U.S. patent application number 12/566238 was filed with the patent office on 2010-01-28 for alpha-amylase mutants with altered properties.
This patent application is currently assigned to NOVOZYMES A/S. Invention is credited to CARSTEN ANDERSEN, CLAUS CRONE FUGLSANG, SOREN KJAERULFF, THOMAS THISTED.
Application Number | 20100022433 12/566238 |
Document ID | / |
Family ID | 27576033 |
Filed Date | 2010-01-28 |
United States Patent
Application |
20100022433 |
Kind Code |
A1 |
THISTED; THOMAS ; et
al. |
January 28, 2010 |
Alpha-Amylase Mutants with Altered Properties
Abstract
The present invention relates to variants (mutants) of parent
Termamyl-like alpha-amylases, which variant has alpha-amylase
activity and exhibits altered stability, in particular at high
temperatures and/or at low pH relative, and/or low Ca.sup.2+ to the
parent alpha-amylase.
Inventors: |
THISTED; THOMAS; (RUNGSTED
KYST, DK) ; KJAERULFF; SOREN; (VANLOSE, DK) ;
ANDERSEN; CARSTEN; (VAERLOSE, DK) ; FUGLSANG; CLAUS
CRONE; (NIVA, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE, SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
NOVOZYMES A/S
BAGSVAERD
DK
|
Family ID: |
27576033 |
Appl. No.: |
12/566238 |
Filed: |
September 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10630203 |
Jul 29, 2003 |
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12566238 |
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09918543 |
Jul 31, 2001 |
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10630203 |
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60225140 |
Aug 14, 2000 |
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60233986 |
Sep 20, 2000 |
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60249104 |
Nov 16, 2000 |
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60286869 |
Apr 26, 2001 |
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Current U.S.
Class: |
510/392 ;
435/201 |
Current CPC
Class: |
C12Y 302/01001 20130101;
C11D 3/38681 20130101; D06M 16/003 20130101; C12P 7/14 20130101;
Y02E 50/17 20130101; C12P 19/02 20130101; C12P 19/14 20130101; Y02E
50/10 20130101; C12N 9/2417 20130101; C11D 3/38618 20130101; C11D
3/386 20130101 |
Class at
Publication: |
510/392 ;
435/201 |
International
Class: |
C11D 7/42 20060101
C11D007/42; C12N 9/26 20060101 C12N009/26; C12S 9/00 20060101
C12S009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 1, 2000 |
DK |
2000 01160 |
Sep 12, 2000 |
DK |
2000 01354 |
Nov 10, 2000 |
DK |
2000 01687 |
Apr 26, 2001 |
DK |
2001 00655 |
Claims
1-26. (canceled)
27. A variant of a parent alpha-amylase, comprising a substitution
of the amino acid at position 239, wherein (a) the parent
alpha-amylase has at least 90% sequence identity to SEQ ID NO: 8,
(b) the variant has alpha-amylase activity, and (c) the variant has
1-15 mutations relative to the parent alpha-amylase, and (d) each
position corresponds to a position of the amino acid sequence of
SEQ ID NO: 8.
28. The variant of claim 27, wherein the parent alpha-amylase has
at least 95% identity to SEQ ID NO: 8.
29. The variant of claim 27, wherein the parent alpha-amylase has
at least 97% identity to SEQ ID NO: 8.
30. The variant of claim 27, wherein the parent alpha-amylase has
at least 99% identity to SEQ ID NO: 8.
31. The variant of claim 27, wherein the parent alpha-amylase
comprises SEQ ID NO: 8.
32. The variant of claim 27, wherein the 1-15 mutations are
substitutions.
33. The variant of claim 27, wherein the substitution of the amino
acid at position 239 is the only mutation.
34. The variant of claim 27, which contains two mutations.
35. The variant of claim 27, which contains three, four or five
mutations.
36. The variant of claim 27, which contains 6-12 mutations.
37. A composition comprising an alpha-amylase variant of claim 27
and one or more enzymes selected from the group consisting of
glucoamylase, phytase and pullulanase.
38. A detergent composition comprising an alpha-amylase variant of
claim 27 and a surfactant.
39. A variant of a parent alpha-amylase, comprising a substitution
of the amino acid at position 239, wherein (a) the parent
alpha-amylase has at least 90% sequence identity to SEQ ID NO: 6,
(b) the variant has alpha-amylase activity, and (c) the variant has
1-15 mutations relative to the parent alpha-amylase, and (d) each
position corresponds to a position of the amino acid sequence of
SEQ ID NO: 8.
40. The variant of claim 39, wherein the parent alpha-amylase has
at least 95% identity to SEQ ID NO: 6.
41. The variant of claim 39, wherein the parent alpha-amylase has
at least 97% identity to SEQ ID NO: 6.
42. The variant of claim 39, wherein the parent alpha-amylase has
at least 99% identity to SEQ ID NO: 6.
43. The variant of claim 39, wherein the parent alpha-amylase
comprises SEQ ID NO: 6.
44. The variant of claim 39, wherein the 1-15 mutations are
substitutions.
45. A composition comprising an alpha-amylase variant of claim 39
and one or more enzymes selected from the group consisting of
glucoamylase, phytase and pullulanase.
46. A detergent composition comprising an alpha-amylase variant of
claim 39 and a surfactant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/630,203 filed Jul. 29, 2003, which is a continuation of U.S.
application Ser. No. 09/918,543 filed Jul. 31, 2001, now abandoned,
which claims the benefit or priority under 35 U.S.C. 119 of Danish
Application Nos. PA2000 01160, PA2000 01354, PA2000 01687 and
PA2001 00655 filed Aug. 1, 2000, Sep. 12, 2000, Nov. 10, 2000, and
Apr. 26, 2001, respectively, and U.S. Provisional Application Nos.
60/225,140, 60/233,986, 60/249,104 and 60/286,869 filed on Aug. 14,
2000, Sep. 20, 2000, Nov. 16, 2000, and Apr. 26, 2001,
respectively, the contents of which are fully incorporated herein
by reference.
SEQUENCE LISTING
[0002] The present application contains a Sequence Listing in the
form of a text file, which is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The present invention relates to variants (mutants) of
parent Termamyl-like alpha-amylases, which variant has
alpha-amylase activity and exhibits an alteration in at least one
of the following properties relative to said parent alpha-amylase:
stability under, e.g., high temperature and/or low pH conditions,
in particular at low calcium concentrations. The variant of the
invention are suitable for starch conversion, ethanol production,
laundry wash, dish wash, hard surface cleaning, textile desizing,
and/or sweetner production.
BACKGROUND OF THE INVENTION
[0004] Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C.
3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis
of starch and other linear and branched 1,4-glucosidic oligo- and
polysaccharides.
BRIEF DISCLOSURE OF THE INVENTION
[0005] The object of the present invention is to provide
Termamyl-like amylases which variants in comparison to the
corresponding parent alpha-amylase, i.e., un-mutated alpha-amylase,
has alpha-amylase activity and exhibits an alteration in at least
one of the following properties relative to said parent
alpha-amylase: stability under, e.g., high temperature and/or low
pH conditions, in particular at low calcium concentrations.
Nomenclature
[0006] In the present description and claims, the conventional
one-letter and three-letter codes for amino acid residues are used.
For ease of reference, alpha-amylase variants of the invention are
described by use of the following nomenclature: [0007] Original
amino acid(s): position(s): substituted amino acid(s)
[0008] According to this nomenclature, for instance the
substitution of alanine for asparagine in position 30 is shown as:
[0009] Ala30Asn or A30N a deletion of alanine in the same position
is shown as: [0010] Ala30* or A30* and an insertion of an
additional amino acid residue, such as lysine, is shown as: [0011]
Ala30AlaLys or A30AK
[0012] A deletion of a consecutive stretch of amino acid residues,
such as amino acid residues 30-33, is indicated as (30-33)* or
.DELTA.(A30-N33).
[0013] Where a specific alpha-amylase contains a "deletion" in
comparison with other alpha-amylases and an insertion is made in
such a position this is indicated as: [0014] *36Asp or *36D for an
insertion of an aspartic acid in position 36.
[0015] Multiple mutations are separated by plus signs, i.e.: [0016]
Ala30Asp+Glu34Ser or A30N+E34S representing mutations in positions
30 and 34 substituting alanine and glutamic acid for asparagine and
serine, respectively.
[0017] When one or more alternative amino acid residues may be
inserted in a given position it is indicated as [0018] A30N,E or
A30N or A30E
[0019] Furthermore, when a position suitable for modification is
identified herein without any specific modification being
suggested, it is to be understood that any amino acid residue may
be substituted for the amino acid residue present in the position.
Thus, for instance, when a modification of an alanine in position
30 is mentioned, but not specified, it is to be understood that the
alanine may be deleted or substituted for any other amino acid,
i.e., any one of:
R, N, D, A, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.
[0020] Further, "A30X" means any one of the following
substitutions:
A30R, A30N, A30D, A30C, A30Q, A30E, A30G, A30H, A30I, A30L, A30K,
A30M, A30F, A30P, A30S, A30T, A30W, A30Y, or A30V; or in short:
A30R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V.
[0021] If the parent enzyme--used for the numbering--already has
the amino acid residue in question suggested for substitution in
that position the following nomenclature is used: [0022] "X30N" or
"X30N,V" in the case where for instance one or N or V is present in
the wildtype.
[0023] Thus, it means that other corresponding parent enzymes are
substituted to an "Asn" or "Val" in position 30.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is an alignment of the amino acid sequences of five
parent Termamyl-like alpha-amylases. The numbers on the extreme
left designate the respective amino acid sequences as follows:
1: SEQ ID NO: 4 (SP722)
2: SEQ ID NO: 2 (SP690)
3: SEQ ID NO: 10 (BAN)
4: SEQ ID NO: 8 (BLA)
5: SEQ ID NO: 6 (BSG).
DETAILED DISCLOSURE OF THE INVENTION
[0025] The object of the present invention is to provide
Termamyl-like amylases, which variants have alpha-amylase activity
and exhibits altered stability at high temperatures and/or at low
pH, in particular at low calcium concentrations.
Termamyl-Like Alpha-Amylases
[0026] A number of alpha-amylases produced by Bacillus spp. are
highly homologous (identical) on the amino acid level.
[0027] The identity of a number of known Bacillus alpha-amylases
can be found in the below Table 1:
TABLE-US-00001 TABLE 1 Percent identity 707 AP1378 BAN BSG SP690
SP722 AA560 Termamyl 707 100.0 86.4 66.9 66.5 87.6 86.2 95.5 68.1
AP1378 86.4 100.0 67.1 68.1 95.1 86.6 86.0 69.4 BAN 66.9 67.1 100.0
65.6 67.1 68.8 66.9 80.7 BSG 66.5 68.1 65.6 100.0 67.9 67.1 66.3
65.4 SP690 87.6 95.1 67.1 67.9 100.0 87.2 87.0 69.2 SP722 86.2 86.6
68.8 67.1 87.2 100.0 86.8 70.8 AA560 95.5 86.0 66.9 66.3 87.0 86.8
100.0 68.3 Termamyl 68.1 69.4 80.7 65.4 69.2 70.8 68.3 100.0
[0028] For instance, the B. licheniformis alpha-amylase comprising
the amino acid sequence shown in SEQ ID NO: 8 (commercially
available as Termamyl.TM.) has been found to be about 81%
homologous with the B. amyloliquefaciens alpha-amylase comprising
the amino acid sequence shown in SEQ ID NO: 10 and about 65%
homologous with the B. stearothermophilus alpha-amylase (BSG)
comprising the amino acid sequence shown in SEQ ID NO: 6. Further
homologous alpha-amylases include SP690 and SP722 disclosed in WO
95/26397 and further depicted in SEQ ID NO: 2 and SEQ ID NO: 4,
respectively, herein. Other amylases are the AA560 alpha-amylase
derived from Bacillus sp. and shown in SEQ ID NO: 12, and the #707
alpha-amylase derived from Bacillus sp., shown in SEQ ID NO: 13 and
described by Tsukamoto et al., 1988, Biochemical and Biophysical
Research Communications 151: 25-31.
[0029] The KSM AP1378 alpha-amylase is disclosed in WO 97/00324
(from KAO Corporation).
[0030] Still further homologous alpha-amylases include the
alpha-amylase produced by the B. licheniformis strain described in
EP 0252666 (ATCC 27811), and the alpha-amylases identified in WO
91/00353 and WO 94/18314. Other commercial Termamyl-like
alpha-amylases are comprised in the products sold under the
following tradenames: Optitherm.TM. and Takatherm.TM. (Solvay);
MaxaMyl.TM. (available from Gist-brocades/Genencor), Spezym AA.TM.
and Spezyme Delta AA.TM. (available from Genencor), and
Keistase.TM. (available from Daiwa), Dex Io, GC 521 (available from
Genencor) and Ultraphlow (from Enzyme Biosystems).
[0031] Because of the substantial homology found between these
alpha-amylases, they are considered to belong to the same class of
alpha-amylases, namely the class of "Termamyl-like
alpha-amylases".
[0032] Accordingly, in the present context, the term
"Termamyl-like" alpha-amylase" is intended to indicate an
alpha-amylase, in particular Bacillus alpha-amylase, which, at the
amino acid level, exhibits a substantial identity to Termamyl.TM.,
i.e., the B. licheniformis alpha-amylase having the amino acid
sequence shown in SEQ ID NO: 8, herein.
[0033] In other words, all the following alpha-amylases, which has
the amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12
and 13 herein are considered to be "Termamyl-like alpha-amylase".
Other Termamyl-like alpha-amylases are alpha-amylases i) which
displays at least 60%, such as at least 70%, e.g., at least 75%, or
at least 80%, at least 85%, at least 90%, at least 95%, at least
97%, at least 99% homology (identity) with at least one of said
amino acid sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10, 12, and
13, and/or is encoded by a DNA sequence which hybridizes to the DNA
sequences encoding the above-specified alpha-amylases which are
apparent from SEQ ID NOS: 1, 3, 5, 7, 9, and of the present
specification (which encoding sequences encode the amino acid
sequences shown in SEQ ID NOS: 2, 4, 6, 8, 10 and 12 herein,
respectively).
Homology
[0034] The homology may be determined as the degree of identity
between the two sequences indicating a derivation of the first
sequence from the second. The homology may suitably be determined
by means of computer programs known in the art such as GAP provided
in the GCG program package (described above). Thus, Gap GCGv8 may
be used with the default scoring matrix for identity and the
following default parameters: GAP creation penalty of 5.0 and GAP
extension penalty of 0.3, respectively for nucleic acidic sequence
comparison, and GAP creation penalty of 3.0 and GAP extension
penalty of 0.1, respectively, for protein sequence comparison. GAP
uses the method of Needleman and Wunsch, 1970, J. Mol. Biol. 48:
443-453, to make alignments and to calculate the identity.
[0035] A structural alignment between Termamyl (SEQ ID NO: 8) and,
e.g., another alpha-amylase may be used to identify
equivalent/corresponding positions in other Termamyl-like
alpha-amylases. One method of obtaining said structural alignment
is to use the Pile Up programme from the GCG package using default
values of gap penalties, i.e., a gap creation penalty of 3.0 and
gap extension penalty of 0.1. Other structural alignment methods
include the hydrophobic cluster analysis (Gaboriaud et al., 1987,
FEBS Letters 224: 149-155) and reverse threading (Huber and Torda,
1998, Protein Science 7(1): 142-149).
Hybridization
[0036] The oligonucleotide probe used in the characterization of
the Termamyl-like alpha-amylase above may suitably be prepared on
the basis of the full or partial nucleotide or amino acid sequence
of the alpha-amylase in question.
[0037] Suitable conditions for testing hybridization involve
pre-soaking in 5.times.SSC and prehybridizing for 1 hour at
40.degree. C. in a solution of 20% formamide, 5.times. Denhardt's
solution, 50 mM sodium phosphate, pH 6.8, and 50 mg of denatured
sonicated calf thymus DNA, followed by hybridization in the same
solution supplemented with 100 mM ATP for 18 hours at 40.degree.
C., followed by three times washing of the filter in 2.times.SSC,
0.2% SDS at 40.degree. C. for 30 minutes (low stringency),
preferably at 50.degree. C. (medium stringency), more preferably at
65.degree. C. (high stringency), even more preferably at 75.degree.
C. (very high stringency). More details about the hybridization
method can be found in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
[0038] In the present context, "derived from" is intended not only
to indicate an alpha-amylase produced or producible by a strain of
the organism in question, but also an alpha-amylase encoded by a
DNA sequence isolated from such strain and produced in a host
organism transformed with said DNA sequence. Finally, the term is
intended to indicate an alpha-amylase, which is encoded by a DNA
sequence of synthetic and/or cDNA origin and which has the
identifying characteristics of the alpha-amylase in question. The
term is also intended to indicate that the parent alpha-amylase may
be a variant of a naturally occurring alpha-amylase, i.e., a
variant, which is the result of a modification (insertion,
substitution, deletion) of one or more amino acid residues of the
naturally occurring alpha-amylase.
Parent Termamyl-Like Alpha-Amylases
[0039] According to the invention all Termamyl-like alpha-amylases,
as defined above, may be used as the parent (i.e., backbone)
alpha-amylase. In a preferred embodiment of the invention the
parent alpha-amylase is derived from B. licheniformis, e.g., one of
those referred to above, such as the B. licheniformis alpha-amylase
having the amino acid sequence shown in SEQ ID NO: 8.
Parent Hybrid Termamyl-Like Alpha-Amylases
[0040] The parent alpha-amylase (i.e., backbone alpha-amylase) may
also be a hybrid alpha-amylase, i.e., an alpha-amylase, which
comprises a combination of partial amino acid sequences derived
from at least two alpha-amylases.
[0041] The parent hybrid alpha-amylase may be one, which on the
basis of amino acid homology (identity) and/or DNA hybridization
(as defined above) can be determined to belong to the Termamyl-like
alpha-amylase family. In this case, the hybrid alpha-amylase is
typically composed of at least one part of a Termamyl-like
alpha-amylase and part(s) of one or more other alpha-amylases
selected from Termamyl-like alpha-amylases or non-Termamyl-like
alpha-amylases of microbial (bacterial or fungal) and/or mammalian
origin.
[0042] Thus, the parent hybrid alpha-amylase may comprise a
combination of partial amino acid sequences deriving from at least
two Termamyl-like alpha-amylases, or from at least one
Termamyl-like and at least one non-Termamyl-like bacterial
alpha-amylase, or from at least one Termamyl-like and at least one
fungal alpha-amylase. The Termamyl-like alpha-amylase from which a
partial amino acid sequence derives, may be any of the specific
Termamyl-like alpha-amylase referred to herein.
[0043] For instance, the parent alpha-amylase may comprise a
C-terminal part of an alpha-amylase derived from a strain of B.
licheniformis, and a N-terminal part of an alpha-amylase derived
from a strain of B. amyloliquefaciens or from a strain of B.
stearothermophilus. For instance, the parent alpha-amylase may
comprise at least 430 amino acid residues of the C-terminal part of
the B. licheniformis alpha-amylase, and may, e.g., comprise a) an
amino acid segment corresponding to the 37 N-terminal amino acid
residues of the B. amyloliquefaciens alpha-amylase having the amino
acid sequence shown in SEQ ID NO: 10 and an amino acid segment
corresponding to the 445 C-terminal amino acid residues of the B.
licheniformis alpha-amylase having the amino acid sequence shown in
SEQ ID NO: 8, or a hybrid Termamyl-like alpha-amylase being
identical to the Termamyl sequence, i.e., the Bacillus
licheniformis alpha-amylase shown in SEQ ID NO: 8, except that the
N-terminal 35 amino acid residues (of the mature protein) has been
replaced by the N-terminal 33 residues of BAN (mature protein),
i.e., the Bacillus amyloliquefaciens alpha-amylase shown in SEQ ID
NO: 10; or b) an amino acid segment corresponding to the 68
N-terminal amino acid residues of the B. stearothermophilus
alpha-amylase having the amino acid sequence shown in SEQ ID NO: 6
and an amino acid segment corresponding to the 415 C-terminal amino
acid residues of the B. licheniformis alpha-amylase having the
amino acid sequence shown in SEQ ID NO: 8.
[0044] Another suitable parent hybrid alpha-amylase is the one
previously described in WO 96/23874 (from Novo Nordisk)
constituting the N-terminus of BAN, Bacillus amyloliquefaciens
alpha-amylase (amino acids 1-300 of the mature protein) and the
C-terminus from Termamyl (amino acids 301-483 of the mature
protein).
[0045] In a preferred embodiment of the invention the parent
Termamyl-like alpha-amylase is a hybrid alpha-amylase of SEQ ID NO:
8 and SEQ ID NO: 10. Specifically, the parent hybrid Termamyl-like
alpha-amylase may be a hybrid alpha-amylase comprising the 445
C-terminal amino acid residues of the B. licheniformis
alpha-amylase shown in SEQ ID NO: 8 and the 37 N-terminal amino
acid residues of the alpha-amylase derived from B.
amyloliquefaciens shown in SEQ ID NO: 10, which may suitably
further have the following mutations:
H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO:
8). The latter mentioned hybrid is used in the examples below and
is referred to as LE174.
[0046] Other specifically contemplated parent alpha-amylase include
LE174 with fewer mutations, i.e., the right above mentioned hydrid
having the following mutations:
A181T+N190F+A209V+Q264S; N190F+A209V+Q264S; A209V+Q264S; Q264S;
H156Y+N190F+A209V+Q264S; H156Y+A209V+Q264S; H156Y+Q264S;
H156Y+A181T+A209V+Q264S; H156Y+A181T+Q264S; H156Y+Q264S;
H156Y+A181T+N190F+Q264S; H156Y+A181T+N190F;
H156Y+A181T+N190F+A209V. These hybrids are also considered to be
part of the invention.
[0047] In a preferred embodiment the parent Termamyl-like alpha
amylase is LE174, SP722, or AA560 including any of
LE174+G48A+T49I+G107A+I201F;
LE174+M197L;
LE174+G48A+T49I+G107A+M197L+I201F;
SP722+D183*+G184*;
SP722+D183*+G184*+N195F;
SP722+D183*+G184*+M202L;
SP722+D183*+G184*+N195F+M202L;
BSG+I181*+G182*;
BSG+I181*+G182*+N193F;
BSG+I181*+G182*+M200L;
BSG+I181*+G182*+N193F+M200L;
AA560+D183*+G184*;
AA560+D183*+G184*+N195F; AA560+D183*+G184*+M202L;
AA560+D183*+G184*+N195F+M202L.
[0048] Other parent alpha-amylases contemplated include LE429,
which is LE174 with an additional substitution in I201F. According
to the invention LE335 is the alpha-amylase, which in comparison to
LE429 has additional substitutions in T49I+G107A; LE399 is
LE335+G48A, i.e., LE174, with G48A+T49I+G107A+I201F.
Altered Properties
[0049] The following section discusses the relationship between
mutations, which are present in variants of the invention, and
desirable alterations in properties (relative to those of a parent
Termamyl-like alpha-amylase), which may result therefrom.
[0050] As mentioned above the invention relates to Termamyl-like
alpha-amylases with altered properties (as mentioned above), in
particular at high temperatures and/or at low pH, in particular at
low calcium concentrations.
[0051] In the context of the present invention "high temperature"
means temperatures from 70-120.degree. C., preferably
80-100.degree. C., especially 85-95.degree. C.
[0052] In the context of the present invention the term "low pH"
means from a pH in the range from 4-6, preferably 4.2-5.5,
especially 4.5-5.
[0053] In the context of the present invention the term "high pH"
means from a pH in the range from 8-11, especially 8.5-10.6.
[0054] In the context of the present invention the term "low
calcium concentration" means free calcium levels lower than 60 ppm,
preferably 40 ppm, more preferably 25 ppm, especially 5 ppm
calcium.
[0055] Parent Termamyl-like alpha-amylase specifically contemplated
in connection with going through the specifically contemplated
altered properties are the above mentioned parent Termamyl-like
alpha-amylase and parent hydrid Termamyl-like alpha-amylases.
[0056] The Termamyl.RTM. alpha-amylase is used as the starting
point, but corresponding positions in, e.g., the SP722, BSG, BAN,
AA560, SP690, KSM AP1378, and #707 should be understood as
disclosed and specifically comtemplated too.
[0057] In a preferred embodiment the variant of the invention has
in particular at high temperatures and/or at low pH.
[0058] In an aspect the invention relates to variant with altered
properties as mentioned above.
[0059] In the first aspect a variant of a parent Termamyl-like
alpha-amylase, comprising an alteration at one or more positions
(using SEQ ID NO: 8 for the amino acid numbering) selected from the
group of:
49, 60, 104, 132, 161, 170, 176, 179, 180, 181, 183, 200, 203, 204,
207, 212, 237, 239, 250, 280, 298, 318, 374, 385, 393, 402, 406,
427, 430, 440, 444, 447, 482, wherein (a) the alteration(s) are
independently
[0060] (i) an insertion of an amino acid downstream of the amino
acid which occupies the position,
[0061] (ii) a deletion of the amino acid which occupies the
position, or
[0062] (iii) a substitution of the amino acid which occupies the
position with a different amino acid,
(b) the variant has alpha-amylase activity and (c) each position
corresponds to a position of the amino acid sequence of the parent
Termamyl-like alpha-amylase having the amino acid sequence shown in
SEQ ID NO: 8.
[0063] In Termamyl.RTM. (SEQ ID NO: 8) such corresponding positions
are:
T49; D60; N104; E132; D161; K170; K176; G179; K180; A181; D183;
D200; Y203; D204; D207; I212; K237; S239; E250; N280; Q298; L318;
Q374; E385; Q393; Y402; H406; L427 D430; V440; N444; E447;
Q482.
[0064] In SP722 (SEQ ID NO: 4) the corresponding positions are:
T51; D62; N106; D134; D163; Q172; K179; G184; K185; A186; D188;
D205; M208; D209; X212; L217, K242, S244, N255, N285, S303, M323;
D387, N395; Y404; H408; I429; D432; V442; K446; Q449; K484.
[0065] Corresponding positions in other parent alpha-amylases can
be found by alignment as described above and shown in the alignment
in FIG. 1.
[0066] In a preferred embodiment the variant of the invention
(using SEQ ID NO: 8 (Termamyl.TM.) for the numbering) has one or
more of the following substitutions:
T49I; D60N; N104D; E132A, V, P; D161N; K170Q; K176R; G179N; K180T;
A181N; D183N; D200N; X203Y; D204S; D207V, E, L, G; X212I; K237P;
S239W; E250G,F; N280S; X298Q; L318M; Q374R; E385V; Q393R; Y402F;
H406L,W; L427I D430N; V440A; N444R,K; E447Q,K; Q482K.
[0067] In a preferred embodiment the variant of the invention
(using SEQ ID NO: 4 (SP722) for the numbering) has one or more of
the following substitutions:
T51I; D62N; N106D; D134A, V, P; D163N; X172Q; K179R; G184N; K185T;
A186N; D188N; D205N; M208Y; D209S; X212V, E, L, G; L2171, K242P,
S244W, N255G,F, N285S, S303Q, X323M; D387V, N395R; Y404F; H408L,W;
X4291; D432N; V442A; X446R,K; X449Q,K; X484K, using SEQ ID NO: 4
(SP722) for numbering.
[0068] Preferred double, triple and multi-mutations--using SEQ ID
NO: 8 as the basis for the numbering--are selected from the group
consisting of:
T49I+D60N; T49I+D60N+E132A; T49I+D60N+E132V;
T49I+D60N+E132V+K170Q;
T49I+D60N+E132A+K170Q; T49I+D60N+E132V+K170Q+K176R;
T49I+D60N+E132A+K170Q+K176R;
T49I+D60N+E132V+K170Q+K176R+D207V;
T49I+D60N+E132A+K170Q+K176R+D207V;
T49I+D60N+E132V+K170Q+K176R+D207E;
T49I+D60N+E132A+K170Q+K176R+D207E;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L+L427I;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L+L427I;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L+L427I;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L+L427I;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L+L427I+V440A;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L+L427I+V440A;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L+L427I+V440A;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L+L427I+V440A;
D60N+E132A; D60N+E132V; D60N+E132V+K170Q; D60N+E132A+K170Q;
D60N+E132V+K170Q+K176R; T49I+D60N+E132A+K170Q+K176R;
D60N+E132V+K170Q+K176R+D207V;
T49I+D60N+E132A+K170Q+K176R+D207V;
D60N+E132V+K170Q+K176R+D207E;
T49I+D60N+E132A+K170Q+K176R+D207E;
D60N+E132V+K170Q+K176R+D207V+E250G;
D60N+E132A+K170Q+K176R+D207V+E250G;
D60N+E132V+K170Q+K176R+D207E+E250G;
D60N+E132A+K170Q+K176R+D207E+E250G;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L+L427I;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L+L427I;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L+L427I;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L+L427I;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L+L427I+V440A;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L+L427I+V440A;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L+L427I+V440A;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H40-
6L+L427I+V440A;
E132V+K170Q; E132A+K170Q; E132V+K170Q+K176R; E132A+K170Q+K176R;
E132V+K170Q+K176R+D207V; E132A+K170Q+K176R+D207V;
E132V+K170Q+K176R+D207E; E132A+K170Q+K176R+D207E;
E132V+K170Q+K176R+D207V+E250G; E132A+K170Q+K176R+D207V+E250G;
E132V+K170Q+K176R+D207E+E250G; E132A+K170Q+K176R+D207E+E250G;
E132V+K170Q+K176R+D207E+E250G+N280S;
E132A+K170Q+K176R+D207E+E250G+N280S;
E132V+K170Q+K176R+D207V+E250G+N280S;
E132A+K170Q+K176R+D207V+E250G+N280S;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I+V440A;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I+V440A;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I+V440A;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I+V440A;
K170Q+K176R; K170Q+K176R+D207V; K170Q+K176R+D207E;
K170Q+K176R+D207V+E250G; K170Q+K176R+D207E+E250G;
K170Q+K176R+D207V+E250G+N280S; K170Q+K176R+D207E+E250G+N280S;
K170Q+K176R+D207E+E250G+N280S+L318M;
K170Q+K176R+D207V+E250G+N280S+L318M;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V4-
40A;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V4-
40A;
K176R+D207V; K176R+D207E; K176R+D207V+E250G;
K176R+D207E+E250G; K176R+D207V+E250G+N280S;
K176R+D207E+E250G+N280S; K176R+D207E+E250G+N280S+L318M;
K176R+D207V+E250G+N280S+L318M;
K176R+D207E+E250G+N280S+L318M+Q374R;
K176R+D207V+E250G+N280S+L318M+Q374R;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
D207V+E250G; D207E+E250G;
D207V+E250G+N280S; D207E+E250G+N280S+L318M;
D207V+E250G+N280S+L318M; D207E+E250G+N280S+L318M+Q374R;
D207V+E250G+N280S+L318M+Q374R;
D207E+E250G+N280S+L318M+Q374R+E385V;
D207V+E250G+N280S+L318M+Q374R+E385V;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
E250G+N280S; E250G+N280S+L318M; E250G+N280S+L318M+Q374R;
E250G+N280S+L318M+Q374R+E385V;
E250G+N280S+L318M+Q374R+E385V+Q393R;
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F;
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
N280S+L318M; N280S+L318M+Q374R; N280S+L318M+Q374R+E385V;
N280S+L318M+Q374R+E385V+Q393R;
N280S+L318M+Q374R+E385V+Q393R+Y402F;
N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
L318M+Q374R; L318M+Q374R+E385V; L318M+Q374R+E385V+Q393R;
L318M+Q374R+E385V+Q393R+Y402F;
L318M+Q374R+E385V+Q393R+Y402F+H406L;
L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
Q374R+E385V; Q374R+E385V+Q393R; Q374R+E385V+Q393R+Y402F;
Q374R+E385V+Q393R+Y402F+H406L;
Q374R+E385V+Q393R+Y402F+H406L+L427I;
Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
E385V+Q393R; E385V+Q393R+Y402F; E385V+Q393R+Y402F+H406L;
E385V+Q393R+Y402F+H406L+L427I;
E385V+Q393R+Y402F+H406L+L427I+V440A;
Q393R+Y402F; Q393R+Y402F+H406L; Q393R+Y402F+H406L+L427I;
Q393R+Y402F+H406L+L427I+V440A; Y402F+H406L;
Y402F+H406L+L427I; Y402F+H406L+L427I+V440A; H406L+L427I;
H406L+L427I+V440A; L427I+V440A;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N4-
44K+E447Q+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E4-
47Q+Q482K;
D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K;
D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E4-
47Q+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q4-
82K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N;
H406W+D430N; N444K+E447Q+Q482K; E447Q+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N4-
44R+N444K+E447K+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N4-
44K+E447K+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W;
H406W+D430N; N444K+E447K+Q482K; E447K+Q482K;
N104D+D161N+A181N+D183N+D200N+D204S+K237P+S239W;
N104D+D161N+A181N+D183N+D200N+D204S+K237P;
N104D+D161N+A181N+D183N+D200N+D204S;
D161N+A181N+D183N+D200N+D204S+K237P+S239W;
D161N+A181N+D183N+D200N+D204S+K237P;
[0069] D161N+A181N+D183N+D200N+D204S; K237P+S239W, using SEQ ID NO:
8 for numbering.
[0070] In a preferred embodiment the variant has the following
substitutions:
K170Q+D207V+N280S; E132A+D207V; D207E+E250G+H406L+L427I;
D207V+L318M;
D60N+D207V+L318M; T49I+E132V+V440A; T49I+K176R+D207V+Y402F;
Q374R+E385V+Q393R; N190F+A209V+Q264S; G48A+T49I+G107A+I201F;
T49I+G107A+I201F; G48A+T49I+I201F; G48A+T49I+G107A; T49I+I201F;
T49I+G107A;
G48A+T49I;
[0071]
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N-
4 44K+E447Q+Q482K using SEQ ID NO: 8 for numbering.
[0072] Specific variant include: LE399; LE174+G48A+T49I+G107A;
LE174+G48A+T49I+I201F; LE174+G48A+G107A+I201F;
LE174+T49I+G107A+I201F; LE174+G48A+T49I; LE174+G48A;
LE174+G107A+I201F; LE174+I201F, are specifically contemplated
variants of the invention.
Stability
[0073] In the context of the present invention, mutations
(including amino acid substitutions and deletions) of importance
with respect to achieving altered stability, in particular improved
stability (i.e., higher or lower), at especially high temperatures
(i.e., 70-120.degree. C.) and/or extreme pH (i.e., low or high pH,
i.e., pH 4-6 or pH 8-11, respectively), in particular at free
(i.e., unbound, therefore in solution) calcium concentrations below
60 ppm, include any of the mutations listed in the "Altered
properties" section. The stability may be determined as described
in the "Materials & Methods" section below.
General Mutations in Variants of the Invention
[0074] A variant of the invention may in one embodiment comprise
one or more modifications in addition to those outlined above.
Thus, it may be advantageous that one or more Proline (Pro)
residues present in the part of the alpha-amylase variant which is
modified is/are replaced with a non-Proline residue which may be
any of the possible, naturally occurring non-Proline residues, and
which preferably is an Alanine, Glycine, Serine, Threonine, Valine
or Leucine.
[0075] Analogously, in one embodiment one or more Cysteine residues
present in the parent alpha-amylase may be replaced with a
non-Cysteine residue such as Serine, Alanine, Threonine, Glycine,
Valine or Leucine.
[0076] Furthermore, a variant of the invention may--either as the
only modification or in combination with any of the above outlined
modifications--be modified so that one or more Asp and/or Glu
present in an amino acid fragment corresponding to the amino acid
fragment 185-209 of SEQ ID NO: 10 is replaced by an Asn and/or Gln,
respectively. Also of interest is the replacement, in the
Termamyl-like alpha-amylase, of one or more of the Lys residues
present in an amino acid fragment corresponding to the amino acid
fragment 185-209 of SEQ ID NO: 10 by an Arg.
[0077] It is to be understood that the present invention
encompasses variants incorporating two or more of the above
outlined modifications.
[0078] Furthermore, it may be advantageous to introduce mutations
in one or more of the following positions (using SEQ ID NO: 8
(Termamyl) for numbering):
M15, V128, A111, H133, W138, T149, M197, N188, A209, A210, H405,
T412, in particular the following single, double or triple or multi
mutations: M15X, in particular M15T,L; V128X, in particular V128E;
H133X, in particular H133Y; N188X, in particular N188S,T,P; M197X,
in particular M197T,L; A209X, in particular A209V;
M197T/W138F; M197T/W138Y; M15T/H133Y/N188S;
M15/V128E/H133Y/N188S; E119C/S130C; D124C/R127C; H133Y/T149I;
G475R, H133Y/S187D; H133Y/A209V.
Methods for Preparing Alpha-Amylase Variants of the Invention
[0079] Several methods for introducing mutations into genes are
known in the art. After a brief description of cloning of
alpha-amylase-encoding DNA sequences, methods for generating
mutations at specific sites within the alpha-amylase-encoding
sequence will be described.
Cloning a DNA Sequence Encoding an Alpha-Amylase
[0080] The DNA sequence encoding a parent alpha-amylase may be
isolated from any cell or microorganism producing the alpha-amylase
in question, using various methods well known in the art. First, a
genomic DNA and/or cDNA library should be constructed using
chromosomal DNA or messenger RNA from the organism that produces
the alpha-amylase to be studied. Then, if the amino acid sequence
of the alpha-amylase is known, homologous, labeled oligonucleotide
probes may be synthesized and used to identify
alpha-amylase-encoding clones from a genomic library prepared from
the organism in question. Alternatively, a labeled oligonucleotide
probe containing sequences homologous to a known alpha-amylase gene
could be used as a probe to identify alpha-amylase-encoding clones,
using hybridization and washing conditions of lower stringency.
[0081] Yet another method for identifying alpha-amylase-encoding
clones would involve inserting fragments of genomic DNA into an
expression vector, such as a plasmid, transforming
alpha-amylase-negative bacteria with the resulting genomic DNA
library, and then plating the transformed bacteria onto agar
containing a substrate for alpha-amylase, thereby allowing clones
expressing the alpha-amylase to be identified.
[0082] Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g., the
phosphoroamidite method described by Beaucage and Caruthers, 1981,
Tetrahedron Letters 22: 1859-1869, or the method described by
Matthes et al., 1984, The EMBO J. 3: 801-805. In the
phosphoroamidite method, oligonucleotides are synthesized, e.g., in
an automatic DNA synthesizer, purified, annealed, ligated and
cloned in appropriate vectors.
[0083] Finally, the DNA sequence may be of mixed genomic and
synthetic origin, mixed synthetic and cDNA origin or mixed genomic
and cDNA origin, prepared by ligating fragments of synthetic,
genomic or cDNA origin (as appropriate, the fragments corresponding
to various parts of the entire DNA sequence), in accordance with
standard techniques. The DNA sequence may also be prepared by
polymerase chain reaction (PCR) using specific primers, for
instance as described in U.S. Pat. No. 4,683,202 or Saiki et al.,
1988, Science 239: 487-491.
Site-Directed Mutagenesis
[0084] Once an alpha-amylase-encoding DNA sequence has been
isolated, and desirable sites for mutation identified, mutations
may be introduced using synthetic oligonucleotides. These
oligonucleotides contain nucleotide sequences flanking the desired
mutation sites; mutant nucleotides are inserted during
oligonucleotide synthesis. In a specific method, a single-stranded
gap of DNA, bridging the alpha-amylase-encoding sequence, is
created in a vector carrying the alpha-amylase gene. Then the
synthetic nucleotide, bearing the desired mutation, is annealed to
a homologous portion of the single-stranded DNA. The remaining gap
is then filled in with DNA polymerase I (Klenow fragment) and the
construct is ligated using T4 ligase. A specific example of this
method is described in Morinaga et al. (1984). U.S. Pat. No.
4,760,025 discloses the introduction of oligonucleotides encoding
multiple mutations by performing minor alterations of the cassette.
However, an even greater variety of mutations can be introduced at
any one time by the Morinaga method, because a multitude of
oligonucleotides, of various lengths, can be introduced.
[0085] Another method for introducing mutations into
alpha-amylase-encoding DNA sequences is described in Nelson and
Long (1989). It involves the 3-step generation of a PCR fragment
containing the desired mutation introduced by using a chemically
synthesized DNA strand as one of the primers in the PCR reactions.
From the PCR-generated fragment, a DNA fragment carrying the
mutation may be isolated by cleavage with restriction endonucleases
and reinserted into an expression plasmid.
[0086] Alternative methods for providing variants of the invention
include gene shuffling, e.g., as described in WO 95/22625 (from
Affymax Technologies N.V.) or in WO 96/00343 (from Novo Nordisk
A/S), or other corresponding techniques resulting in a hybrid
enzyme comprising the mutation(s), e.g., substitution(s) and/or
deletion(s), in question. Examples of parent alpha-amylases, which
suitably may be used for providing a hybrid with the desired
mutations(s) according to the invention include the KSM-K36 and
KSM-K38 alpha-amylases disclosed in EP 1,022,334 (hereby
incorporated by reference).
Expression of Alpha-Amylase Variants
[0087] According to the invention, a DNA sequence encoding the
variant produced by methods described above, or by any alternative
methods known in the art, can be expressed, in enzyme form, using
an expression vector which typically includes control sequences
encoding a promoter, operator, ribosome binding site, translation
initiation signal, and, optionally, a repressor gene or various
activator genes.
[0088] The recombinant expression vector carrying the DNA sequence
encoding an alpha-amylase variant of the invention may be any
vector, which may conveniently be subjected to recombinant DNA
procedures, and the choice of vector will often depend on the host
cell into which it is to be introduced. Thus, the vector may be an
autonomously replicating vector, i.e., a vector which exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g., a plasmid, a bacteriophage or an
extrachromosomal element, minichromosome or an artificial
chromosome. Alternatively, the vector may be one which, when
introduced into a host cell, is integrated into the host cell
genome and replicated together with the chromosome(s) into which it
has been integrated.
[0089] In the vector, the DNA sequence should be operably connected
to a suitable promoter sequence. The promoter may be any DNA
sequence, which shows transcriptional activity in the host cell of
choice and may be derived from genes encoding proteins either
homologous or heterologous to the host cell. Examples of suitable
promoters for directing the transcription of the DNA sequence
encoding an alpha-amylase variant of the invention, especially in a
bacterial host, are the promoter of the lac operon of E. coli, the
Streptomyces coelicolor agarase gene dagA promoters, the promoters
of the Bacillus licheniformis alpha-amylase gene (amyL), the
promoters of the Bacillus stearothermophilus maltogenic amylase
gene (amyM), the promoters of the Bacillus amyloliquefaciens
alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA
and xylB genes etc. For transcription in a fungal host, examples of
useful promoters are those derived from the gene encoding A. oryzae
TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger
neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger
glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline
protease, A. oryzae triose phosphate isomerase or A. nidulans
acetamidase.
[0090] The expression vector of the invention may also comprise a
suitable transcription terminator and, in eukaryotes,
polyadenylation sequences operably connected to the DNA sequence
encoding the alpha-amylase variant of the invention. Termination
and polyadenylation sequences may suitably be derived from the same
sources as the promoter.
[0091] The vector may further comprise a DNA sequence enabling the
vector to replicate in the host cell in question. Examples of such
sequences are the origins of replication of plasmids pUC19,
pACYC177, pUB110, pE194, pAMB1 and pIJ702.
[0092] The vector may also comprise a selectable marker, e.g., a
gene the product of which complements a defect in the host cell,
such as the dal genes from B. subtilis or B. licheniformis, or one
which confers antibiotic resistance such as ampicillin, kanamycin,
chloramphenicol or tetracyclin resistance. Furthermore, the vector
may comprise Aspergillus selection markers such as amdS, argB, niaD
and sC, a marker giving rise to hygromycin resistance, or the
selection may be accomplished by co-transformation, e.g., as
described in WO 91/17243.
[0093] While intracellular expression may be advantageous in some
respects, e.g., when using certain bacteria as host cells, it is
generally preferred that the expression is extracellular. In
general, the Bacillus alpha-amylases mentioned herein comprise a
preregion permitting secretion of the expressed protease into the
culture medium. If desirable, this preregion may be replaced by a
different preregion or signal sequence, conveniently accomplished
by substitution of the DNA sequences encoding the respective
preregions.
[0094] The procedures used to ligate the DNA construct of the
invention encoding an alpha-amylase variant, the promoter,
terminator and other elements, respectively, and to insert them
into suitable vectors containing the information necessary for
replication, are well known to persons skilled in the art (cf., for
instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,
2nd Ed., Cold Spring Harbor, 1989).
[0095] The cell of the invention, either comprising a DNA construct
or an expression vector of the invention as defined above, is
advantageously used as a host cell in the recombinant production of
an alpha-amylase variant of the invention. The cell may be
transformed with the DNA construct of the invention encoding the
variant, conveniently by integrating the DNA construct (in one or
more copies) in the host chromosome. This integration is generally
considered to be an advantage as the DNA sequence is more likely to
be stably maintained in the cell. Integration of the DNA constructs
into the host chromosome may be performed according to conventional
methods, e.g., by homologous or heterologous recombination.
Alternatively, the cell may be transformed with an expression
vector as described above in connection with the different types of
host cells.
[0096] The cell of the invention may be a cell of a higher organism
such as a mammal or an insect, but is preferably a microbial cell,
e.g., a bacterial or a fungal (including yeast) cell.
[0097] Examples of suitable bacteria are gram-positive bacteria
such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentus,
Bacillus brevis, Bacillus stearothermophilus, Bacillus
alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans,
Bacillus circulans, Bacillus lautus, Bacillus megaterium, Bacillus
thuringiensis, or Streptomyces lividans or Streptomyces murinus, or
gram-negative bacteria such as E. coli. The transformation of the
bacteria may, for instance, be effected by protoplast
transformation or by using competent cells in a manner known per
se.
[0098] The yeast organism may favorably be selected from a species
of Saccharomyces or Schizosaccharomyces, e.g., Saccharomyces
cerevisiae. The filamentous fungus may advantageously belong to a
species of Aspergillus, e.g., Aspergillus oryzae or Aspergillus
niger. Fungal cells may be transformed by a process involving
protoplast formation and transformation of the protoplasts followed
by regeneration of the cell wall in a manner known per se. A
suitable procedure for transformation of Aspergillus host cells is
described in EP 238 023.
[0099] In a yet further aspect, the present invention relates to a
method of producing an alpha-amylase variant of the invention,
which method comprises cultivating a host cell as described above
under conditions conducive to the production of the variant and
recovering the variant from the cells and/or culture medium.
[0100] The medium used to cultivate the cells may be any
conventional medium suitable for growing the host cell in question
and obtaining expression of the alpha-amylase variant of the
invention. Suitable media are available from commercial suppliers
or may be prepared according to published recipes (e.g., as
described in catalogues of the American Type Culture
Collection).
[0101] The alpha-amylase variant secreted from the host cells may
conveniently be recovered from the culture medium by well-known
procedures, including separating the cells from the medium by
centrifugation or filtration, and precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulphate, followed by the use of chromatographic procedures such as
ion exchange chromatography, affinity chromatography, or the
like.
INDUSTRIAL APPLICATIONS
[0102] The alpha-amylase variants of this invention possess
valuable properties allowing for a variety of industrial
applications. In particular, enzyme variants of the invention are
applicable as a component in washing, dishwashing, and hard surface
cleaning detergent compositions.
[0103] Variant of the invention with altered properties may be used
for starch processes, in particular starch conversion, especially
liquefaction of starch (see, e.g., U.S. Pat. No. 3,912,590, EP
patent publications Nos. 252 730 and 63 909, WO 99/19467, and WO
96/28567, which are all hereby incorporated by reference). Also
contemplated are compositions for starch conversion purposes, which
may beside the variant of the invention also comprise an AMG,
pullulanase, and other alpha-amylases.
[0104] Further, variants of the invention are also particularly
useful in the production of sweeteners and ethanol (see, e.g., U.S.
Pat. No. 5,231,017 hereby incorporated by reference), such as fuel,
drinking and industrial ethanol, from starch or whole grains.
[0105] A variant of the invention may also be used for textile
desizing (see, e.g., WO 95/21247, U.S. Pat. No. 4,643,736, and EP
119,920, which are hereby incorporated by reference).
Detergent Compositions
[0106] As mentioned above, variants of the invention may suitably
be incorporated in detergent compositions. Reference is made, for
example, to WO 96/23874 and WO 97/07202 for further details
concerning relevant ingredients of detergent compositions (such as
laundry or dishwashing detergents), appropriate methods of
formulating the variants in such detergent compositions, and for
examples of relevant types of detergent compositions.
[0107] Detergent compositions comprising a variant of the invention
may additionally comprise one or more other enzymes, such as a
protease, a lipase, a peroxidase, another amylolytic enzyme,
glucoamylase, maltogenic amylase, CGTase and/or a cellulase,
mannanase (such as Mannaway.TM. from Novozymes, Denmark)),
pectinase, pectine lyase, cutinase, laccase, and/or another
alpha-amylase.
[0108] Alpha-amylase variants of the invention may be incorporated
in detergents at conventionally employed concentrations. It is at
present contemplated that a variant of the invention may be
incorporated in an amount corresponding to 0.00001-10 mg
(calculated as pure, active enzyme protein) of alpha-amylase per
liter of wash/dishwash liquor using conventional dosing levels of
detergent.
Compositions
[0109] The invention also related to composition comprising a
variant of the invention, and in a preferred embodiment also a B.
stearothermophilus alpha-amylase (BSG), in particular a variant
thereof.
[0110] In another embodiment the composition comprises beside a
variant of the invention a glucoamylase, in particular a
glucoamylase originating from Aspergillus niger (e.g., the G1 or G2
A. niger AMG disclosed in Boel et al., 1984, "Glucoamylases G1 and
G2 from Aspergillus niger are synthesized from two different but
closely related mRNAs", EMBO J. 3(5): 1097-1102, or a variant
therefore, in particular a variant disclosed in WO 00/04136 or WO
01/04273 or the Talaromyces emersonii AMG disclosed in WO
99/28448.
[0111] A specific combination is LE399 and a variant disclosed in
WO 00/04136 or WO 01/04273, in particular a variant with one or
more of the following substitutions:
N9A, S56A, V59A, S119P, A246T, N313G, E342T, A393R, S394R, Y402F,
E408R, in particular a variant with all mutation.
[0112] In an embodiment the composition of the invention also
comprises a pullulanase, in particular a Bacillus pullulanase.
Materials and Methods
Enzymes:
[0113] Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8
and also available from Novozymes. AA560: SEQ ID NO: 12; disclosed
in WO 00/60060; deposited on 25 Jan. 1999 at DSMZ and assigned the
DSMZ no. 12649. AA560 was deposited by the inventors under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Microorganisms for the Purposes of Patent Procedure
at Deutshe Sammmlung von Microorganismen und Zellkulturen GmbH
(DSMZ), MascheroderWeg 1b, D-38124 Braunschweig DE. LB medium (In 1
liter H2O: 10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl,
pH adjusted to 7.0 w. NaOH, autoclaved). TY agar plates (In 1 liter
H2O: 16 g bacto-tryptone, 10 g bacto-yeast extract, 5 g NaCl, pH
adjusted to 7.0 w. NaOH, and 15 g bacto-agar is added prior to
autoclaving). 10% Lugol solution (Iodine/Potassium iodine solution;
made by 10-fold dil. in H2O of stock: Sigma Cat. no. L 6146).
Bacillus subtilis SHA273: see WO 95/10603
Plasmids
[0114] pDN1528 contains the complete gene encoding Termamyl, amyL,
the expression of which is directed by its own promoter. Further,
the plasmid contains the origin of replication, ori, from plasmid
pUB110 and the cat gene from plasmid pC194 conferring resistance
towards chloramphenicol. pDN1528 is shown in FIG. 9 of WO
96/23874.
Methods:
Low pH Filter Assay
[0115] Bacillus libraries are plated on a sandwich of cellulose
acetate (OE 67, Schleicher & Schuell, Dassel, Germany)--and
nitrocellulose filters (Protran-Ba 85, Schleicher & Schuell,
Dassel, Germany) on TY agar plates with 10 micro g/ml
chloramphenicol at 37.degree. C. for at least 21 hours. The
cellulose acetate layer is located on the TY agar plate.
[0116] Each filter sandwich is specifically marked with a needle
after plating, but before incubation in order to be able to
localize positive variants on the filter, and the nitrocellulose
filter with bound variants is transferred to a container with
citrate buffer, pH 4.5 and incubated at 80.degree. C. for 20
minutes (when screening for variants in the wild type backbone) or
85.degree. C. for 60 minutes (when screening for variants in the
LE399 backbone). The cellulose acetate filters with colonies are
stored on the TY-plates at room temperature until use. After
incubation, residual activity is detected on assay plates
containing 1% agarose, 0.2% starch in citrate buffer, pH 6.0. The
assay plates with nitrocellulose filters are marked the same way as
the filter sandwich and incubated for 2 hours at 50.degree. C.
After removal of the filters the assay plates are stained with 10%
Lugol solution. Starch degrading variants are detected as white
spots on dark blue background and then identified on the storage
plates. Positive variants are re-screened twice under the same
conditions as the first screen.
Secondary Screening
[0117] Positive transformants after rescreening are picked from the
storage plate and tested in a secondary plate assay. Positive
transformants are grown for 22 hours at 37.degree. C. in 5 ml
LB+chloramphenicol. The Bacillus culture of each positive
transformant and as a control a clone expressing the corresponding
backbone are incubated in citrate buffer, pH 4.5 at 90.degree. C.
and samples are taken at 0, 10, 20, 30, 40, 60 and 80 minutes. A 3
microliter sample is spotted on an assay plate. The assay plate is
stained with 10% Lugol solution. Improved variants are seen as
variants with higher residual activity (detected as halos on the
assay plate) than the backbone. The improved variants are
determined by nucleotide sequencing.
Stability Assay of Unpurified Variants:
[0118] Bacillus cultures expressing the variants to be analyzed are
grown for 21 hours at 37.degree. C. in 10 ml LB+chloramphenicol.
800 microliter culture is mixed with 200 micro l citrate buffer, pH
4.5. A number of 70 microliter aliquots corresponding to the number
of sample time points are made in PCR tubes and incubated at
70.degree. C. (for variants in the wt backbone) or 90.degree. C.
(for variants in LE399) for various time points (typically 5, 10,
15, 20, 25 and 30 minutes) in a PCR machine. The 0 min sample is
not incubated at high temperature. Activity in the sample is
measured by transferring 20 microliters to 200 microliters of the
alpha-amylase PNP-G7 substrate MPR3 ((Boehringer Mannheim Cat. no.
1660730) as described below under "Assays for Alpha-Amylase
Activity". Results are plotted as percentage activity (relative to
the 0 time point) versus time, or stated as percentage residual
activity after incubation for a certain period of time.
Fermentation and Purification of Alpha-Amylase Variants
[0119] A B. subtilis strain harboring the relevant expression
plasmid is streaked on an LB-agar plate with 10 micro g/ml
kanamycin from -80.degree. C. stock, and grown overnight at
37.degree. C.
[0120] The colonies are transferred to 100 ml PS-1 media
supplemented with 10 micro g/ml chloamphinicol in a 500 ml shaking
flask.
Composition of PS-1 Medium:
TABLE-US-00002 [0121] Pearl sugar 100 g/l Soy Bean Meal 40 g/l
Na.sub.2HPO.sub.4, 12 H.sub.2O 10 g/l PluronicTM PE 6100 0.1 g/l
CaCO.sub.3 5 g/l
[0122] The culture is shaken at 37.degree. C. at 270 rpm for 5
days.
[0123] Cells and cell debris are removed from the fermentation
broth by centrifugation at 4500 rpm in 20-25 minutes. Afterwards
the supernatant is filtered to obtain a completely clear solution.
The filtrate is concentrated and washed on a UF-filter (10000 cut
off membrane) and the buffer is changed to 20 mM Acetate pH 5.5.
The UF-filtrate is applied on a S-sepharose F.F. and elution is
carried out by step elution with 0.2 M NaCl in the same buffer. The
eluate is dialysed against 10 mM Tris, pH 9.0 and applied on a
Q-sepharose F.F. and eluted with a linear gradient from 0-0.3 M
NaCl over 6 column volumes. The fractions that contain the activity
(measured by the Phadebas assay) are pooled, pH was adjusted to pH
7.5 and remaining color was removed by a treatment with 0.5% W/vol.
active coal in 5 minutes.
Stability Determination of Purified Variants
[0124] All stability trials of purified variants are made using the
same set up. The method is as follows:
[0125] The enzyme is incubated under the relevant conditions (1-4).
Samples are taken at various time points, e.g., after 0, 5, 10, 15
and 30 minutes and diluted 25 times (same dilution for all taken
samples) in assay buffer (0.1 M 50 mM Britton buffer pH 7.3) and
the activity is measured using the Phadebas assay (Pharmacia) under
standard conditions pH 7.3, 37.degree. C.
[0126] The activity measured before incubation (0 minutes) is used
as reference (100%). The decline in percent is calculated as a
function of the incubation time. The table shows the residual
activity after, e.g., 30 minutes of incubation.
Specific Activity Determination
[0127] The specific activity is determined using the Phadebas assay
(Pharmacia) as activity/mg enzyme. The manufacturer's instructions
are followed (see also below under "Assay for .alpha.-amylase
activity").
Assays for Alpha-Amylase Activity
1. Phadebas Assay
[0128] Alpha-amylase activity is determined by a method employing
Phadebas.RTM. tablets as substrate. Phadebas tablets (Phadebas.RTM.
Amylase Test, supplied by Pharmacia Diagnostic) contain a
cross-linked insoluble blue-colored starch polymer, which has been
mixed with bovine serum albumin and a buffer substance and
tabletted.
[0129] For every single measurement one tablet is suspended in a
tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic
acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl.sub.2,
pH adjusted to the value of interest with NaOH). The test is
performed in a water bath at the temperature of interest. The
alpha-amylase to be tested is diluted in x ml of 50 mM
Britton-Robinson buffer. 1 ml of this alpha-amylase solution is
added to the 5 ml 50 mM Britton-Robinson buffer. The starch is
hydrolyzed by the alpha-amylase giving soluble blue fragments. The
absorbance of the resulting blue solution, measured
spectrophotometrically at 620 nm, is a function of the
alpha-amylase activity.
[0130] It is important that the measured 620 nm absorbance after 10
or 15 minutes of incubation (testing time) is in the range of 0.2
to 2.0 absorbance units at 620 nm. In this absorbance range there
is linearity between activity and absorbance (Lambert-Beer law).
The dilution of the enzyme must therefore be adjusted to fit this
criterion. Under a specified set of conditions (temp., pH, reaction
time, buffer conditions) 1 mg of a given alpha-amylase will
hydrolyze a certain amount of substrate and a blue color will be
produced. The color intensity is measured at 620 nm. The measured
absorbance is directly proportional to the specific activity
(activity/mg of pure alpha-amylase protein) of the alpha-amylase in
question under the given set of conditions.
2. Alternative Method
[0131] Alpha-amylase activity is determined by a method employing
the PNP-G7 substrate. PNP-G7 which is a abbreviation for
p-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide
which can be cleaved by an endo-amylase. Following the cleavage,
the alpha-Glucosidase included in the kit digest the substrate to
liberate a free PNP molecule which has a yellow colour and thus can
be measured by visible spectophometry at .lamda.=405 nm (400-420
nm). Kits containing PNP-G7 substrate and alpha-Glucosidase is
manufactured by Boehringer-Mannheim (cat. No. 1054635).
[0132] To prepare the reagent solution 10 ml of substrate/buffer
solution is added to 50 ml enzyme/buffer solution as recommended by
the manufacturer. The assay is performed by transferring 20
microliter sample to a 96 well microtitre plate and incubating at
25.degree. C. 200 micro l reagent solution pre-equilibrated to
25.degree. C. is added. The solution is mixed and pre-incubated 1
minute and absorption is measured every 30 sec. over 4 minutes at
OD 405 nm in an ELISA reader.
[0133] The slope of the time dependent absorption-curve is directly
proportional to the activity of the alpha-amylase in question under
the given set of conditions.
EXAMPLES
Example 1
Construction, by Error-Prone PCR Mutagenesis, of Bacillus
licheniformis Alpha-Amylase Variants Having an Improved Stability
at Low pH, High Temperature and Low Calcium Ion Concentration
Compared to the Parent Enzyme
Error-Prone PCR Mutagenesis and Library Construction
[0134] To improve the stability at low pH and low calcium
concentration of the parent Bacillus licheniformis alpha-amylase,
error-prone PCR mutagenesis was performed. The plasmid pDN1528
encoding the wild-type Bacillus licheniformis alpha-amylase gene
was utilized as template to amplify this gene with primers: 22149:
5'-CGA TTG CTG ACG CTG TTA TTT GCG-3' (SEQ ID NO: 14) and 24814:
5'-GAT CAC CCG CGA TAC CGT C-3' (SEQ ID NO: 15) under PCR
conditions where increased error rates leads to introduction of
random point mutations. The PCR conditions utilized were: 10 mM
Tris-HCl, pH 8.3, 50 mM KCl, 4 mM MgCl.sub.2, 0.3 mM MnCl.sub.2,
0.1 mM dGTP/dATP, 0.5 mM dTTP/dCTP, and 2.5 units Taq polymerase
per 100 microliter reaction.
[0135] The resultant PCR fragment was purified on gel and used in a
PCR-based multimerization step with a gel purified vector fragment
created by PCR amplification of pDN1528 with primers #24: 5'-GAA
TGT ATG TCG GCC GGC AAA ACG CCG GTG A-3' (SEQ ID NO: 16) and #27:
5'-GCC GCC GCT GCT GCA GAA TGA GGC AGC AAG-3' (SEQ ID NO: 17)
forming an overlap to the insert fragment. The multimerization
reaction was subsequently introduced into B. subtilis (Shafikhani
et al., 1997, Biotechniques 23: 304-310).
Screening
[0136] The error-prone library described above was screened in the
low pH filter assay (see "Materials & Methods"). Clones testing
positive upon rescreening was submitted to secondary screening for
stability in the liquid assay described in Materials and
Methods.
Results:
Increased Stability at pH 4.5, 5 ppm Calcium Incubated at
90.degree. C.
TABLE-US-00003 [0137] Name wt LE488 LE489 7.19.1 8.9.1 Mutations --
D207V K170Q E132A D207E D207V D207V E250G N280S H406L L427I
Stability1) - + + + + 1)A "+" indicates significant increase in
stability relative to wild type.
Increased Stability at pH 4.5, 5 ppm Calcium Incubated at
90.degree. C.
TABLE-US-00004 [0138] Name wt LE491 LE492 LE493 LE494 19.3.1
Mutations -- D60N T49I T49I Q374R N190F D207V E132V K176R E385V
A209V L318M V440A D207V Q393R Q264S Y402F Stability1) - + + + + +
1)A "+" indicates significant increase in stability relative to
wt.
Increased Stability at pH 4.5, 5 ppm Calcium Incubated at
90.degree. C.
TABLE-US-00005 [0139] Name wt E132-1 D207-7 D207-6 E250-8 Mutations
-- E132P D207L D207G E250F Stability1) - + + + + 1)A "+" indicates
significant increase in stability relative to wt.
Example 2
Transfer, by Site-Directed Mutagenesis, of a Selection of Mutations
from Example 1 to a New (Non-Wild Type) Backbone to Improve
Stability at Low pH and Low Calcium Ion Concentration Compared to
the Parent Enzyme
Site-Directed Mutagenesis
[0140] Mutations from LE493 (K176R+D207V+Y402F) were transferred to
LE399 yielding LE495. This was performed by the overlap PCR method
(Kirchhoff and Desrosiers, 1993, PCR Methods and Applications, 2:
301-304). 2 overlapping PCR fragments were generated by
amplification of the LE399 template with the primers: Fragment A:
#312 Mut176 5'-CCC GAA AGC TGA ACC GCA TCT ATA GGT TTC AAG GGA AGA
CTT GGG ATT-3' (SEQ ID NO: 18) (mutated codon indicated in bold)
and #290 D207overlap 5'-AGG ATG GTC ATA ATC AAA GTC GG-3'(SEQ ID
NO: 19); Fragment B: #313 Mut207 5'-CCG ACT TTG ATT ATG ACC ATC CTG
TTG TCG TAG CAG AGA TTA AGA GAT GGG G-3' (SEQ ID NO: 20) and #314
Mut402 5'-CGA CAA TGT CAT GGT GGT CGA AAA AAT CAT GCT GTG CTC CGT
ACG-3' (SEQ ID NO: 21). Fragments A and B were mixed in equimolar
ratios and subsequently the full-length fragment was amplified with
the external primers: #312 Mut176 and #314 Mut402. This fragment
was used in a multimerization reaction with the vector PCR fragment
created with the primers #296 Y402multi 5'-TTT CGA CCA CCA TGA CAT
TGT CG-3' (SEQ ID NO: 22) and #305 399Multi176 5'-TAT AGA TGC GGT
TCA GCT TTC GGG-3' (SEQ ID NO: 23) on template LE399 as described
above. The multimerization reaction was subsequently transformed
into B. subtilis. Clones were screened for stability in the assay
mentioned above. The presence of the mutations from LE493 in
several clones with increased stability was confirmed by
sequencing.
[0141] LE 497 was obtained in a similar manner by amplifying the
LE399 encoding template with primers #312 Mut176 and #314 Mut402
and using the resulting PCR fragment in a multimerization reaction
with a vector fragment obtained by PCR amplification of the LE399
template with the primers #296 Y402multi and #305 399Multi176.
Results:
Stabilization of LE399 Variant at pH 4.5, 5 ppm Calcium Incubated
at 90.degree. C.
TABLE-US-00006 [0142] Name LE399 LE495 LE497 Mutations -- K176R
K176R (backbone) D207V Y402F Y402F Stability1) -- + + 1)A "+"
indicates significant increase in stability relative to backbone.
Sequence CWU 1
1
3011455DNABacillus speciesCDS(1)..(1455) 1cat cat aat gga aca aat
ggt act atg atg caa tat ttc gaa tgg tat 48His His Asn Gly Thr Asn
Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr1 5 10 15ttg cca aat gac ggg
aat cat tgg aac agg ttg agg gat gac gca gct 96Leu Pro Asn Asp Gly
Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30aac tta aag agt
aaa ggg ata aca gct gta tgg atc cca cct gca tgg 144Asn Leu Lys Ser
Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45aag ggg act
tcc cag aat gat gta ggt tat gga gcc tat gat tta tat 192Lys Gly Thr
Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60gat ctt
gga gag ttt aac cag aag ggg acg gtt cgt aca aaa tat gga 240Asp Leu
Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75
80aca cgc aac cag cta cag gct gcg gtg acc tct tta aaa aat aac ggc
288Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly
85 90 95att cag gta tat ggt gat gtc gtc atg aat cat aaa ggt gga gca
gat 336Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala
Asp 100 105 110ggt acg gaa att gta aat gcg gta gaa gtg aat cgg agc
aac cga aac 384Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser
Asn Arg Asn 115 120 125cag gaa acc tca gga gag tat gca ata gaa gcg
tgg aca aag ttt gat 432Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala
Trp Thr Lys Phe Asp 130 135 140ttt cct gga aga gga aat aac cat tcc
agc ttt aag tgg cgc tgg tat 480Phe Pro Gly Arg Gly Asn Asn His Ser
Ser Phe Lys Trp Arg Trp Tyr145 150 155 160cat ttt gat ggg aca gat
tgg gat cag tca cgc cag ctt caa aac aaa 528His Phe Asp Gly Thr Asp
Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170 175ata tat aaa ttc
agg gga aca ggc aag gcc tgg gac tgg gaa gtc gat 576Ile Tyr Lys Phe
Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190aca gag
aat ggc aac tat gac tat ctt atg tat gca gac gtg gat atg 624Thr Glu
Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200
205gat cac cca gaa gta ata cat gaa ctt aga aac tgg gga gtg tgg tat
672Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr
210 215 220acg aat aca ctg aac ctt gat gga ttt aga ata gat gca gtg
aaa cat 720Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val
Lys His225 230 235 240ata aaa tat agc ttt acg aga gat tgg ctt aca
cat gtg cgt aac acc 768Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr
His Val Arg Asn Thr 245 250 255aca ggt aaa cca atg ttt gca gtg gct
gag ttt tgg aaa aat gac ctt 816Thr Gly Lys Pro Met Phe Ala Val Ala
Glu Phe Trp Lys Asn Asp Leu 260 265 270ggt gca att gaa aac tat ttg
aat aaa aca agt tgg aat cac tcg gtg 864Gly Ala Ile Glu Asn Tyr Leu
Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285ttt gat gtt cct ctc
cac tat aat ttg tac aat gca tct aat agc ggt 912Phe Asp Val Pro Leu
His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300ggt tat tat
gat atg aga aat att tta aat ggt tct gtg gtg caa aaa 960Gly Tyr Tyr
Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys305 310 315
320cat cca aca cat gcc gtt act ttt gtt gat aac cat gat tct cag ccc
1008His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
325 330 335ggg gaa gca ttg gaa tcc ttt gtt caa caa tgg ttt aaa cca
ctt gca 1056Gly Glu Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro
Leu Ala 340 345 350tat gca ttg gtt ctg aca agg gaa caa ggt tat cct
tcc gta ttt tat 1104Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro
Ser Val Phe Tyr 355 360 365ggg gat tac tac ggt atc cca acc cat ggt
gtt ccg gct atg aaa tct 1152Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly
Val Pro Ala Met Lys Ser 370 375 380aaa ata gac cct ctt ctg cag gca
cgt caa act ttt gcc tat ggt acg 1200Lys Ile Asp Pro Leu Leu Gln Ala
Arg Gln Thr Phe Ala Tyr Gly Thr385 390 395 400cag cat gat tac ttt
gat cat cat gat att atc ggt tgg aca aga gag 1248Gln His Asp Tyr Phe
Asp His His Asp Ile Ile Gly Trp Thr Arg Glu 405 410 415gga aat agc
tcc cat cca aat tca ggc ctt gcc acc att atg tca gat 1296Gly Asn Ser
Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430ggt
cca ggt ggt aac aaa tgg atg tat gtg ggg aaa aat aaa gcg gga 1344Gly
Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440
445caa gtt tgg aga gat att acc gga aat agg aca ggc acc gtc aca att
1392Gln Val Trp Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile
450 455 460aat gca gac gga tgg ggt aat ttc tct gtt aat gga ggg tcc
gtt tcg 1440Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser
Val Ser465 470 475 480gtt tgg gtg aag caa 1455Val Trp Val Lys
Gln4852485PRTBacillus species 2His His Asn Gly Thr Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp
Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30Asn Leu Lys Ser Lys Gly Ile
Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Asn
Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95Ile Gln
Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105
110Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
115 120 125Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys
Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys
Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Thr Asp Trp Asp Gln
Ser Arg Gln Leu Gln Asn Lys 165 170 175Ile Tyr Lys Phe Arg Gly Thr
Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190Thr Glu Asn Gly Asn
Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205Asp His Pro
Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220Thr
Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230
235 240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn
Thr 245 250 255Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys
Asn Asp Leu 260 265 270Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser
Trp Asn His Ser Val 275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu
Tyr Asn Ala Ser Asn Ser Gly 290 295 300Gly Tyr Tyr Asp Met Arg Asn
Ile Leu Asn Gly Ser Val Val Gln Lys305 310 315 320His Pro Thr His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Gly Glu
Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345
350Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met
Lys Ser 370 375 380Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe
Ala Tyr Gly Thr385 390 395 400Gln His Asp Tyr Phe Asp His His Asp
Ile Ile Gly Trp Thr Arg Glu 405 410 415Gly Asn Ser Ser His Pro Asn
Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Pro Gly Gly Asn
Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445Gln Val Trp
Arg Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460Asn
Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470
475 480Val Trp Val Lys Gln 48531455DNABacillus
speciesCDS(1)..(1455) 3cat cat aat ggg aca aat ggg acg atg atg caa
tac ttt gaa tgg cac 48His His Asn Gly Thr Asn Gly Thr Met Met Gln
Tyr Phe Glu Trp His1 5 10 15ttg cct aat gat ggg aat cac tgg aat aga
tta aga gat gat gct agt 96Leu Pro Asn Asp Gly Asn His Trp Asn Arg
Leu Arg Asp Asp Ala Ser 20 25 30aat cta aga aat aga ggt ata acc gct
att tgg att ccg cct gcc tgg 144Asn Leu Arg Asn Arg Gly Ile Thr Ala
Ile Trp Ile Pro Pro Ala Trp 35 40 45aaa ggg act tcg caa aat gat gtg
ggg tat gga gcc tat gat ctt tat 192Lys Gly Thr Ser Gln Asn Asp Val
Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60gat tta ggg gaa ttt aat caa
aag ggg acg gtt cgt act aag tat ggg 240Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr Lys Tyr Gly65 70 75 80aca cgt agt caa ttg
gag tct gcc atc cat gct tta aag aat aat ggc 288Thr Arg Ser Gln Leu
Glu Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95gtt caa gtt tat
ggg gat gta gtg atg aac cat aaa gga gga gct gat 336Val Gln Val Tyr
Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110gct aca
gaa aac gtt ctt gct gtc gag gtg aat cca aat aac cgg aat 384Ala Thr
Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125
caa gaa ata tct ggg gac tac aca att gag gct tgg act aag ttt gat
432Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140ttt cca ggg agg ggt aat aca tac tca gac ttt aaa tgg cgt
tgg tat 480Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg
Trp Tyr145 150 155 160cat ttc gat ggt gta gat tgg gat caa tca cga
caa ttc caa aat cgt 528His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg
Gln Phe Gln Asn Arg 165 170 175atc tac aaa ttc cga ggt gat ggt aag
gca tgg gat tgg gaa gta gat 576Ile Tyr Lys Phe Arg Gly Asp Gly Lys
Ala Trp Asp Trp Glu Val Asp 180 185 190tcg gaa aat gga aat tat gat
tat tta atg tat gca gat gta gat atg 624Ser Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 gat cat ccg gag gta
gta aat gag ctt aga aga tgg gga gaa tgg tat 672Asp His Pro Glu Val
Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215 220aca aat aca
tta aat ctt gat gga ttt agg atc gat gcg gtg aag cat 720Thr Asn Thr
Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235
240att aaa tat agc ttt aca cgt gat tgg ttg acc cat gta aga aac gca
768Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala
245 250 255acg gga aaa gaa atg ttt gct gtt gct gaa ttt tgg aaa aat
gat tta 816Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn
Asp Leu 260 265 270ggt gcc ttg gag aac tat tta aat aaa aca aac tgg
aat cat tct gtc 864Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp
Asn His Ser Val 275 280 285 ttt gat gtc ccc ctt cat tat aat ctt tat
aac gcg tca aat agt gga 912Phe Asp Val Pro Leu His Tyr Asn Leu Tyr
Asn Ala Ser Asn Ser Gly 290 295 300ggc aac tat gac atg gca aaa ctt
ctt aat gga acg gtt gtt caa aag 960Gly Asn Tyr Asp Met Ala Lys Leu
Leu Asn Gly Thr Val Val Gln Lys305 310 315 320cat cca atg cat gcc
gta act ttt gtg gat aat cac gat tct caa cct 1008His Pro Met His Ala
Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335ggg gaa tca
tta gaa tca ttt gta caa gaa tgg ttt aag cca ctt gct 1056Gly Glu Ser
Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350tat
gcg ctt att tta aca aga gaa caa ggc tat ccc tct gtc ttc tat 1104Tyr
Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360
365 ggt gac tac tat gga att cca aca cat agt gtc cca gca atg aaa gcc
1152Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala
370 375 380aag att gat cca atc tta gag gcg cgt caa aat ttt gca tat
gga aca 1200Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr
Gly Thr385 390 395 400caa cat gat tat ttt gac cat cat aat ata atc
gga tgg aca cgt gaa 1248Gln His Asp Tyr Phe Asp His His Asn Ile Ile
Gly Trp Thr Arg Glu 405 410 415gga aat acc acg cat ccc aat tca gga
ctt gcg act atc atg tcg gat 1296Gly Asn Thr Thr His Pro Asn Ser Gly
Leu Ala Thr Ile Met Ser Asp 420 425 430ggg cca ggg gga gag aaa tgg
atg tac gta ggg caa aat aaa gca ggt 1344Gly Pro Gly Gly Glu Lys Trp
Met Tyr Val Gly Gln Asn Lys Ala Gly 435 440 445 caa gtt tgg cat gac
ata act gga aat aaa cca gga aca gtt acg atc 1392Gln Val Trp His Asp
Ile Thr Gly Asn Lys Pro Gly Thr Val Thr Ile 450 455 460aat gca gat
gga tgg gct aat ttt tca gta aat gga gga tct gtt tcc 1440Asn Ala Asp
Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470 475
480att tgg gtg aaa cga 1455Ile Trp Val Lys Arg 4854485PRTBacillus
species 4His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu
Trp His1 5 10 15Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp
Asp Ala Ser 20 25 30Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile
Pro Pro Ala Trp 35 40 45Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly
Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr
Val Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Ser Gln Leu Glu Ser Ala
Ile His Ala Leu Lys Asn Asn Gly 85 90 95Val Gln Val Tyr Gly Asp Val
Val Met Asn His Lys Gly Gly Ala Asp 100 105 110Ala Thr Glu Asn Val
Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125Gln Glu Ile
Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140Phe
Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr145 150
155 160His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn
Arg 165 170 175Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp
Glu Val Asp 180 185 190Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr
Ala Asp Val Asp Met 195 200 205Asp His Pro Glu Val Val Asn Glu Leu
Arg Arg Trp Gly Glu Trp Tyr 210 215 220Thr Asn Thr Leu Asn Leu Asp
Gly Phe Arg Ile Asp Ala Val Lys His225 230 235 240Ile Lys Tyr Ser
Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala 245 250 255Thr Gly
Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265
270Gly Ala Leu Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val
275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn
Ser Gly 290 295 300Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr
Val Val Gln Lys305 310 315 320His Pro Met His Ala Val Thr Phe Val
Asp Asn His Asp Ser Gln Pro 325 330 335Gly Glu Ser Leu Glu Ser Phe
Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350Tyr Ala Leu Ile Leu
Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365Gly Asp Tyr
Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380Lys
Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr385 390
395 400Gln His
Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410
415Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp
420 425 430Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys
Ala Gly 435 440 445Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly
Thr Val Thr Ile 450 455 460Asn Ala Asp Gly Trp Ala Asn Phe Ser Val
Asn Gly Gly Ser Val Ser465 470 475 480Ile Trp Val Lys Arg
48551548DNABacillus stearothermophilusCDS(1)..(1548) 5gcc gca ccg
ttt aac ggc acc atg atg cag tat ttt gaa tgg tac ttg 48Ala Ala Pro
Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu1 5 10 15ccg gat
gat ggc acg tta tgg acc aaa gtg gcc aat gaa gcc aac aac 96Pro Asp
Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30tta
tcc agc ctt ggc atc acc gct ctt tgg ctg ccg ccc gct tac aaa 144Leu
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40
45gga aca agc cgc agc gac gta ggg tac gga gta tac gac ttg tat gac
192Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60ctc ggc gaa ttc aat caa aaa ggg acc gtc cgc aca aaa tac gga
aca 240Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
Thr65 70 75 80aaa gct caa tat ctt caa gcc att caa gcc gcc cac gcc
gct gga atg 288Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala
Ala Gly Met 85 90 95caa gtg tac gcc gat gtc gtg ttc gac cat aaa ggc
ggc gct gac ggc 336Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly
Gly Ala Asp Gly 100 105 110acg gaa tgg gtg gac gcc gtc gaa gtc aat
ccg tcc gac cgc aac caa 384Thr Glu Trp Val Asp Ala Val Glu Val Asn
Pro Ser Asp Arg Asn Gln 115 120 125gaa atc tcg ggc acc tat caa atc
caa gca tgg acg aaa ttt gat ttt 432Glu Ile Ser Gly Thr Tyr Gln Ile
Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140ccc ggg cgg ggc aac acc
tac tcc agc ttt aag tgg cgc tgg tac cat 480Pro Gly Arg Gly Asn Thr
Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His145 150 155 160ttt gac ggc
gtt gat tgg gac gaa agc cga aaa ttg agc cgc att tac 528Phe Asp Gly
Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175aaa
ttc cgc ggc atc ggc aaa gcg tgg gat tgg gaa gta gac acg gaa 576Lys
Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185
190aac gga aac tat gac tac tta atg tat gcc gac ctt gat atg gat cat
624Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205ccc gaa gtc gtg acc gag ctg aaa aac tgg ggg aaa tgg tat
gtc aac 672Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr
Val Asn 210 215 220aca acg aac att gat ggg ttc cgg ctt gat gcc gtc
aag cat att aag 720Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val
Lys His Ile Lys225 230 235 240ttc agt ttt ttt cct gat tgg ttg tcg
tat gtg cgt tct cag act ggc 768Phe Ser Phe Phe Pro Asp Trp Leu Ser
Tyr Val Arg Ser Gln Thr Gly 245 250 255aag ccg cta ttt acc gtc ggg
gaa tat tgg agc tat gac atc aac aag 816Lys Pro Leu Phe Thr Val Gly
Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270ttg cac aat tac att
acg aaa aca gac gga acg atg tct ttg ttt gat 864Leu His Asn Tyr Ile
Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp 275 280 285gcc ccg tta
cac aac aaa ttt tat acc gct tcc aaa tca ggg ggc gca 912Ala Pro Leu
His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300ttt
gat atg cgc acg tta atg acc aat act ctc atg aaa gat caa ccg 960Phe
Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro305 310
315 320aca ttg gcc gtc acc ttc gtt gat aat cat gac acc gaa ccc ggc
caa 1008Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly
Gln 325 330 335gcg ctg cag tca tgg gtc gac cca tgg ttc aaa ccg ttg
gct tac gcc 1056Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu
Ala Tyr Ala 340 345 350ttt att cta act cgg cag gaa gga tac ccg tgc
gtc ttt tat ggt gac 1104Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys
Val Phe Tyr Gly Asp 355 360 365tat tat ggc att cca caa tat aac att
cct tcg ctg aaa agc aaa atc 1152Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile
Pro Ser Leu Lys Ser Lys Ile 370 375 380gat ccg ctc ctc atc gcg cgc
agg gat tat gct tac gga acg caa cat 1200Asp Pro Leu Leu Ile Ala Arg
Arg Asp Tyr Ala Tyr Gly Thr Gln His385 390 395 400gat tat ctt gat
cac tcc gac atc atc ggg tgg aca agg gaa ggg ggc 1248Asp Tyr Leu Asp
His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405 410 415act gaa
aaa cca gga tcc gga ctg gcc gca ctg atc acc gat ggg ccg 1296Thr Glu
Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425
430gga gga agc aaa tgg atg tac gtt ggc aaa caa cac gct gga aaa gtg
1344Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445ttc tat gac ctt acc ggc aac cgg agt gac acc gtc acc atc
aac agt 1392Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
Asn Ser 450 455 460gat gga tgg ggg gaa ttc aaa gtc aat ggc ggt tcg
gtt tcg gtt tgg 1440Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser
Val Ser Val Trp465 470 475 480gtt cct aga aaa acg acc gtt tct acc
atc gct cgg ccg atc aca acc 1488Val Pro Arg Lys Thr Thr Val Ser Thr
Ile Ala Arg Pro Ile Thr Thr 485 490 495cga ccg tgg act ggt gaa ttc
gtc cgt tgg acc gaa cca cgg ttg gtg 1536Arg Pro Trp Thr Gly Glu Phe
Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510gca tgg cct tga
1548Ala Trp Pro 5156515PRTBacillus stearothermophilus 6Ala Ala Pro
Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu1 5 10 15Pro Asp
Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30Leu
Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40
45Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp
50 55 60Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
Thr65 70 75 80Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala His Ala
Ala Gly Met 85 90 95Gln Val Tyr Ala Asp Val Val Phe Asp His Lys Gly
Gly Ala Asp Gly 100 105 110Thr Glu Trp Val Asp Ala Val Glu Val Asn
Pro Ser Asp Arg Asn Gln 115 120 125Glu Ile Ser Gly Thr Tyr Gln Ile
Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140Pro Gly Arg Gly Asn Thr
Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His145 150 155 160Phe Asp Gly
Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165 170 175Lys
Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185
190Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His
195 200 205Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly Lys Trp Tyr
Val Asn 210 215 220Thr Thr Asn Ile Asp Gly Phe Arg Leu Asp Ala Val
Lys His Ile Lys225 230 235 240Phe Ser Phe Phe Pro Asp Trp Leu Ser
Tyr Val Arg Ser Gln Thr Gly 245 250 255Lys Pro Leu Phe Thr Val Gly
Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270Leu His Asn Tyr Ile
Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp 275 280 285Ala Pro Leu
His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290 295 300Phe
Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln Pro305 310
315 320Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly
Gln 325 330 335Ala Leu Gln Ser Trp Val Asp Pro Trp Phe Lys Pro Leu
Ala Tyr Ala 340 345 350Phe Ile Leu Thr Arg Gln Glu Gly Tyr Pro Cys
Val Phe Tyr Gly Asp 355 360 365Tyr Tyr Gly Ile Pro Gln Tyr Asn Ile
Pro Ser Leu Lys Ser Lys Ile 370 375 380Asp Pro Leu Leu Ile Ala Arg
Arg Asp Tyr Ala Tyr Gly Thr Gln His385 390 395 400Asp Tyr Leu Asp
His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405 410 415Thr Glu
Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425
430Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala Gly Lys Val
435 440 445Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile
Asn Ser 450 455 460Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser
Val Ser Val Trp465 470 475 480Val Pro Arg Lys Thr Thr Val Ser Thr
Ile Ala Arg Pro Ile Thr Thr 485 490 495Arg Pro Trp Thr Gly Glu Phe
Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510Ala Trp Pro
51571920DNABacillus licheniformisCDS(421)..(1872) 7cggaagattg
gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60gagacggaaa
aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag
120agattattaa aaagctgaaa gcaaaaggct atcaattggt aactgtatct
cagcttgaag 180aagtgaagaa gcagagaggc tattgaataa atgagtagaa
gcgccatatc ggcgcttttc 240ttttggaaga aaatataggg aaaatggtac
ttgttaaaaa ttcggaatat ttatacaaca 300tcatatgttt cacattgaaa
ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360cgattgctga
cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420gca aat
ctt aat ggg acg ctg atg cag tat ttt gaa tgg tac atg ccc 468Ala Asn
Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro1 5 10 15aat
gac ggc caa cat tgg agg cgt ttg caa aac gac tcg gca tat ttg 516Asn
Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25
30gct gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag gga
564Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45acg agc caa gcg gat gtg ggc tac ggt gct tac gac ctt tat gat
tta 612Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp
Leu 50 55 60ggg gag ttt cat caa aaa ggg acg gtt cgg aca aag tac ggc
aca aaa 660Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
Thr Lys65 70 75 80gga gag ctg caa tct gcg atc aaa agt ctt cat tcc
cgc gac att aac 708Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser
Arg Asp Ile Asn 85 90 95 gtt tac ggg gat gtg gtc atc aac cac aaa
ggc ggc gct gat gcg acc 756Val Tyr Gly Asp Val Val Ile Asn His Lys
Gly Gly Ala Asp Ala Thr 100 105 110gaa gat gta acc gcg gtt gaa gtc
gat ccc gct gac cgc aac cgc gta 804Glu Asp Val Thr Ala Val Glu Val
Asp Pro Ala Asp Arg Asn Arg Val 115 120 125att tca gga gaa cac cta
att aaa gcc tgg aca cat ttt cat ttt ccg 852Ile Ser Gly Glu His Leu
Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140ggg cgc ggc agc
aca tac agc gat ttt aaa tgg cat tgg tac cat ttt 900Gly Arg Gly Ser
Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe145 150 155 160gac
gga acc gat tgg gac gag tcc cga aag ctg aac cgc atc tat aag 948Asp
Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170
175ttt caa gga aag gct tgg gat tgg gaa gtt tcc aat gaa aac ggc aac
996Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190tat gat tat ttg atg tat gcc gac atc gat tat gac cat cct
gat gtc 1044Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro
Asp Val 195 200 205gca gca gaa att aag aga tgg ggc act tgg tat gcc
aat gaa ctg caa 1092Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala
Asn Glu Leu Gln 210 215 220ttg gac ggt ttc cgt ctt gat gct gtc aaa
cac att aaa ttt tct ttt 1140Leu Asp Gly Phe Arg Leu Asp Ala Val Lys
His Ile Lys Phe Ser Phe225 230 235 240ttg cgg gat tgg gtt aat cat
gtc agg gaa aaa acg ggg aag gaa atg 1188Leu Arg Asp Trp Val Asn His
Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255ttt acg gta gct gaa
tat tgg cag aat gac ttg ggc gcg ctg gaa aac 1236Phe Thr Val Ala Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270tat ttg aac
aaa aca aat ttt aat cat tca gtg ttt gac gtg ccg ctt 1284Tyr Leu Asn
Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285cat
tat cag ttc cat gct gca tcg aca cag gga ggc ggc tat gat atg 1332His
Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295
300agg aaa ttg ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg
1380Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys
Ser305 310 315 320gtt aca ttt gtc gat aac cat gat aca cag ccg ggg
caa tcg ctt gag 1428Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335tcg act gtc caa aca tgg ttt aag ccg ctt
gct tac gct ttt att ctc 1476Ser Thr Val Gln Thr Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350aca agg gaa tct gga tac cct cag
gtt ttc tac ggg gat atg tac ggg 1524Thr Arg Glu Ser Gly Tyr Pro Gln
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365acg aaa gga gac tcc cag
cgc gaa att cct gcc ttg aaa cac aaa att 1572Thr Lys Gly Asp Ser Gln
Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380gaa ccg atc tta
aaa gcg aga aaa cag tat gcg tac gga gca cag cat 1620Glu Pro Ile Leu
Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His385 390 395 400gat
tat ttc gac cac cat gac att gtc ggc tgg aca agg gaa ggc gac 1668Asp
Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410
415agc tcg gtt gca aat tca ggt ttg gcg gca tta ata aca gac gga ccc
1716Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430ggt ggg gca aag cga atg tat gtc ggc cgg caa aac gcc ggt
gag aca 1764Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly
Glu Thr 435 440 445tgg cat gac att acc gga aac cgt tcg gag ccg gtt
gtc atc aat tcg 1812Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val
Val Ile Asn Ser 450 455 460gaa ggc tgg gga gag ttt cac gta aac ggc
ggg tcg gtt tca att tat 1860Glu Gly Trp Gly Glu Phe His Val Asn Gly
Gly Ser Val Ser Ile Tyr465 470 475 480gtt caa aga tag aagagcagag
aggacggatt tcctgaagga aatccgtttt 1912Val Gln Argtttatttt
19208483PRTBacillus licheniformis 8Ala Asn Leu Asn Gly Thr Leu Met
Gln Tyr Phe Glu Trp Tyr Met Pro1 5 10 15Asn Asp Gly Gln His Trp Arg
Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30Ala Glu His Gly Ile Thr
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45 Thr Ser Gln Ala
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60Gly Glu Phe
His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75 80Gly
Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn 85 90
95Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn
Arg Val 115 120 125Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His
Phe His Phe Pro 130 135 140Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys
Trp His Trp Tyr His Phe145
150 155 160Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile
Tyr Lys 165 170 175Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn
Glu Asn Gly Asn 180 185 190Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp
Tyr Asp His Pro Asp Val 195 200 205Ala Ala Glu Ile Lys Arg Trp Gly
Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220Leu Asp Gly Phe Arg Leu
Asp Ala Val Lys His Ile Lys Phe Ser Phe225 230 235 240Leu Arg Asp
Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255Phe
Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265
270Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu
275 280 285His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr
Asp Met 290 295 300Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His
Pro Leu Lys Ser305 310 315 320Val Thr Phe Val Asp Asn His Asp Thr
Gln Pro Gly Gln Ser Leu Glu 325 330 335Ser Thr Val Gln Thr Trp Phe
Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340 345 350Thr Arg Glu Ser Gly
Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365Thr Lys Gly
Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380Glu
Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His385 390
395 400Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly
Asp 405 410 415Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr
Asp Gly Pro 420 425 430Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln
Asn Ala Gly Glu Thr 435 440 445Trp His Asp Ile Thr Gly Asn Arg Ser
Glu Pro Val Val Ile Asn Ser 450 455 460Glu Gly Trp Gly Glu Phe His
Val Asn Gly Gly Ser Val Ser Ile Tyr465 470 475 480Val Gln
Arg92084DNABacillus amyloliquefaciensCDS(343)..(1794) 9gccccgcaca
tacgaaaaga ctggctgaaa acattgagcc tttgatgact gatgatttgg 60ctgaagaagt
ggatcgattg tttgagaaaa gaagaagacc ataaaaatac cttgtctgtc
120atcagacagg gtatttttta tgctgtccag actgtccgct gtgtaaaaat
aaggaataaa 180ggggggttgt tattatttta ctgatatgta aaatataatt
tgtataagaa aatgagaggg 240agaggaaaca tgattcaaaa acgaaagcgg
acagtttcgt tcagacttgt gcttatgtgc 300acgctgttat ttgtcagttt
gccgattaca aaaacatcag cc gta aat ggc acg 354Val Asn Gly Thr1ctg atg
cag tat ttt gaa tgg tat acg ccg aac gac ggc cag cat tgg 402Leu Met
Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp Gly Gln His Trp5 10 15
20aaa cga ttg cag aat gat gcg gaa cat tta tcg gat atc gga atc act
450Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp Ile Gly Ile Thr
25 30 35gcc gtc tgg att cct ccc gca tac aaa gga ttg agc caa tcc gat
aac 498Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser Gln Ser Asp
Asn 40 45 50gga tac gga cct tat gat ttg tat gat tta gga gaa ttc cag
caa aaa 546Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Gln
Gln Lys 55 60 65ggg acg gtc aga acg aaa tac ggc aca aaa tca gag ctt
caa gat gcg 594Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu
Gln Asp Ala 70 75 80atc ggc tca ctg cat tcc cgg aac gtc caa gta tac
gga gat gtg gtt 642Ile Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr
Gly Asp Val Val85 90 95 100ttg aat cat aag gct ggt gct gat gca aca
gaa gat gta act gcc gtc 690Leu Asn His Lys Ala Gly Ala Asp Ala Thr
Glu Asp Val Thr Ala Val 105 110 115gaa gtc aat ccg gcc aat aga aat
cag gaa act tcg gag gaa tat caa 738Glu Val Asn Pro Ala Asn Arg Asn
Gln Glu Thr Ser Glu Glu Tyr Gln 120 125 130atc aaa gcg tgg acg gat
ttt cgt ttt ccg ggc cgt gga aac acg tac 786Ile Lys Ala Trp Thr Asp
Phe Arg Phe Pro Gly Arg Gly Asn Thr Tyr 135 140 145agt gat ttt aaa
tgg cat tgg tat cat ttc gac gga gcg gac tgg gat 834Ser Asp Phe Lys
Trp His Trp Tyr His Phe Asp Gly Ala Asp Trp Asp 150 155 160gaa tcc
cgg aag atc agc cgc atc ttt aag ttt cgt ggg gaa gga aaa 882Glu Ser
Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg Gly Glu Gly Lys165 170 175
180gcg tgg gat tgg gaa gta tca agt gaa aac ggc aac tat gac tat tta
930Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu
185 190 195atg tat gct gat gtt gac tac gac cac cct gat gtc gtg gca
gag aca 978Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala
Glu Thr 200 205 210aaa aaa tgg ggt atc tgg tat gcg aat gaa ctg tca
tta gac ggc ttc 1026Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser
Leu Asp Gly Phe 215 220 225cgt att gat gcc gcc aaa cat att aaa ttt
tca ttt ctg cgt gat tgg 1074Arg Ile Asp Ala Ala Lys His Ile Lys Phe
Ser Phe Leu Arg Asp Trp 230 235 240gtt cag gcg gtc aga cag gcg acg
gga aaa gaa atg ttt acg gtt gcg 1122Val Gln Ala Val Arg Gln Ala Thr
Gly Lys Glu Met Phe Thr Val Ala245 250 255 260gag tat tgg cag aat
aat gcc ggg aaa ctc gaa aac tac ttg aat aaa 1170Glu Tyr Trp Gln Asn
Asn Ala Gly Lys Leu Glu Asn Tyr Leu Asn Lys 265 270 275aca agc ttt
aat caa tcc gtg ttt gat gtt ccg ctt cat ttc aat tta 1218Thr Ser Phe
Asn Gln Ser Val Phe Asp Val Pro Leu His Phe Asn Leu 280 285 290cag
gcg gct tcc tca caa gga ggc gga tat gat atg agg cgt ttg ctg 1266Gln
Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg Arg Leu Leu 295 300
305gac ggt acc gtt gtg tcc agg cat ccg gaa aag gcg gtt aca ttt gtt
1314Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala Val Thr Phe Val
310 315 320gaa aat cat gac aca cag ccg gga cag tca ttg gaa tcg aca
gtc caa 1362Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu Ser Thr
Val Gln325 330 335 340act tgg ttt aaa ccg ctt gca tac gcc ttt att
ttg aca aga gaa tcc 1410Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile
Leu Thr Arg Glu Ser 345 350 355ggt tat cct cag gtg ttc tat ggg gat
atg tac ggg aca aaa ggg aca 1458Gly Tyr Pro Gln Val Phe Tyr Gly Asp
Met Tyr Gly Thr Lys Gly Thr 360 365 370tcg cca aag gaa att ccc tca
ctg aaa gat aat ata gag ccg att tta 1506Ser Pro Lys Glu Ile Pro Ser
Leu Lys Asp Asn Ile Glu Pro Ile Leu 375 380 385aaa gcg cgt aag gag
tac gca tac ggg ccc cag cac gat tat att gac 1554Lys Ala Arg Lys Glu
Tyr Ala Tyr Gly Pro Gln His Asp Tyr Ile Asp 390 395 400cac ccg gat
gtg atc gga tgg acg agg gaa ggt gac agc tcc gcc gcc 1602His Pro Asp
Val Ile Gly Trp Thr Arg Glu Gly Asp Ser Ser Ala Ala405 410 415
420aaa tca ggt ttg gcc gct tta atc acg gac gga ccc ggc gga tca aag
1650Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys
425 430 435cgg atg tat gcc ggc ctg aaa aat gcc ggc gag aca tgg tat
gac ata 1698Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp Tyr
Asp Ile 440 445 450acg ggc aac cgt tca gat act gta aaa atc gga tct
gac ggc tgg gga 1746Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser
Asp Gly Trp Gly 455 460 465gag ttt cat gta aac gat ggg tcc gtc tcc
att tat gtt cag aaa taa 1794Glu Phe His Val Asn Asp Gly Ser Val Ser
Ile Tyr Val Gln Lys 470 475 480ggtaataaaa aaacacctcc aagctgagtg
cgggtatcag cttggaggtg cgtttatttt 1854ttcagccgta tgacaaggtc
ggcatcaggt gtgacaaata cggtatgctg gctgtcatag 1914gtgacaaatc
cgggttttgc gccgtttggc tttttcacat gtctgatttt tgtataatca
1974acaggcacgg agccggaatc tttcgccttg gaaaaataag cggcgatcgt
agctgcttcc 2034aatatggatt gttcatcggg atcgctgctt ttaatcacaa
cgtgggatcc 208410483PRTBacillus amyloliquefaciens 10Val Asn Gly Thr
Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn Asp1 5 10 15Gly Gln His
Trp Lys Arg Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30Ile Gly
Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45Gln
Ser Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55
60Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu65
70 75 80Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln Val
Tyr 85 90 95Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp Ala Thr
Glu Asp 100 105 110Val Thr Ala Val Glu Val Asn Pro Ala Asn Arg Asn
Gln Glu Thr Ser 115 120 125Glu Glu Tyr Gln Ile Lys Ala Trp Thr Asp
Phe Arg Phe Pro Gly Arg 130 135 140Gly Asn Thr Tyr Ser Asp Phe Lys
Trp His Trp Tyr His Phe Asp Gly145 150 155 160Ala Asp Trp Asp Glu
Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg 165 170 175Gly Glu Gly
Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185 190Tyr
Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val 195 200
205Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser
210 215 220Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile Lys Phe
Ser Phe225 230 235 240Leu Arg Asp Trp Val Gln Ala Val Arg Gln Ala
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Ala Glu Tyr Trp Gln Asn
Asn Ala Gly Lys Leu Glu Asn 260 265 270Tyr Leu Asn Lys Thr Ser Phe
Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285His Phe Asn Leu Gln
Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300Arg Arg Leu
Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala305 310 315
320Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Thr Ser Pro Lys Glu Ile Pro
Ser Leu Lys Asp Asn Ile 370 375 380Glu Pro Ile Leu Lys Ala Arg Lys
Glu Tyr Ala Tyr Gly Pro Gln His385 390 395 400Asp Tyr Ile Asp His
Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Ala
Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435 440
445Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser
450 455 460Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val Ser
Ile Tyr465 470 475 480Val Gln Lys111458DNABacillus
speciesCDS(1)..(1458) 11cac cat aat ggt acg aac ggc aca atg atg cag
tac ttt gaa tgg tat 48His His Asn Gly Thr Asn Gly Thr Met Met Gln
Tyr Phe Glu Trp Tyr1 5 10 15cta cca aat gac gga aac cat tgg aat aga
tta agg tct gat gca agt 96Leu Pro Asn Asp Gly Asn His Trp Asn Arg
Leu Arg Ser Asp Ala Ser 20 25 30aac cta aaa gat aaa ggg atc tca gcg
gtt tgg att cct cct gca tgg 144Asn Leu Lys Asp Lys Gly Ile Ser Ala
Val Trp Ile Pro Pro Ala Trp 35 40 45aag ggt gcc tct caa aat gat gtg
ggg tat ggt gct tat gat ctg tat 192Lys Gly Ala Ser Gln Asn Asp Val
Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60gat tta gga gaa ttc aat caa
aaa gga acc att cgt aca aaa tat gga 240Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Ile Arg Thr Lys Tyr Gly65 70 75 80acg cgc aat cag tta
caa gct gca gtt aac gcc ttg aaa agt aat gga 288Thr Arg Asn Gln Leu
Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95att caa gtg tat
ggc gat gtt gta atg aat cat aaa ggg gga gca gac 336Ile Gln Val Tyr
Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110gct acc
gaa atg gtt agg gca gtt gaa gta aac ccg aat aat aga aat 384Ala Thr
Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120
125caa gaa gtg tcc ggt gaa tat aca att gag gct tgg aca aag ttt gac
432Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140ttt cca gga cga ggt aat act cat tca aac ttc aaa tgg aga
tgg tat 480Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg
Trp Tyr145 150 155 160cac ttt gat gga gta gat tgg gat cag tca cgt
aag ctg aac aat cga 528His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg
Lys Leu Asn Asn Arg 165 170 175att tat aaa ttt aga ggt gat gga aaa
ggg tgg gat tgg gaa gtc gat 576Ile Tyr Lys Phe Arg Gly Asp Gly Lys
Gly Trp Asp Trp Glu Val Asp 180 185 190aca gaa aac ggt aac tat gat
tac cta atg tat gca gat att gac atg 624Thr Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205gat cac cca gag gta
gtg aat gag cta aga aat tgg ggt gtt tgg tat 672Asp His Pro Glu Val
Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220acg aat aca
tta ggc ctt gat ggt ttt aga ata gat gca gta aaa cat 720Thr Asn Thr
Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230 235
240ata aaa tac agc ttt act cgt gat tgg att aat cat gtt aga agt gca
768Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala
245 250 255act ggc aaa aat atg ttt gcg gtt gcg gaa ttt tgg aaa aat
gat tta 816Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn
Asp Leu 260 265 270ggt gct att gaa aac tat tta aac aaa aca aac tgg
aac cat tca gtc 864Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp
Asn His Ser Val 275 280 285ttt gat gtt ccg ctg cac tat aac ctc tat
aat gct tca aaa agc gga 912Phe Asp Val Pro Leu His Tyr Asn Leu Tyr
Asn Ala Ser Lys Ser Gly 290 295 300ggg aat tat gat atg agg caa ata
ttt aat ggt aca gtc gtg caa aga 960Gly Asn Tyr Asp Met Arg Gln Ile
Phe Asn Gly Thr Val Val Gln Arg305 310 315 320cat cca atg cat gct
gtt aca ttt gtt gat aat cat gat tcg caa cct 1008His Pro Met His Ala
Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335gaa gaa gct
tta gag tct ttt gtt gaa gaa tgg ttc aaa cca tta gcg 1056Glu Glu Ala
Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350tat
gct ttg aca tta aca cgt gaa caa ggc tac cct tct gta ttt tat 1104Tyr
Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360
365gga gat tat tat ggc att cca acg cat ggt gta cca gcg atg aaa tcg
1152Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser
370 375 380aaa att gac ccg att cta gaa gcg cgt caa aag tat gca tat
gga aga 1200Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr
Gly Arg385 390 395 400caa aat gac tac tta gac cat cat aat atc atc
ggt tgg aca cgt gaa 1248Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile
Gly Trp Thr Arg Glu 405 410 415ggg aat aca gca cac ccc aac tcc ggt
tta gct act atc atg tcc gat 1296Gly Asn Thr Ala His Pro Asn Ser Gly
Leu Ala Thr Ile Met Ser Asp 420 425 430ggg gca gga gga aat aag tgg
atg ttt gtt ggg cgt aat aaa gct ggt 1344Gly Ala Gly Gly Asn Lys Trp
Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445caa gtt tgg acc gat
atc act gga aat cgt gca ggt act gtt acg att 1392Gln Val Trp Thr Asp
Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455 460aat gct gat
gga tgg ggt aat ttt tct gta aat gga gga tca gtt tct 1440Asn Ala Asp
Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470
475 480att tgg gta aac aaa taa 1458Ile Trp Val Asn Lys
48512485PRTBacillus species 12His His Asn Gly Thr Asn Gly Thr Met
Met Gln Tyr Phe Glu Trp Tyr1 5 10 15Leu Pro Asn Asp Gly Asn His Trp
Asn Arg Leu Arg Ser Asp Ala Ser 20 25 30Asn Leu Lys Asp Lys Gly Ile
Ser Ala Val Trp Ile Pro Pro Ala Trp 35 40 45Lys Gly Ala Ser Gln Asn
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly65 70 75 80Thr Arg Asn
Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95Ile Gln
Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105
110Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn
115 120 125Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys
Phe Asp 130 135 140Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys
Trp Arg Trp Tyr145 150 155 160His Phe Asp Gly Val Asp Trp Asp Gln
Ser Arg Lys Leu Asn Asn Arg 165 170 175Ile Tyr Lys Phe Arg Gly Asp
Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190Thr Glu Asn Gly Asn
Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205Asp His Pro
Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220Thr
Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His225 230
235 240Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser
Ala 245 250 255Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys
Asn Asp Leu 260 265 270Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn
Trp Asn His Ser Val 275 280 285Phe Asp Val Pro Leu His Tyr Asn Leu
Tyr Asn Ala Ser Lys Ser Gly 290 295 300Gly Asn Tyr Asp Met Arg Gln
Ile Phe Asn Gly Thr Val Val Gln Arg305 310 315 320His Pro Met His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335Glu Glu
Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345
350Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr
355 360 365Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met
Lys Ser 370 375 380Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr
Ala Tyr Gly Arg385 390 395 400Gln Asn Asp Tyr Leu Asp His His Asn
Ile Ile Gly Trp Thr Arg Glu 405 410 415Gly Asn Thr Ala His Pro Asn
Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430Gly Ala Gly Gly Asn
Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445Gln Val Trp
Thr Asp Ile Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455 460Asn
Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser465 470
475 480Ile Trp Val Asn Lys 48513197PRTBacillus species 13Phe Asp
Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly1 5 10 15Gly
Asn Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg 20 25
30His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro
35 40 45Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu
Ala 50 55 60Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val
Phe Tyr65 70 75 80Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro
Ala Met Arg Ser 85 90 95Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys
Tyr Ala Tyr Gly Lys 100 105 110Gln Asn Asp Tyr Leu Asp His His Asn
Ile Ile Gly Trp Thr Arg Glu 115 120 125Gly Asn Thr Ala His Pro Asn
Ser Gly Leu Ala Thr Ile Met Ser Asp 130 135 140Gly Ala Gly Gly Ser
Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly145 150 155 160Gln Val
Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 165 170
175Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser
180 185 190Ile Trp Val Asn Lys 1951424DNAArtificial
SequenceSynthetic construct 14cgattgctga cgctgttatt tgcg
241519DNAArtificial SequenceSynthetic construct 15gatcacccgc
gataccgtc 191631DNAArtificial SequenceSynthetic construct
16gaatgtatgt cggccggcaa aacgccggtg a 311730DNAArtificial
SequenceSynthetic construct 17gccgccgctg ctgcagaatg aggcagcaag
301848DNAArtificial SequenceSynthetic construct 18cccgaaagct
gaaccgcatc tataggtttc aagggaagac ttgggatt 481923DNAArtificial
SequenceSynthetic construct 19aggatggtca taatcaaagt cgg
232052DNAArtificial SequenceSynthetic construct 20ccgactttga
ttatgaccat cctgttgtcg tagcagagat taagagatgg gg 522145DNAArtificial
SequenceSynthetic construct 21cgacaatgtc atggtggtcg aaaaaatcat
gctgtgctcc gtacg 452223DNAArtificial SequenceSynthetic construct
22tttcgaccac catgacattg tcg 232324DNAArtificial SequenceSynthetic
construct 23tatagatgcg gttcagcttt cggg 24241650DNABacillus species
24cttgaatcat tatttaaagc tggttatgat atatgtaagc gttatcatta aaaggaggta
60tttgatgaaa agatgggtag tagcaatgct ggcagtgtta tttttatttc cttcggtagt
120agttgcagat ggcttgaatg gaacgatgat gcagtattat gagtggcatc
tagagaatga 180tgggcaacac tggaatcggt tgcatgatga tgccgaagct
ttaagtaatg cgggtattac 240agctatttgg atacccccag cctacaaagg
aaatagtcag gctgatgttg ggtatggtgc 300atacgacctt tatgatttag
gggagtttaa tcaaaaaggt accgttcgaa cgaaatacgg 360gacaaaggct
cagcttgagc gagctatagg gtccctaaag tcgaatgata tcaatgttta
420tggggatgtc gtaatgaatc ataaattagg agctgatttc acggaggcag
tgcaagctgt 480tcaagtaaat ccttcgaacc gttggcagga tatttcaggt
gtctacacga ttgatgcatg 540gacgggattt gactttccag ggcgcaacaa
tgcctattcc gattttaaat ggagatggtt 600ccattttaat ggcgttgact
gggatcaacg ctatcaagaa aaccatcttt ttcgctttgc 660aaatacgaac
tggaactggc gagtggatga agagaatggt aattatgact atttattagg
720atcgaacatt gactttagcc acccagaggt tcaagaggaa ttaaaggatt
gggggagctg 780gtttacggat gagctagatt tagatgggta tcgattggat
gctattaagc atattccatt 840ctggtatacg tcagattggg ttaggcatca
gcgaagtgaa gcagaccaag atttatttgt 900cgtaggggag tattggaagg
atgacgtagg tgctctcgaa ttttatttag atgaaatgaa 960ttgggagatg
tctctattcg atgttccgct caattataat ttttaccggg cttcaaagca
1020aggcggaagc tatgatatgc gtaatatttt acgaggatct ttagtagaag
cacatccgat 1080tcatgcagtt acgtttgttg ataatcatga tactcagcca
ggagagtcat tagaatcatg 1140ggtcgctgat tggtttaagc cacttgctta
tgcgacaatc ttgacgcgtg aaggtggtta 1200tccaaatgta ttttacggtg
actactatgg gattcctaac gataacattt cagctaagaa 1260ggatatgatt
gatgagttgc ttgatgcacg tcaaaattac gcatatggca cacaacatga
1320ctattttgat cattgggata tcgttggatg gacaagagaa ggtacatcct
cacgtcctaa 1380ttcgggtctt gctactatta tgtccaatgg tcctggagga
tcaaaatgga tgtacgtagg 1440acagcaacat gcaggacaaa cgtggacaga
tttaactggc aatcacgcgg cgtcggttac 1500gattaatggt gatggctggg
gcgaattctt tacaaatgga ggatctgtat ccgtgtatgt 1560gaaccaataa
taaaaagcct tgagaaggga ttcctcccta actcaaggct ttctttatgt
1620cgtttagctc aacgcttcta cgaagcttta 165025501PRTBacillus species
25Met Lys Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro1
5 10 15Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr
Tyr 20 25 30Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu
His Asp 35 40 45Asp Ala Glu Ala Leu Ser Asn Ala Gly Ile Thr Ala Ile
Trp Ile Pro 50 55 60Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly
Tyr Gly Ala Tyr65 70 75 80Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Val Arg Thr 85 90 95 Lys Tyr Gly Thr Lys Ala Gln Leu
Glu Arg Ala Ile Gly Ser Leu Lys 100 105 110Ser Asn Asp Ile Asn Val
Tyr Gly Asp Val Val Met Asn His Lys Leu 115 120 125Gly Ala Asp Phe
Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Ser 130 135 140Asn Arg
Trp Gln Asp Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp Thr145 150 155
160Gly Phe Asp Phe Pro Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys Trp
165 170 175 Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg Tyr
Gln Glu 180 185 190Asn His Leu Phe Arg Phe Ala Asn Thr Asn Trp Asn
Trp Arg Val Asp 195 200 205Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu
Gly Ser Asn Ile Asp Phe 210 215 220Ser His Pro Glu Val Gln Glu Glu
Leu Lys Asp Trp Gly Ser Trp Phe225 230 235 240Thr Asp Glu Leu Asp
Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 245 250 255 Ile Pro Phe
Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Ser Glu 260 265 270Ala
Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val 275 280
285Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu
290 295 300Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Lys
Gln Gly305 310 315 320Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly
Ser Leu Val Glu Ala 325 330 335 His Pro Ile His Ala Val Thr Phe Val
Asp Asn His Asp Thr Gln Pro 340 345 350Gly Glu Ser Leu Glu Ser Trp
Val Ala Asp Trp Phe Lys Pro Leu Ala 355 360 365Tyr Ala Thr Ile Leu
Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 370 375 380Gly Asp Tyr
Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp385 390 395
400Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr
405 410 415 Gln His Asp Tyr Phe Asp His Trp Asp Ile Val Gly Trp Thr
Arg Glu 420 425 430Gly Thr Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr
Ile Met Ser Asn 435 440 445Gly Pro Gly Gly Ser Lys Trp Met Tyr Val
Gly Gln Gln His Ala Gly 450 455 460Gln Thr Trp Thr Asp Leu Thr Gly
Asn His Ala Ala Ser Val Thr Ile465 470 475 480Asn Gly Asp Gly Trp
Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 485 490 495 Val Tyr Val
Asn Gln 500261745DNABacillus
speciesCDS(190)..(1692)sig_peptide(190)..(253)mat_peptide(253)..()
26aactaagtaa catcgattca ggataaaagt atgcgaaacg atgcgcaaaa ctgcgcaact
60actagcactc ttcagggact aaaccacctt ttttccaaaa atgacatcat ataaacaaat
120ttgtctacca atcactattt aaagctgttt atgatatatg taagcgttat
cattaaaagg 180aggtatttg atg aga aga tgg gta gta gca atg ttg gca gtg
tta ttt tta 231Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe
Leu-20 -15 -10ttt cct tcg gta gta gtt gca gat gga ttg aac ggt acg
atg atg cag 279Phe Pro Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr
Met Met Gln -5 -1 1 510tat tat gag tgg cat ttg gaa aac gac ggg cag
cat tgg aat cgg ttg 327Tyr Tyr Glu Trp His Leu Glu Asn Asp Gly Gln
His Trp Asn Arg Leu 15 20 25cac gat gat gcc gca gct ttg agt gat gct
ggt att aca gct att tgg 375His Asp Asp Ala Ala Ala Leu Ser Asp Ala
Gly Ile Thr Ala Ile Trp 30 35 40att ccg cca gcc tac aaa ggt aat agt
cag gcg gat gtt ggg tac ggt 423Ile Pro Pro Ala Tyr Lys Gly Asn Ser
Gln Ala Asp Val Gly Tyr Gly 45 50 55gca tac gat ctt tat gat tta gga
gag ttc aat caa aag ggt act gtt 471Ala Tyr Asp Leu Tyr Asp Leu Gly
Glu Phe Asn Gln Lys Gly Thr Val 60 65 70cga acg aaa tac gga act aag
gca cag ctt gaa cga gct att ggg tcc 519Arg Thr Lys Tyr Gly Thr Lys
Ala Gln Leu Glu Arg Ala Ile Gly Ser75 80 8590ctt aaa tct aat gat
atc aat gta tac gga gat gtc gtg atg aat cat 567Leu Lys Ser Asn Asp
Ile Asn Val Tyr Gly Asp Val Val Met Asn His 95 100 105aaa atg gga
gct gat ttt acg gag gca gtg caa gct gtt caa gta aat 615Lys Met Gly
Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn 110 115 120cca
acg aat cgt tgg cag gat att tca ggt gcc tac acg att gat gcg 663Pro
Thr Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala 125 130
135tgg acg ggt ttc gac ttt tca ggg cgt aac aac gcc tat tca gat ttt
711Trp Thr Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe
140 145 150aag tgg aga tgg ttc cat ttt aat ggt gtt gac tgg gat cag
cgc tat 759Lys Trp Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln
Arg Tyr155 160 165170caa gaa aat cat att ttc cgc ttt gca aat acg
aac tgg aac tgg cga 807Gln Glu Asn His Ile Phe Arg Phe Ala Asn Thr
Asn Trp Asn Trp Arg 175 180 185gtg gat gaa gag aac ggt aat tat gat
tac ctg tta gga tcg aat atc 855Val Asp Glu Glu Asn Gly Asn Tyr Asp
Tyr Leu Leu Gly Ser Asn Ile 190 195 200gac ttt agt cat cca gaa gta
caa gat gag ttg aag gat tgg ggt agc 903Asp Phe Ser His Pro Glu Val
Gln Asp Glu Leu Lys Asp Trp Gly Ser 205 210 215tgg ttt acc gat gag
tta gat ttg gat ggt tat cgt tta gat gct att 951Trp Phe Thr Asp Glu
Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile 220 225 230aaa cat att
cca ttc tgg tat aca tct gat tgg gtt cgg cat cag cgc 999Lys His Ile
Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg235 240
245250aac gaa gca gat caa gat tta ttt gtc gta ggg gaa tat tgg aag
gat 1047Asn Glu Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys
Asp 255 260 265gac gta ggt gct ctc gaa ttt tat tta gat gaa atg aat
tgg gag atg 1095Asp Val Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn
Trp Glu Met 270 275 280tct cta ttc gat gtt cca ctt aat tat aat ttt
tac cgg gct tca caa 1143Ser Leu Phe Asp Val Pro Leu Asn Tyr Asn Phe
Tyr Arg Ala Ser Gln 285 290 295caa ggt gga agc tat gat atg cgt aat
att tta cga gga tct tta gta 1191Gln Gly Gly Ser Tyr Asp Met Arg Asn
Ile Leu Arg Gly Ser Leu Val 300 305 310gaa gcg cat ccg atg cat gca
gtt acg ttt gtt gat aat cat gat act 1239Glu Ala His Pro Met His Ala
Val Thr Phe Val Asp Asn His Asp Thr315 320 325330cag cca ggg gag
tca tta gag tca tgg gtt gct gat tgg ttt aag cca 1287Gln Pro Gly Glu
Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro 335 340 345ctt gct
tat gcg aca att ttg acg cgt gaa ggt ggt tat cca aat gta 1335Leu Ala
Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val 350 355
360ttt tac ggt gat tac tat ggg att cct aac gat aac att tca gct aaa
1383Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys
365 370 375aaa gat atg att gat gag ctg ctt gat gca cgt caa aat tac
gca tat 1431Lys Asp Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr
Ala Tyr 380 385 390ggc acg cag cat gac tat ttt gat cat tgg gat gtt
gta gga tgg act 1479Gly Thr Gln His Asp Tyr Phe Asp His Trp Asp Val
Val Gly Trp Thr395 400 405410agg gaa gga tct tcc tcc aga cct aat
tca ggc ctt gcg act att atg 1527Arg Glu Gly Ser Ser Ser Arg Pro Asn
Ser Gly Leu Ala Thr Ile Met 415 420 425tcg aat gga cct ggt ggt tcc
aag tgg atg tat gta gga cgt cag aat 1575Ser Asn Gly Pro Gly Gly Ser
Lys Trp Met Tyr Val Gly Arg Gln Asn 430 435 440gca gga caa aca tgg
aca gat tta act ggt aat aac gga gcg tcc gtt 1623Ala Gly Gln Thr Trp
Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val 445 450 455aca att aat
ggc gat gga tgg ggc gaa ttc ttt acg aat gga gga tct 1671Thr Ile Asn
Gly
Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser 460 465 470gta tcc
gtg tac gtg aac caa taacaaaaag ccttgagaag ggattcctcc 1722Val Ser
Val Tyr Val Asn Gln475 480ctaactcaag gctttcttta tgt
174527501PRTBacillus species 27Met Arg Arg Trp Val Val Ala Met Leu
Ala Val Leu Phe Leu Phe Pro-20 -15 -10-5Ser Val Val Val Ala Asp Gly
Leu Asn Gly Thr Met Met Gln Tyr Tyr -1 1 5 10Glu Trp His Leu Glu
Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 15 20 25Asp Ala Ala Ala
Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 30 35 40Pro Ala Tyr
Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr 45 50 55Asp Leu
Tyr Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr60 65 70
75Lys Tyr Gly Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys
80 85 90Ser Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His Lys
Met 95 100 105Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val
Asn Pro Thr 110 115 120Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr
Ile Asp Ala Trp Thr 125 130 135Gly Phe Asp Phe Ser Gly Arg Asn Asn
Ala Tyr Ser Asp Phe Lys Trp140 145 150 155Arg Trp Phe His Phe Asn
Gly Val Asp Trp Asp Gln Arg Tyr Gln Glu 160 165 170Asn His Ile Phe
Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val Asp 175 180 185Glu Glu
Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile Asp Phe 190 195
200Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe
205 210 215Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile
Lys His220 225 230 235Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg
His Gln Arg Asn Glu 240 245 250Ala Asp Gln Asp Leu Phe Val Val Gly
Glu Tyr Trp Lys Asp Asp Val 255 260 265Gly Ala Leu Glu Phe Tyr Leu
Asp Glu Met Asn Trp Glu Met Ser Leu 270 275 280Phe Asp Val Pro Leu
Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly 285 290 295Gly Ser Tyr
Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val Glu Ala300 305 310
315His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr Gln Pro
320 325 330Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys Pro
Leu Ala 335 340 345Tyr Ala Thr Ile Leu Thr Arg Glu Gly Gly Tyr Pro
Asn Val Phe Tyr 350 355 360Gly Asp Tyr Tyr Gly Ile Pro Asn Asp Asn
Ile Ser Ala Lys Lys Asp 365 370 375Met Ile Asp Glu Leu Leu Asp Ala
Arg Gln Asn Tyr Ala Tyr Gly Thr380 385 390 395Gln His Asp Tyr Phe
Asp His Trp Asp Val Val Gly Trp Thr Arg Glu 400 405 410Gly Ser Ser
Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn 415 420 425Gly
Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn Ala Gly 430 435
440Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser Val Thr Ile
445 450 455Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser
Val Ser460 465 470 475Val Tyr Val Asn Gln 48028501PRTBacillus
species 28Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu
Phe Pro1 5 10 15Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met
Gln Tyr Tyr 20 25 30Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn
Arg Leu His Asp 35 40 45Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr
Ala Ile Trp Ile Pro 50 55 60Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp
Val Gly Tyr Gly Ala Tyr65 70 75 80Asp Leu Tyr Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr 85 90 95Lys Tyr Gly Thr Lys Ala Gln
Leu Glu Arg Ala Ile Gly Ser Leu Lys 100 105 110Ser Asn Asp Ile Asn
Val Tyr Gly Asp Val Val Met Asn His Lys Met 115 120 125Gly Ala Asp
Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 130 135 140Asn
Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr145 150
155 160Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys
Trp 165 170 175Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln Arg
Tyr Gln Glu 180 185 190Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp
Asn Trp Arg Val Asp 195 200 205Glu Glu Asn Gly Asn Tyr Asp Tyr Leu
Leu Gly Ser Asn Ile Asp Phe 210 215 220Ser His Pro Glu Val Gln Asp
Glu Leu Lys Asp Trp Gly Ser Trp Phe225 230 235 240Thr Asp Glu Leu
Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 245 250 255Ile Pro
Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 260 265
270Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val
275 280 285Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met
Ser Leu 290 295 300Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala
Ser Gln Gln Gly305 310 315 320Gly Ser Tyr Asp Met Arg Asn Ile Leu
Arg Gly Ser Leu Val Glu Ala 325 330 335His Pro Met His Ala Val Thr
Phe Val Asp Asn His Asp Thr Gln Pro 340 345 350Gly Glu Ser Leu Glu
Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 355 360 365Tyr Ala Thr
Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 370 375 380Gly
Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp385 390
395 400Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly
Thr 405 410 415Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp
Thr Arg Glu 420 425 430Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala
Thr Ile Met Ser Asn 435 440 445Gly Pro Gly Gly Ser Lys Trp Met Tyr
Val Gly Arg Gln Asn Ala Gly 450 455 460Gln Thr Trp Thr Asp Leu Thr
Gly Asn Asn Gly Ala Ser Val Thr Ile465 470 475 480Asn Gly Asp Gly
Trp Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 485 490 495Val Tyr
Val Asn Gln 500291920DNABacillus licheniformisCDS(421)..(1872)
29cggaagattg gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg
60gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag
120agattattaa aaagctgaaa gcaaaaggct atcaattggt aactgtatct
cagcttgaag 180aagtgaagaa gcagagaggc tattgaataa atgagtagaa
gcgccatatc ggcgcttttc 240ttttggaaga aaatataggg aaaatggtac
ttgttaaaaa ttcggaatat ttatacaaca 300tcatatgttt cacattgaaa
ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360cgattgctga
cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420gca aat
ctt aat ggg acg ctg atg cag tat ttt gaa tgg tac atg ccc 468Ala Asn
Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro1 5 10 15aat
gac ggc caa cat tgg agg cgt ttg caa aac gac tcg gca tat ttg 516Asn
Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25
30gct gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag gga
564Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly
35 40 45acg agc caa gcg gat gtg ggc tac ggt gct tac gac ctt tat gat
tta 612Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp
Leu 50 55 60ggg gag ttt cat caa aaa ggg acg gtt cgg aca aag tac ggc
aca aaa 660Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly
Thr Lys65 70 75 80gga gag ctg caa tct gcg atc aaa agt ctt cat tcc
cgc gac att aac 708Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser
Arg Asp Ile Asn 85 90 95gtt tac ggg gat gtg gtc atc aac cac aaa ggc
ggc gct gat gcg acc 756Val Tyr Gly Asp Val Val Ile Asn His Lys Gly
Gly Ala Asp Ala Thr 100 105 110gaa gat gta acc gcg gtt gaa gtc gat
ccc gct gac cgc aac cgc gta 804Glu Asp Val Thr Ala Val Glu Val Asp
Pro Ala Asp Arg Asn Arg Val 115 120 125att tca gga gaa cac cta att
aaa gcc tgg aca cat ttt cat ttt ccg 852Ile Ser Gly Glu His Leu Ile
Lys Ala Trp Thr His Phe His Phe Pro 130 135 140ggg cgc ggc agc aca
tac agc gat ttt aaa tgg cat tgg tac cat ttt 900Gly Arg Gly Ser Thr
Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe145 150 155 160gac gga
acc gat tgg gac gag tcc cga aag ctg aac cgc atc tat aag 948Asp Gly
Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170
175ttt caa gga aag gct tgg gat tgg gaa gtt tcc aat gaa aac ggc aac
996Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn
180 185 190tat gat tat ttg atg tat gcc gac atc gat tat gac cat cct
gat gtc 1044Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro
Asp Val 195 200 205gca gca gaa att aag aga tgg ggc act tgg tat gcc
aat gaa ctg caa 1092Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala
Asn Glu Leu Gln 210 215 220ttg gac ggt ttc cgt ctt gat gct gtc aaa
cac att aaa ttt tct ttt 1140Leu Asp Gly Phe Arg Leu Asp Ala Val Lys
His Ile Lys Phe Ser Phe225 230 235 240ttg cgg gat tgg gtt aat cat
gtc agg gaa aaa acg ggg aag gaa atg 1188Leu Arg Asp Trp Val Asn His
Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255ttt acg gta gct gaa
tat tgg cag aat gac ttg ggc gcg ctg gaa aac 1236Phe Thr Val Ala Glu
Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270tat ttg aac
aaa aca aat ttt aat cat tca gtg ttt gac gtg ccg ctt 1284Tyr Leu Asn
Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285cat
tat cag ttc cat gct gca tcg aca cag gga ggc ggc tat gat atg 1332His
Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295
300agg aaa ttg ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg
1380Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys
Ser305 310 315 320gtt aca ttt gtc gat aac cat gat aca cag ccg ggg
caa tcg ctt gag 1428Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335tcg act gtc caa aca tgg ttt aag ccg ctt
gct tac gct ttt att ctc 1476Ser Thr Val Gln Thr Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350aca agg gaa tct gga tac cct cag
gtt ttc tac ggg gat atg tac ggg 1524Thr Arg Glu Ser Gly Tyr Pro Gln
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365acg aaa gga gac tcc cag
cgc gaa att cct gcc ttg aaa cac aaa att 1572Thr Lys Gly Asp Ser Gln
Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380gaa ccg atc tta
aaa gcg aga aaa cag tat gcg tac gga gca cag cat 1620Glu Pro Ile Leu
Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His385 390 395 400gat
tat ttc gac cac cat gac att gtc ggc tgg aca agg gaa ggc gac 1668Asp
Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410
415agc tcg gtt gca aat tca ggt ttg gcg gca tta ata aca gac gga ccc
1716Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro
420 425 430ggt ggg gca aag cga atg tat gtc ggc cgg caa aac gcc ggt
gag aca 1764Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly
Glu Thr 435 440 445tgg cat gac att acc gga aac cgt tcg gag ccg gtt
gtc atc aat tcg 1812Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val
Val Ile Asn Ser 450 455 460gaa ggc tgg gga gag ttt cac gta aac ggc
ggg tcg gtt tca att tat 1860Glu Gly Trp Gly Glu Phe His Val Asn Gly
Gly Ser Val Ser Ile Tyr465 470 475 480gtt caa aga tag aagagcagag
aggacggatt tcctgaagga aatccgtttt 1912Val Gln Argtttatttt
192030483PRTBacillus licheniformis 30Ala Asn Leu Asn Gly Thr Leu
Met Gln Tyr Phe Glu Trp Tyr Met Pro1 5 10 15Asn Asp Gly Gln His Trp
Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30Ala Glu His Gly Ile
Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly 35 40 45Thr Ser Gln Ala
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu 50 55 60 Gly Glu
Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys65 70 75
80Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His Ser Arg Asp Ile Asn
85 90 95Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala
Thr 100 105 110Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg
Asn Arg Val 115 120 125Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr
His Phe His Phe Pro 130 135 140Gly Arg Gly Ser Thr Tyr Ser Asp Phe
Lys Trp His Trp Tyr His Phe145 150 155 160Asp Gly Thr Asp Trp Asp
Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175Phe Gln Gly Lys
Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190Tyr Asp
Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200
205Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln
210 215 220Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe
Ser Phe225 230 235 240Leu Arg Asp Trp Val Asn His Val Arg Glu Lys
Thr Gly Lys Glu Met 245 250 255Phe Thr Val Ala Glu Tyr Trp Gln Asn
Asp Leu Gly Ala Leu Glu Asn 260 265 270Tyr Leu Asn Lys Thr Asn Phe
Asn His Ser Val Phe Asp Val Pro Leu 275 280 285His Tyr Gln Phe His
Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300Arg Lys Leu
Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser305 310 315
320Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu
325 330 335Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe
Ile Leu 340 345 350Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro
Ala Leu Lys His Lys Ile 370 375 380Glu Pro Ile Leu Lys Ala Arg Lys
Gln Tyr Ala Tyr Gly Ala Gln His385 390 395 400Asp Tyr Phe Asp His
His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415Ser Ser Val
Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430Gly
Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440
445Trp His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser
450 455 460Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser
Ile Tyr465 470 475 480Val Gln Arg
* * * * *