccccagc cgccaaacgg
aggcaatcgg 2220accgtgcttt ga 223218743PRTCochliobolus
heterostrophus ATCC 48331DOMAIN(47)...(297)Glycosyl hydrolase
family 3 N terminal domainDOMAIN(367)...(594)Glycosyl hydrolase
family 3 C terminal domainSITE(82)...(85)N-glycosylation site.
Prosite id = PS00001SITE(252)...(255)N-glycosylation site. Prosite
id = PS00001SITE(314)...(317)N-glycosylation site. Prosite id =
PS00001SITE(344)...(347)N-glycosylation site. Prosite id =
PS00001SITE(394)...(397)N-glycosylation site. Prosite id =
PS00001SITE(434)...(437)N-glycosylation site. Prosite id =
PS00001SITE(440)...(443)N-glycosylation site. Prosite id =
PS00001SITE(580)...(583)N-glycosylation site. Prosite id =
PS00001SITE(638)...(641)N-glycosylation site. Prosite id =
PS00001SITE(739)...(742)N-glycosylation site. Prosite id = PS00001
18Ala Ile Gly Pro Asp Cys Thr Asn Gly Pro Leu Ser Thr Asn Ala Ile 1
5 10 15 Cys Asp Val Asn Ala Pro Pro His Glu Arg Ala Ala Ala Leu Val
Ala 20 25 30 Ala Met Glu Pro Gln Glu Lys Leu Asp Asn Leu Val Ser
Lys Ser Lys 35 40 45 Gly Val Ser Arg Leu Gly Leu Pro Ala Tyr Asn
Trp Trp Gly Glu Ala 50 55 60 Leu His Gly Val Ala Gly Ala Pro Gly
Ile Lys Phe Val Glu Pro Tyr 65 70 75 80 Lys Asn Ala Thr Ser Phe Pro
Met Pro Ile Leu Met Ser Ala Ala Phe 85 90 95 Asp Asp Asp Leu Ile
Phe Lys Ile Ala Asn Ile Ile Gly Asn Glu Ala 100 105 110 Arg Ala Phe
Gly Asn Gly Gly Val Ala Pro Met Asp Tyr Trp Thr Pro 115 120 125 Asp
Ile Asn Pro Val Arg Asp Ile Arg Trp Gly Arg Ala Ser Glu Ser 130 135
140 Pro Gly Glu Asp Ile Arg Arg Ile Lys Gly Tyr Thr Lys Ala Leu Leu
145 150 155 160 Ala Gly Leu Glu Gly Asp Gln Ala Gln Arg Lys Ile Ile
Ala Thr Cys 165 170 175 Lys His Tyr Val Gly Tyr Asp Met Glu Ala Trp
Gly Gly Tyr Asp Arg 180 185 190 His Asn Phe Ser Ala Lys Ile Thr Met
Gln Asp Leu Ala Glu Tyr Tyr 195 200 205 Met Pro Pro Phe Gln Gln Cys
Ala Arg Asp Ser Lys Val Gly Ser Phe 210 215 220 Met Cys Ser Tyr Asn
Ala Val Asn Gly Val Pro Thr Cys Ala Asp Thr 225 230 235 240 Tyr Val
Leu Gln Thr Ile Leu Arg Asp His Trp Asn Trp Thr Asp Ser 245 250 255
Asn Asn Tyr Ile Thr Ser Asp Cys Glu Ala Val Ala Asp Ile Ser Glu 260
265 270 Asn His Lys Tyr Val Glu Thr Leu Ala Gln Gly Thr Ala Leu Ala
Phe 275 280 285 Ala Lys Gly Met Asp Leu Ser Cys Glu Tyr Ser Gly Ser
Ser Asp Ile 290 295 300 Pro Gly Ala Trp Ser Gln Gly Leu Leu Asn Leu
Ser Val Ile Asp Lys 305 310 315 320 Ala Leu Thr Arg Gln Tyr Glu Gly
Leu Val His Ala Gly Tyr Phe Asp 325 330 335 Gly Ala Lys Ala Thr Tyr
Ala Asn Leu Ser Tyr Asn Asp Ile Asn Thr 340 345 350 Pro Glu Ala Arg
Gln Leu Ser Leu Gln Val Thr Ser Glu Gly Leu Val 355 360 365 Met Leu
Lys Asn Asp His Thr Leu Pro Leu Pro Leu Thr Lys Gly Ser 370 375 380
Lys Val Ala Met Ile Gly Phe Trp Ala Asn Asp Ser Ser Lys Leu Gln 385
390 395 400 Gly Ile Tyr Ser Gly Pro Pro Pro Tyr Arg His Ser Pro Val
Phe Ala 405 410 415 Gly Glu Gln Met Gly Leu Asp Met Ala Ile Ala Trp
Gly Pro Met Ile 420 425 430 Gln Asn Ser Ser Val Pro Asp Asn Trp Thr
Thr Asn Ala Leu Asp Ala 435 440 445 Ala Glu Lys Ser Asp Tyr Ile Leu
Tyr Phe Gly Gly Gln Asp Trp Thr 450 455 460 Val Ala Gln Glu Gly Tyr
Asp Arg Thr Thr Ile Ser Phe Pro Gln Val 465 470 475 480 Gln Ile Asp
Leu Leu Ala Lys Leu Ala Lys Leu Gly Lys Pro Leu Val 485 490 495 Val
Ile Thr Leu Gly Asp Met Thr Asp His Ser Pro Leu Leu Ser Met 500 505
510 Glu Gly Ile Asn Ser Ile Ile Trp Ala Asn Trp Pro Gly Gln Asp Gly
515 520 525 Gly Pro Ala Ile Leu Asn Val Ile Ser Gly Val His Ala Pro
Ala Gly 530 535 540 Arg Leu Pro Ile Thr Glu Tyr Pro Ala Asp Tyr Val
Lys Leu Ser Met 545 550 555 560 Leu Asp Met Asn Leu Arg Pro His Ala
Glu Ser Pro Gly Arg Thr Tyr 565 570 575 Arg Trp Phe Asn Glu Ser Val
Gln Pro Phe Gly Phe Gly Leu His Tyr 580 585 590 Thr Thr Phe Glu Ala
Gly Phe Ala Ser Glu Glu Gly Leu Thr Tyr Asp 595 600 605 Ile Gln Glu
Thr Leu Asp Ser Cys Thr Gln Gln Tyr Lys Asp Leu Cys 610 615 620 Glu
Val Ala Pro Leu Glu Val Thr Val Ala Asn Lys Gly Asn Arg Thr 625 630
635 640 Ser Asp Phe Val Ala Leu Ala Phe Ile Lys Gly Glu Val Gly Pro
Lys 645 650 655 Pro Tyr Pro Leu Lys Thr Leu Ile Thr Tyr Gly Arg Leu
Arg Asp Ile 660 665 670 His Gly Gly Ala Lys Lys Ser Ala Ser Leu Pro
Leu Thr Leu Gly Glu 675 680 685 Leu Ala Arg Val Asp Gln Ser Gly Asn
Thr Val Ile Tyr Pro Gly Glu 690 695 700 Tyr Thr Leu Leu Leu Asp Glu
Pro Thr Gln Ala Glu Leu Lys Leu Thr 705 710 715 720 Ile Thr Gly Glu
Glu Thr Ile Leu Asp Lys Trp Pro Gln Pro Pro Asn 725 730 735 Gly Gly
Asn Arg Thr Val Leu 740 191362DNAUnknownBacterial DNA 19atgcttcagt
ttccgaaaga ttttatttgg ggagctgcaa cttcatcgta tcaaattgaa 60ggaacagcga
ctggagaaga taaaatttac tcgatctggg atcacttttc ccgcattcct
120ggcaaagtag cgaatgggga taatggcgat atcgcaattg atcattacaa
tcgttatgtt 180gaagacatcg cattaatgaa agcgcttcat ttgaaagcgt
atcgattttc gactagttgg 240gcgagacttt attgtgaaac gccagggaag
tttaacgaaa aaggtttaga tttttataag 300cgtcttgtac atgaattgct
agagaacggt atcgagccaa tgttgaccat ttatcattgg 360gatatgccac
aagctcttca agagaaaggt ggctgggaaa atcgtgatat cgttcactac
420ttccaagaat acgctgcttt cctttacgag aatcttgggg atgtcgtgaa
aaaatggatt 480acgcataatg agccgtgggt tgtcacctat ttaggatatg
ggaatggcga acatgcccca 540gggattcaaa actttacatc atttttaaaa
gcagcacatc atgttcttct ctcacacggg 600gaagcggtaa aagcgtttcg
agcaatcggt tcgaaagatg gggaaattgg tattacgttg 660aatttgacac
ctggatatgc ggtcgatccg aaagatgaaa aagcagttga tgccgctcga
720aaatgggacg gctttatgaa tcgttggttt ttagatcctg tatttaaggg
acaatatcca 780gcagatatgt tagaagtgta taaagattat ttaccagacg
tttacaaaga gggagattta 840caaacgattc agcaaccgat cgactttttc
ggatttaact attattcaac agcaacatta 900aaagattgga aaacaggtga
ccgtgaaccg atcgtatttg aacatgtgag cacaggaaga 960cctgtgacgg
atatgaattg ggaagtgaat ccaaacggtt tgtttgattt aatggtgcga
1020ttgaaaaaag attatggcga tattccatta tacattaccg aaaacggtgc
tgcatacaaa 1080gatcgcgtca acgaacaagg tgaagtagaa gatgatgagc
gagttgctta tatacgggag 1140catttaatcg cttgccaccg cgcgattgaa
caaggcgtca atttaaaagg atattatgta 1200tggtcgctgt tcgataattt
tgagtgggca tttggatatg ataagcgctt tgggattgta 1260tacgtggatt
atgaaacgct agagcgcatc ccgaaaaaga gtgcattatg gtacaaggaa
1320acgattataa acaacggatt gcaagtagac aatgacaaat aa
136220453PRTUnknownBacterial proteinDOMAIN(1)...(447)Glycosyl
hydrolase family 1SITE(8)...(22)Glycosyl hydrolases family 1
N-terminal signature. Prosite id =
PS00653SITE(184)...(187)N-glycosylation site. Prosite id =
PS00001SITE(350)...(358)Glycosyl hydrolases family 1 active site.
Prosite id = PS00572 20Met Leu Gln Phe Pro Lys Asp Phe Ile Trp Gly
Ala Ala Thr Ser Ser 1 5 10 15 Tyr Gln Ile Glu Gly Thr Ala Thr Gly
Glu Asp Lys Ile Tyr Ser Ile 20 25 30 Trp Asp His Phe Ser Arg Ile
Pro Gly Lys Val Ala Asn Gly Asp Asn 35 40 45 Gly Asp Ile Ala Ile
Asp His Tyr Asn Arg Tyr Val Glu Asp Ile Ala 50 55 60 Leu Met Lys
Ala Leu His Leu Lys Ala Tyr Arg Phe Ser Thr Ser Trp 65 70 75 80 Ala
Arg Leu Tyr Cys Glu Thr Pro Gly Lys Phe Asn Glu Lys Gly Leu 85 90
95 Asp Phe Tyr Lys Arg Leu Val His Glu Leu Leu Glu Asn Gly Ile Glu
100 105 110 Pro Met Leu Thr Ile Tyr His Trp Asp Met Pro Gln Ala Leu
Gln Glu 115 120 125 Lys Gly Gly Trp Glu Asn Arg Asp Ile Val His Tyr
Phe Gln Glu Tyr 130 135 140 Ala Ala Phe Leu Tyr Glu Asn Leu Gly Asp
Val Val Lys Lys Trp Ile 145 150 155 160 Thr His Asn Glu Pro Trp Val
Val Thr Tyr Leu Gly Tyr Gly Asn Gly 165 170 175 Glu His Ala Pro Gly
Ile Gln Asn Phe Thr Ser Phe Leu Lys Ala Ala 180 185 190 His His Val
Leu Leu Ser His Gly Glu Ala Val Lys Ala Phe Arg Ala 195 200 205 Ile
Gly Ser Lys Asp Gly Glu Ile Gly Ile Thr Leu Asn Leu Thr Pro 210 215
220 Gly Tyr Ala Val Asp Pro Lys Asp Glu Lys Ala Val Asp Ala Ala Arg
225 230 235 240 Lys Trp Asp Gly Phe Met Asn Arg Trp Phe Leu Asp Pro
Val Phe Lys 245 250 255 Gly Gln Tyr Pro Ala Asp Met Leu Glu Val Tyr
Lys Asp Tyr Leu Pro 260 265 270 Asp Val Tyr Lys Glu Gly Asp Leu Gln
Thr Ile Gln Gln Pro Ile Asp 275 280 285 Phe Phe Gly Phe Asn Tyr Tyr
Ser Thr Ala Thr Leu Lys Asp Trp Lys 290 295 300 Thr Gly Asp Arg Glu
Pro Ile Val Phe Glu His Val Ser Thr Gly Arg 305 310 315 320 Pro Val
Thr Asp Met Asn Trp Glu Val Asn Pro Asn Gly Leu Phe Asp 325 330 335
Leu Met Val Arg Leu Lys Lys Asp Tyr Gly Asp Ile Pro Leu Tyr Ile 340
345 350 Thr Glu Asn Gly Ala Ala Tyr Lys Asp Arg Val Asn Glu Gln Gly
Glu 355 360 365 Val Glu Asp Asp Glu Arg Val Ala Tyr Ile Arg Glu His
Leu Ile Ala 370 375 380 Cys His Arg Ala Ile Glu Gln Gly Val Asn Leu
Lys Gly Tyr Tyr Val 385 390 395 400 Trp Ser Leu Phe Asp Asn Phe Glu
Trp Ala Phe Gly Tyr Asp Lys Arg 405 410 415 Phe Gly Ile Val Tyr Val
Asp Tyr Glu Thr Leu Glu Arg Ile Pro Lys 420 425 430 Lys Ser Ala Leu
Trp Tyr Lys Glu Thr Ile Ile Asn Asn Gly Leu Gln 435 440 445 Val Asp
Asn Asp Lys 450 21930DNACochliobolus heterostrophus ATCC 48331
21gtgtcgccca agcaggacag ccgtcaaatc cagggtatca aggacccgac gattatccag
60aacaatggtg tataccatgt ctttgccagc acggccaagg aagcgggata caacctagtc
120tacttcaact ttaccgactt cagcagggcc aaccaggcgc cattcttcta
cctcgaccag 180tcaggtatcg gcacaggtta ccgtgctgct cctcaagtct
tctacttcgc cccccagaag 240ctctggtacc tcatctacca aaacggcaat
gcagcataca gcaccaaccc cgacatttcc 300aacccacgag gctggaccgc
cccgcaagtc ttctacccca acggaacccc ccagacgatc 360caaaacggcc
taggaacgac cggctactgg gtcgacatgt gggtaatctg cgacacggcc
420ctctgccacc tgtactcatc cgacgacaac ggcggcctat accgcagcca
aacgcccgtc 480tcgcaattcc cacgcggcat gaacgagccc gtggtaacgc
tcaaggccaa caaaaacgac 540ctctttgaag cctcgaccgt gtacaacatt
gtaaacacca gcacctacct cctcatggtc 600gaatgcatcg gctccggcaa
ctcccccggc ggcctgcgct acttccgctc ctggaccacc 660cagtccctca
ccagcgacaa gtggactccc cttgccgcat cccagcaaac ccctttcctc
720ggcgccgcta acacccagtt ccccgccggc cgctggtccc agagcttgtc
ccacggcgag 780ctcgttcgca caaatgtaga ccagaggctc cagattcgcc
cctgtgaaat gaggtacctc 840taccagggta tcgatcctaa tgctacgggc
acttacaatg ccctgccctg gaaactcgcc 900cttgcaaccc agacaaactc
caagtgttag 93022309PRTCochliobolus heterostrophus ATCC
48331DOMAIN(3)...(276)Glycosyl hydrolase family
62SITE(43)...(46)N-glycosylation site. Prosite id =
PS00001SITE(192)...(195)N-glycosylation site. Prosite id =
PS00001SITE(287)...(290)N-glycosylation site. Prosite id = PS00001
22Met Ser Pro Lys Gln Asp Ser Arg Gln Ile Gln Gly Ile Lys Asp Pro 1
5 10 15 Thr Ile Ile Gln Asn Asn Gly Val Tyr His Val Phe Ala Ser Thr
Ala 20 25 30 Lys Glu Ala Gly Tyr Asn Leu Val Tyr Phe Asn Phe Thr
Asp Phe Ser 35 40 45 Arg Ala Asn Gln Ala Pro Phe Phe Tyr Leu Asp
Gln Ser Gly Ile Gly 50 55 60 Thr Gly Tyr Arg Ala Ala Pro Gln Val
Phe Tyr Phe Ala Pro Gln Lys 65 70 75 80 Leu Trp Tyr Leu Ile Tyr Gln
Asn Gly Asn Ala Ala Tyr Ser Thr Asn 85 90 95 Pro Asp Ile Ser Asn
Pro Arg Gly Trp Thr Ala Pro Gln Val Phe Tyr 100 105 110 Pro Asn Gly
Thr Pro Gln Thr Ile Gln Asn Gly Leu Gly Thr Thr Gly 115 120 125 Tyr
Trp Val Asp Met Trp Val Ile Cys Asp Thr Ala Leu Cys His Leu 130 135
140 Tyr Ser Ser Asp Asp Asn Gly Gly Leu Tyr Arg Ser Gln Thr Pro Val
145 150 155 160 Ser Gln Phe Pro Arg Gly Met Asn Glu Pro Val Val Thr
Leu Lys Ala 165 170 175 Asn Lys Asn Asp Leu Phe Glu Ala Ser Thr Val
Tyr Asn Ile Val Asn 180 185 190 Thr Ser Thr Tyr Leu Leu Met Val Glu
Cys Ile Gly Ser Gly Asn Ser 195 200 205 Pro Gly Gly Leu Arg Tyr Phe
Arg Ser Trp Thr Thr Gln Ser Leu Thr 210 215 220 Ser Asp Lys Trp Thr
Pro Leu Ala Ala Ser Gln Gln Thr Pro Phe Leu 225 230 235 240 Gly Ala
Ala Asn Thr Gln Phe Pro Ala Gly Arg Trp Ser Gln Ser Leu 245 250 255
Ser His Gly Glu Leu Val Arg Thr Asn Val Asp Gln Arg Leu Gln Ile 260
265 270 Arg Pro Cys Glu Met Arg Tyr Leu Tyr Gln Gly Ile Asp Pro Asn
Ala 275 280 285 Thr Gly Thr Tyr Asn Ala Leu Pro Trp Lys Leu Ala Leu
Ala Thr Gln 290 295 300 Thr Asn Ser Lys Cys 305
232163DNAUnknownObtained from environmental sample 23atgaaacatc
acaactataa cgcgcatcat tcgccaatcg gcgctttcgg ctcattcacg 60ctcggttttc
gtggtgctca gggcggcctc ggactggagt taggcggccc ggccaatcac
120aacatgtaca tcggagtgga agacgagcag cgcaccttcc attgccttcc
cttttttggg 180gatgctgctg caggggccga ggaagcactg cgctacgatg
tggaaggcag ccaatccagt 240gacgatccac tggccggcgc ctatgtcgga
cacccagagg atgcgccatc gctgcctccg 300gccaagttgc gtgcgctgga
ccaaagcgcc atctcacggg attttcaact tacgaccgac 360acctggacag
caccggattt ctcactcacg atctattcgc cggtacgcgg cgtgcccgat
420ccgacaacgg ctgcggaaga cgaattgaaa gccattcttg tgcccgctgt
actctgcgag 480ttgacggtgg ataactcgag tgggcagcag tctcggcgtg
cgctctttgg tttcaccggg 540aacgatcctt attggggaac gcggcgcctt
gatgatgtag cgaatagtgc gttcgtgggg 600gttggcgagg gaaatcatct
ggccattgcg tcacgagatg aaggagtgac ggcggcgctg 660ggcttcaaca
tcaatggcgt tatcaacgag actttgcctg agaattacgc ctttggtctg
720ggcaaatgcg cagttttgct ctgcgaggtg cctgccggtg aaaagcgcac
gttccatatc 780gccgtctgtt ttcatcggag cggcatcgcc accaccggtt
tgaagatgcg ctattattac 840acgcgctttt tccctgacat cgaaagcgta
gccgcttatg cactggagca gttcgattct 900ctcaaaagtg cagctctcca
agacaatcaa ctagtggaga acgcgtcgct ttcagaagac 960cagaaatgga
tgttctgcca cgcggtgcgc tcgtactatg gctcaacgga gttgctggag
1020tataacgaca atccggtgtg ggtggtcaac gaaggcgaat atcgtatgat
gaacaccttc 1080gacttgacgg tggatcatct ctactgggaa ctgcgcctga
atccctgggt tgtgaaaaat 1140cagctcgact ggtttgtgga tcgctactcg
tatgaggaca aggtgcgctt tcctggtgac 1200aaaactgagt acccatgcgg
tctctccttc acgcacgata tgggcgtgac gaatgtgtgg 1260tcgcgccccg
gctattcgtc ttatgagaag cagggactca agggtgtctt ttcgtatatg
1320acgcacgagc aactcgtcaa ctggctctgc tgcgccacgg tgtatgtgga
acagaccggt 1380gaccaggagt ggcttgaaca acggtggccg attttcaaca
ggtgctttga gagtttgctc 1440aaccgcgatc accccgatcc tgaaaagcgg
cgcggcttaa tgcaactcga ttcgacccgt 1500tgcgccggtg gtgcggagat
caccacttac gacagccttg atgtctcgct ggggcagtca 1560cgcaacaaca
cctatctggg tgggaaaatc tgggcgagct atctggcact cgaaaaattg
1620ttccgcgagc gcggcgacgt ggaaagagcg caagtggcgc atcaacaggc
gcatcgcacg 1680gcgcaaacgc tgctggagaa tgtcggcgag aatgggacga
ttcccgccgt actcgaaggc 1740agcaatcagt cgagaatcat tcccgtgatt
gaggggttga tcttccccta cttcaccggg 1800cgcaaggatg tcctcagttc
cgatggcgac ttcggcgaga tgttttcggc actcaagcgc 1860catcttgaag
ccgtgctaaa acccggtatc tgtctgtttg aagatggtgg ctggaagtta
1920agttcaacct ctgataactc gtggctgagc aaaatctacc tgtgccagtt
cgtcgcgcgc 1980cagatattgg gccgtgaacg cgatgacatt gacaagcgcg
ccgatgccgc gcacgtgggc 2040tggctgctcg atgagcgcaa tgcctacttc
gcgtggagtg accagatgct ggccggcttt 2100gcggaaggct ccaagtacta
cccgcgcggt gtgaccagcg cattgtggtt gctggaaggc 2160tga
216324720PRTUnknownObtained from environmental
sampleDOMAIN(19)...(456)Glycosyl hydrolase family
52SITE(165)...(168)N-glycosylation site. Prosite id =
PS00001SITE(229)...(232)N-glycosylation site. Prosite id =
PS00001SITE(314)...(317)N-glycosylation site. Prosite id =
PS00001SITE(522)...(525)N-glycosylation site. Prosite id =
PS00001SITE(571)...(574)N-glycosylation site. Prosite id =
PS00001SITE(582)...(585)N-glycosylation site. Prosite id = PS00001
24Met Lys His His Asn Tyr Asn Ala His His Ser Pro Ile Gly Ala Phe 1
5 10 15 Gly Ser Phe Thr Leu Gly Phe Arg Gly Ala Gln Gly Gly Leu Gly
Leu 20 25 30 Glu Leu Gly Gly Pro Ala Asn His Asn Met Tyr Ile Gly
Val Glu Asp 35 40 45 Glu Gln Arg Thr Phe His Cys Leu Pro Phe Phe
Gly Asp Ala Ala Ala 50 55 60 Gly Ala Glu Glu Ala Leu Arg Tyr Asp
Val Glu Gly Ser Gln Ser Ser 65 70 75 80 Asp Asp Pro Leu Ala Gly Ala
Tyr Val Gly His Pro Glu Asp Ala Pro 85 90 95 Ser Leu Pro Pro Ala
Lys Leu Arg Ala Leu Asp Gln Ser Ala Ile Ser 100 105 110 Arg Asp Phe
Gln Leu Thr Thr Asp Thr Trp Thr Ala Pro Asp Phe Ser 115 120 125 Leu
Thr Ile Tyr Ser Pro Val Arg Gly Val Pro Asp Pro Thr Thr Ala 130 135
140 Ala Glu Asp Glu Leu Lys Ala Ile Leu Val Pro Ala Val Leu Cys Glu
145 150 155 160 Leu Thr Val Asp Asn Ser Ser Gly Gln Gln Ser Arg Arg
Ala Leu Phe 165 170 175 Gly Phe Thr Gly Asn Asp Pro Tyr Trp Gly Thr
Arg Arg Leu Asp Asp 180 185 190 Val Ala Asn Ser Ala Phe Val Gly Val
Gly Glu Gly Asn His Leu Ala 195 200 205 Ile Ala Ser Arg Asp Glu Gly
Val Thr Ala Ala Leu Gly Phe Asn Ile 210 215 220 Asn Gly Val Ile Asn
Glu Thr Leu Pro Glu Asn Tyr Ala Phe Gly Leu 225 230 235 240 Gly Lys
Cys Ala Val Leu Leu Cys Glu Val Pro Ala Gly Glu Lys Arg 245 250 255
Thr Phe His Ile Ala Val Cys Phe His Arg Ser Gly Ile Ala Thr Thr 260
265 270 Gly Leu Lys Met Arg Tyr Tyr Tyr Thr Arg Phe Phe Pro Asp Ile
Glu 275 280 285 Ser Val Ala Ala Tyr Ala Leu Glu Gln Phe Asp Ser Leu
Lys Ser Ala 290 295 300 Ala Leu Gln Asp Asn Gln Leu Val Glu Asn Ala
Ser Leu Ser Glu Asp 305 310 315 320 Gln Lys Trp Met Phe Cys His Ala
Val Arg Ser Tyr Tyr Gly Ser Thr 325 330 335 Glu Leu Leu Glu Tyr Asn
Asp Asn Pro Val Trp Val Val Asn Glu Gly 340 345 350 Glu Tyr Arg Met
Met Asn Thr Phe Asp Leu Thr Val Asp His Leu Tyr 355 360 365 Trp Glu
Leu Arg Leu Asn Pro Trp Val Val Lys Asn Gln Leu Asp Trp 370 375 380
Phe Val Asp Arg Tyr Ser Tyr Glu Asp Lys Val Arg Phe Pro Gly Asp 385
390 395 400 Lys Thr Glu Tyr Pro Cys Gly Leu Ser Phe Thr His Asp Met
Gly Val 405 410 415 Thr Asn Val Trp Ser Arg Pro Gly Tyr Ser Ser Tyr
Glu Lys Gln Gly 420 425 430 Leu Lys Gly Val Phe Ser Tyr Met Thr His
Glu Gln Leu Val Asn Trp 435 440 445 Leu Cys Cys Ala Thr Val Tyr Val
Glu Gln Thr Gly Asp Gln Glu Trp 450 455 460 Leu Glu Gln Arg Trp Pro
Ile Phe Asn Arg Cys Phe Glu Ser Leu Leu 465 470 475 480 Asn Arg Asp
His Pro Asp Pro Glu Lys Arg Arg Gly Leu Met Gln Leu 485 490 495 Asp
Ser Thr Arg Cys Ala Gly Gly Ala Glu Ile Thr Thr Tyr Asp Ser 500 505
510 Leu Asp Val Ser Leu Gly Gln Ser Arg Asn Asn Thr Tyr Leu Gly Gly
515 520 525 Lys Ile Trp Ala Ser Tyr Leu Ala Leu Glu Lys Leu Phe Arg
Glu Arg 530 535 540 Gly Asp Val Glu Arg Ala Gln Val Ala His Gln Gln
Ala His Arg Thr 545 550 555 560 Ala Gln Thr Leu Leu Glu Asn Val Gly
Glu Asn Gly Thr Ile Pro Ala 565 570 575 Val Leu Glu Gly Ser Asn Gln
Ser Arg Ile Ile Pro Val Ile Glu Gly 580 585 590 Leu Ile Phe Pro Tyr
Phe Thr Gly Arg Lys Asp Val Leu Ser Ser Asp 595 600 605 Gly Asp Phe
Gly Glu Met Phe Ser Ala Leu Lys Arg His Leu Glu Ala 610 615 620 Val
Leu Lys Pro Gly Ile Cys Leu Phe Glu Asp Gly Gly Trp Lys Leu 625 630
635 640 Ser Ser Thr Ser Asp Asn Ser Trp Leu Ser Lys Ile Tyr Leu Cys
Gln 645 650 655 Phe Val Ala Arg Gln Ile Leu Gly Arg Glu Arg Asp Asp
Ile Asp Lys 660 665 670 Arg Ala Asp Ala Ala His Val Gly Trp Leu Leu
Asp Glu Arg Asn Ala 675 680 685 Tyr Phe Ala Trp Ser Asp Gln Met Leu
Ala Gly Phe Ala Glu Gly Ser 690 695 700 Lys Tyr Tyr Pro Arg Gly Val
Thr Ser Ala Leu Trp Leu Leu Glu Gly 705 710 715 720
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