U.S. patent application number 09/918543 was filed with the patent office on 2002-10-24 for alpha-amylase mutants with altered properties.
This patent application is currently assigned to Novozymes A/S. Invention is credited to Andersen, Carsten, Fuglsang, Claus Crone, Kjaerulff, Soren, Thisted, Thomas.
Application Number | 20020155574 09/918543 |
Document ID | / |
Family ID | 27576033 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155574 |
Kind Code |
A1 |
Thisted, Thomas ; et
al. |
October 24, 2002 |
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 Ca2+ to the
parent alpha-amylase.
Inventors: |
Thisted, Thomas; (Rungsted
Kyst, DK) ; Kjaerulff, Soren; (Vanlose, DK) ;
Andersen, Carsten; (Vaerloese, 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
Krogshoejvej 36
Bagsvaerd
DK
DK-2880
|
Family ID: |
27576033 |
Appl. No.: |
09/918543 |
Filed: |
July 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
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: |
435/202 ;
435/203; 435/320.1; 435/325; 435/69.1 |
Current CPC
Class: |
C12Y 302/01001 20130101;
C12P 19/02 20130101; C12N 9/2417 20130101; C12P 19/14 20130101;
D06M 16/003 20130101; C12P 7/14 20130101; C11D 3/38618 20130101;
Y02E 50/10 20130101; C11D 3/386 20130101; Y02E 50/17 20130101; C11D
3/38681 20130101 |
Class at
Publication: |
435/202 ;
435/69.1; 435/203; 435/320.1; 435/325 |
International
Class: |
C12N 009/28; C12N
009/30; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 1, 2000 |
DK |
PA 2000 01160 |
Sep 12, 2000 |
DK |
PA 2000 01354 |
Nov 10, 2000 |
DK |
PA 2000 01687 |
Apr 26, 2001 |
DK |
PA 2001 00655 |
Claims
1. A variant of an alpha-amylase having at least 60% homology to
SEQ ID NO.8, comprising an alteration at one or more positions
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 (i) an insertion of an
amino acid downstream of the amino acid which occupies the
position, (ii) a deletion of the amino acid which occupies the
position, or (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 alpha-amylase having the
amino acid sequence shown in SEQ ID NO: 8:
2. The variant of claim 1, which variant has one or more of the
following mutations: 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 using SEQ ID NO: 8 for the numbering.
3. The variant of claim 1 or 2, wherein the variant has the
following mutations: 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; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K2-
37P+S239W+H406W+D430N+N444K+E447Q+Q482K;
D161N+G179N+K180T+A181N+D183N+D20-
0N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+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+D161lN+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+-
E447Q+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D-
430N+E447Q+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S2-
39W+H406W+D430N;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H40-
6W+D430N; H406W+D430N; N444K+E447Q+Q482K; E447Q+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N-
444R+N444K+E447K+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S2-
39W+H406W+D430N+N444R+N444K+E447K+Q482K;
N104D+D161N+G179N+K180T+A181N+D18- 3N+D200N+D204S+K237P+S239W;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237- P+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;
D161N+A181N+D183N+D200N+D204S; K237P+S239W, using SEQ ID NO: 8 for
the numbering.
4. The variant of any of claims 1-3, wherein the parent
alpha-amylase is derived from a strain of B. licheniformis (SEQ ID
NO: 8), B. amyloliquefaciens (SEQ ID NO: 10), or B.
stearothermophilus (SEQ ID NO: 6).
5. The variant of any of claims 1-4, wherein the parent
alpha-amylase is any of: LE174; LE174+G48A+T49I+G107A+I201F;
LE174+M197L; LE174+G48A+T49I+G107A+M197L+I201F.
6. The variant of claim 1, wherein the variant is mutated in one or
more of the following positions: T51I; D62N; N106D; D134A,V,P;
D163N; X172Q; K179R; G184N; K185T; A186N; D188N; D205N; M208Y;
D209S; X212V,E,L,G; L217I, K242P, S244W, N255G,F, N285S, S303Q,
X323M; D387V, N395R; Y404F; H408L,W; X429I; D432N; V442A; X446R,K;
X449Q,K; X484K, using SEQ ID NO: 4 for the numbering.
7. The variant of claim 1 or 6, wherein the variant has the
following mutations: E212V+N285S; D134A+E212V; 255G+H408L+X429I;
E212V+X323M; D62N+E212V+X323M; T51I+D134V+V442A;
T51I+K179R+E212V+Y404F; D387V+N395R; N195F+X212V+K269S, when using
SEQ ID NO: 4 for the numbering.
8. The variant of any of claims 1-7, wherein the parent
alpha-amylase is selected from the group comprising: SEQ ID NO: 2;
SEQ ID NO: 4; SEQ ID NO: 12; SEQ ID NO: 13; or KSM-AP1378.
9. The variant of any of claims 1-8, wherein the parent alpha
amylase is any of: SEQ ID NO. 4+D183*+G184*; SEQ ID NO.
4+D183*+G184*+N195F; SP722+D183*+G184*+M202L; SEQ ID NO.
4+D183*+G184*+N195F+M202L; SEQ ID NO.6+I181*+G182*; SEQ ID
NO.6+I181*+G182*+N193F; SEQ ID NO.6+I181*+G182*+M200L; SEQ ID
NO.6+I181*+G182*+N193F+M200L; SEQ ID NO.12+D183*+G184*; SEQ ID
NO.12+D183*+G184*+N195F; SEQ ID NO.12+D183*+G184*+M202L; SEQ ID
NO.12+D183*+G184*+N195F+M202L.
10. The variant of any of claims 1-9, wherein the parent
alpha-amylase has an amino acid sequence which has a degree of
identity to SEQ ID NO: 8 of at least 70%, more preferably at least
80%, even more preferably at least about 90%, even more preferably
at least 95%, even more preferably at least 97%, and even more
preferably at least 99%.
11. The variant of any of claims 1-10, wherein the parent
alpha-amylase is encoded by a nucleic acid sequence, which
hybridizes under low, preferably medium, preferred high stringency
conditions, with the nucleic acid sequence of SEQ ID NO: 7.
12. The variant of any of claims 1-11, which variant has altered
stability, in particular at high temperatures from 70-120.degree.
C. and/or low pH in the range from pH 4-6.
13. A DNA construct comprising a DNA sequence encoding an
alpha-amylase variant according to any one of claims 1-12.
14. A recombinant expression vector which carries a DNA construct
according to claim 13.
15. A cell which is transformed with a DNA construct according to
claim 13 or a vector according to claim 14.
16. The cell according to claim 15, which is a microorganism,
preferably a bacterium or a fungus.
17. The cell according to claim 16, which cell is a gram-positive
bacterium, such as Bacillus subtilis, Bacillus licheniformis,
Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus,
Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus
coagulans, Bacillus circulans, Bacillus lautus or Bacillus
thuringiensis.
18. A composition comprising an alpha-amylase variant of any of
claims 1-12.
19. The composition of claim 18, further comprising a B.
stearothermophilus alpha-amylase, particular in a ratio of 1:10 to
10:1, preferably 1:2.
20. The composition of claim 18 or 19, wherein the composition
further comprises a glucoamylase, pullulanase and/or a phytase.
21. A detergent composition comprising an alpha-amylase variant
according to any of claims 1-12.
22. A detergent composition of claim 21, which additionally
comprises another enzyme such as a protease, a lipase, a
peroxidase, another amylolytic enzyme, glucoamylase, maltogenic
amylase, CGTase, mannanase, cutinase, laccase and/or a
cellulase.
23. Use of an alpha-amylase variant according to any of claims 1-12
or a composition according to any of claims 18-20 for starch
liquefaction.
24. Use of an alpha-amylase variant according to any of claims 1-12
or a composition according to claims 18-20 for ethanol
production.
25. Use of an alpha-amylase variant according to any one of claims
1-12 or a composition according to claims 18-20 for washing and/or
dishwashing.
26. Use of an alpha-amylase variant of any one of claims 1-12 or a
composition according to claims 18-20 for textile desizing.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims, under 35 U.S.C. 119, priority of
Danish application no. PA 2000 01160, filed Aug. 1, 2000,
application no. PA 2000 01354, filed Sep. 12, 2000, application no.
PA 2000 01687, filed Nov. 10, 2000, and application no. PA 2001
00655, filed on Apr. 26, 2001, and this application claims the
benefit of U.S. provisional application No. 60/225140, filed on
Aug. 14, 2000, application No. 60/233986, filed Sep. 30, 2000,
application No. 60/24104, filed Nov. 16, 2000, application No.
60/286869, filed Apr. 26, 2001, the contents of which are fully
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] 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
[0003] 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
[0004] 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.
[0005] 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
[0010] a deletion of alanine in the same position is shown as:
[0011] Ala30* or A30*
[0012] and insertion of an additional amino acid residue, such as
lysine, is shown as:
[0013] Ala30AlaLys or A30AK
[0014] 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).
[0015] 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:
[0016] *36Asp or *36D
[0017] for insertion of an aspartic acid in position 36. Multiple
mutations are separated by plus signs, i.e.:
[0018] Ala30Asp+Glu34Ser or A30N+E34S representing mutations in
positions 30 and 34 substituting alanine and glutamic acid for
asparagine and serine, respectively.
[0019] When one or more alternative amino acid residues may be
inserted in a given position it is indicated as A30N,E or
[0020] A30N or A30E
[0021] 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:
[0022] R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
[0023] Further, "A30X" means any one of the following
substitutions:
[0024] 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.
[0025] 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:
[0026] "X30N" or "X30N,V" in the case where for instance one or N
or V is present in the wildtype. Thus, it means that other
corresponding parent enzymes are substituted to an "Asn" or "Val"
in position 30.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] 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:
[0028] 1: SEQ ID NO: 4 (SP722)
[0029] 2: SEQ ID NO: 2 (SP690)
[0030] 3: SEQ ID NO: 10 (BAN)
[0031] 4: SEQ ID NO: 8 (BLA)
[0032] 5: SEQ ID NO: 6 (BSG).
DETAILED DISCLOSURE OF THE INVENTION
[0033] 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.
[0034] Termamyl-like Alpha-amylases
[0035] A number of alpha-amylases produced by Bacillus spp. are
highly homologous (identical) on the amino acid level. The identity
of a number of known Bacillus alpha-amylases can be found in the
below Table 1:
1TABLE 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
[0036] 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., Biochemical and Biophysical Research
Communications, 151 (1988), pp. 25-31.
[0037] The KSM AP1378 alpha-amylase is disclosed in WO 97/00324
(from KAO Corporation).
[0038] 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 lo, GC 521 (available from
Genencor) and Ultraphlow (from Enzyme Biosystems).
[0039] 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".
[0040] 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,
i.e., the B. licheniformis alpha-amylase having the amino acid
sequence shown in SEQ ID NO: 8, herein.
[0041] 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).
[0042] Homology
[0043] 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,
p.443-453, to make alignments and to calculate the identity.
[0044] 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, pp. 149-155) and reverse threading (Huber, T;
Torda, AE, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).
[0045] Hybridisation
[0046] The oligonucleotide probe used in the characterisation 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.
[0047] Suitable conditions for testing hybridisation 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 hybridisation 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),
preferred 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 hybridisation
method can be found in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989.
[0048] 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.
[0049] Parent Termamyl-like Alpha-amylases
[0050] According to the invention all Termamy-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.
[0051] Parent Hybrid Termamyl-like Alpha-amylases
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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).
[0057] 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.
[0058] 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.
[0059] In a preferred embodiment the parent Termamyl-like alpha
amylase is LE174, SP722, or AA560 including any of
[0060] LE174+G48A+T49I+G107A+I201F; LE174+M197L;
LE174+G48A+T49I+G107A+M19- 7L+I201F, or 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.
[0061] 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.
[0062] Altered Properties
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] In a preferred embodiment the variant of the invention has
in particular at high temperatures and/or at low pH.
[0072] In an aspect the invention relates to variant with altered
properties as mentioned above.
[0073] 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:
[0074] 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,
[0075] wherein
[0076] (a) the alteration(s) are independently
[0077] (i) an insertion of an amino acid downstream of the amino
acid which occupies the position,
[0078] (ii) a deletion of the amino acid which occupies the
position, or
[0079] (iii) a substitution of the amino acid which occupies the
position with a different amino acid,
[0080] (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.
[0081] In Termamyl.RTM. (SEQ ID NO: 8) such corresponding positions
are:
[0082] 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.
[0083] In SP722 (SEQ ID NO: 4) the corresponding positions are:
[0084] 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.
[0085] Corresponding positions in other parent alpha-amylases can
be found by alignment as described above and shown in the alignment
in FIG. 1.
[0086] 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:
[0087] 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; L427T D430N; V440A; N444R,K; E447Q,K; Q482K.
[0088] 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:
[0089] T51I; D62N; N106D; D134A,V,P; D163N; X172Q; K179R; G184N;
K185T; A186N; D188N; D205N; M208Y; D209S; X212V,E,L,G; L217I,
K242P, S244W, N255G,F, N285S, S303Q, X323M; D387V, N395R; Y404F;
H408L,W; X429I; D432N; V442A; X446R,K; X449Q,K; X484K, using SEQ ID
NO: 4 (SP722) for the numbering.
[0090] Preferred double, triple and multi-mutations--using SEQ ID
NO: 8 as the basis for the numbering--are selected from the group
consisting of:
[0091] T49I+D60N; T49I+D60N+E132A; T49I+D60N+E132V;
T491+D60N+E132V+K170Q; T49I+D60N+E132A+K170Q;
T49I+D60N+E132V+K170Q+K176R; T49I+D60N+E132A+K170Q+K176R;
T49I+D60N+El32V+K170Q+K176R+D207V;
T49I+D60N+E132A+K170Q+K176R+D207V;
T49I+D60N+E132V+K170Q+K176R+D207E;
T49I+D60N+E132A+K170Q+K176R+D207E;
T49I+D60N+E132V+K170OQ+K176R+D207V+E25- 0G;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G;
T49I+D60N+E132V+K170Q+K176R+D- 207E+E250G;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S;
T49I+D60N+E132A+K170Q+K176- R+D207E+E250G+N280S;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S;
T49I+D60N+E132V+K170Q+K176- R+D207V+E250G+N280S+L318M;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L- 318M;
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+D6ON+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+N28OS+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+Q393R;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y40-
2F;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+-
Y402F;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q39-
3R+Y402F;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+385V+Q-
393R+Y402F;
T49I+D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385-
V+Q393R+Y402F+H406L;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q-
374R+E385V+Q393R+Y402F+H406L;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280-
S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
T49I+D60N+E132A+K170Q+K176R+D207E+E-
250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
T49I+D60N+E132V+K170Q+K176-
R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y40-
2F+H406L+L427I;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+-
E385V+Q393R+Y402F+H406L+L427I;
T49I+D60N+E132A+K170Q+K176R+D207E+E250G+N28-
0S+L318M+Q374R+E38V+Q393R+Y402F+H406L+L427I;
T49I+D60N+E132V+K170Q+K176R+D-
207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V440A;
T49I+D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y40-
2F+H406L+L427I+V440A;
T49I+D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+-
Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
T49I+D60N+E132A+K170Q+K176R+D20-
7E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+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+E2- 50G+N280S;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S;
D60N+E132V+K170Q+K176R+D207V+E2- 50G+N280S+L318M;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M;
D60N+E132A+K170Q+K176R+D2- 07E+E250G+N280S+L318M;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q374- R;
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+K17 6R+D207E+E250G+N280S+L318M+Q374R+E385V;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+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+Q373R+E385V+Q393R+Y402F+H4-
06L;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402-
F+H406L;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+-
Y402F+H406L;
D60N+E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q3-
93R+Y402F+H406L;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385-
V+Q393R+Y402F+H406L+L427I;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+-
Q374R+E385V+Q393R+Y402F+H406L+L427I;
D60N+E132V+K170Q+K176R+D207E+E250G+N2-
80S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
D60N+E132A+K170Q+K176R+D207-
E+E250G+N280S+L318M+Q374R+E385R+Q393R+Y402F+H406L+L427I;
D60N+E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H4-
06L+L427I+V440A;
D60N+E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385-
V+Q393R+Y402F+H406L+L427I+V440A;
D60N+E132V+K170Q+K176R+D207E+E250G+N280S+-
L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
D60N+E132A+K170Q+K176R+D2-
07E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+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+Q- 374R+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+Q373R+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+Q373R;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+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+N28DS+L318M+Q374R+E385V+Q393R+Y402F;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+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+Q373R+E385V+Q393R+Y402F+H406L+L-
427I;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H4-
06L+L427I;
E132V+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y40-
2F+H406L+L427I;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393-
R+Y402F+H406L+L427I;
E132V+K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V-
+Q393R+Y402F+H406L+L427I+V440A;
E132A+K170Q+K176R+D207V+E250G+N280S+L318M+-
Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
E132V+K170Q+K176R+D207E+E250G+N-
280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I+V440A;
E132A+K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L-
427I+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+Q373R+385V+Q393R+Y402F+H406L;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L;
K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L427I;
K170Q+K176R+D207V+E250G+N280S+L318M+Q373R+E385V+Q393R+Y402F+H406L+L427I+V-
440A;
K170Q+K176R+D207E+E250G+N280S+L318M+Q374R+E385V+Q393R+Y402F+H406L+L4-
27I+V440A; 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+Q- 393R+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+Q373R+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+Q373R+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+N-
444K+E447Q+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H4-
06W+D430N+N444K+E447Q+Q482K;
D161N+A181N+D183N+D200N+D204S+K237P+S239W+H40-
6W+D430N+N444K+E447Q+Q482K;
D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406-
W+D430N+E447Q+Q482K; N104D+D161N+G179N+K180
T+A181N+D183N+D200N+D204S+K237- P+S239W+H406W+D430N+E447Q+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S-
+K237P+S239W+H406W+D430N+E447Q+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+-
D200N+D204S+K237P+S239W+H406W+D430N;
D161N+G179N+K180T+A181N+D183N+D200N+D-
204S+K237P+S239W+H406W+D430N; H406W+D430N; N444K+E447Q+Q482K;
E447Q+Q482K;
N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N-
444R+N444K+E447K+Q482K;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S2-
39W+H406W+D430N+N444R+N444K+E447K+Q482K;
N104D+D161N+G179N+K180T+A181N+D18- 3N+D200N+D204S+K237P+S239W;
D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237- P+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;
D161N+A181N+D183N+D200N+D204S; K237P+S239W, using SEQ ID NO: 8 for
the numbering.
[0092] 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; T491I+K176R+D207V+Y402F; Q374R+E385V+Q393R;
N190F+A209V+Q264S; G48A+T491I+G107A+I201F; T491I+G107A+I201F;
G48A+T49I+I201F; G48A+T491I+G107A; T49I+I201F; T49I+G107A;
G48A+T49I; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S2-
39W+H406W+D430N+N444K+E447Q+Q482K using SEQ ID NO: 8 for the
numbering. 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.
[0093] Stability
[0094] In the context of the present invention, mutations
(including amino acid substitutionsa and deletion) 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.
[0095] General Mutations in Variants of the Invention
[0096] 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.
[0097] 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.
[0098] 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.
[0099] It is to be understood that the present invention
encompasses variants incorporating two or more of the above
outlined modifications.
[0100] Furthermore, it may be advantageous to introduce mutations
in one or more of the following positions (using SEQ ID NO: 8
(Termamyl) for the numbering):
[0101] M15, V128, A111, H133, W138, T149, M197, N188, A209, A210,
H405, T412, in particular the following single, double or triple or
multi mutations:
[0102] M15X, in particular M15T,L;
[0103] V128X, in particular V128E;
[0104] H133X, in particular H133Y;
[0105] N188X, in particular N188S,T,P;
[0106] M197X, in particular M197T,L;
[0107] A209X, in particular A209V;
[0108] M197T/W138F; M197T/W138Y; M15T/H133Y/N188S;
[0109] M15/V128E/H133Y/N188S; E119C/S130C; D124C/R127C;
H133Y/T149I;
[0110] G475R, H133Y/S187D; H133Y/A209V.
[0111] Methods for Preparing Alpha-amylase Variants of the
Invention
[0112] 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 discribed.
[0113] Cloning a DNA Sequence Encoding an Alpha-amylase
[0114] 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.
[0115] 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.
[0116] Alternatively, the DNA sequence encoding the enzyme may be
prepared synthetically by established standard methods, e.g., the
phosphoroamidite method described by S. L. Beaucage and M. H.
Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the
method described by Matthes et al., The EMBO J. 3, 1984, pp.
801-805. In the phosphoroamidite method, oligonucleotides are
synthesized, e.g., in an automatic DNA synthesizer, purified,
annealed, ligated and cloned in appropriate vectors.
[0117] 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 R. K. Saiki et
al., Science 239, 1988, pp. 487-491.
[0118] Site-directed Mutagenesis
[0119] 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 disclose 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.
[0120] 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.
[0121] 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).
[0122] Expression of Alpha-amylase Variants
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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).
[0131] 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.
[0132] 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.
[0133] 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
gramnegative 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.
[0134] 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.
[0135] 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.
[0136] 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).
[0137] 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.
[0138] Industrial Applications
[0139] 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.
[0140] 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 all references hereby incorporated by reference). Also
contemplated are compositions for starch conversion purposes, which
may beside the variant of the invention also comprise a AMG,
pullulanase, and other alpha-amylases.
[0141] 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.
[0142] 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, EP
119,920 hereby in corporate by reference).
[0143] Detergent Compositions
[0144] 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.
[0145] 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.
[0146] 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.
[0147] Compositions
[0148] 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.
[0149] In another embodient 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), p. 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.
[0150] A specific combination is LE399 and a variant disclosed in
WO 00/04136 or Wo 01/04273, in particular a variant with oe or more
of the following substitutions:
N9A,S56A,V59A,S119P,A246T,N313G,E342T,A393R,S394- R,Y402F,E408R, in
particular a variant with all mutation.
[0151] In an embodiment the composition of the invention also
comprises a pullulanase, in particular a Bacillus pullulanase.
[0152] Materials and Methods
[0153] Enzymes:
[0154] Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 8
and also available from Novozymes.
[0155] AA560: SEQ ID NO: 12; disclosed in WO 00/60060; deposited on
Jan. 25, 1999 at DSMZ and assigned the DSMZ no. 12649. AA560 were
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), Mascheroder Weg lb,
D-38124 Braunschweig Del.
[0156] LB medium (In 1 liter H20: 10 g bacto-tryptone, 5 g
bacto-yeast extract, 10 g NaCl, pH adjusted to 7.0 w. NaOH,
autoclaved).
[0157] TY agar plates (In 1 liter H20: 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).
[0158] 10% Lugol solution (Iodine/Potassium iodine solution; made
by 10-fold dil. in H20 of stock: Sigma Cat. no. L 6146).
[0159] Bacillus subtilis SHA273: see WO 95/10603
[0160] Plasmids
[0161] 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.
[0162] Methods:
[0163] Low pH Filter Assay
[0164] 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.
[0165] 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 ay 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.
[0166] Secondary Screening
[0167] 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
micro liter 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.
[0168] Stability Assay of Unpurified Variants:
[0169] Bacillus cultures expressing the variants to be analysed are
grown for 21 hours at 37.degree. C. in 10 ml LB+chloramphenicol.
800 micro liter culture is mixed with 200 micro 1 citrate buffer,
pH 4.5. A number of 70 micro 1 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 micro 1 to 200 micro 1 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.
[0170] Fermentation and Purification of Alpha-amylase Variants
[0171] A B. subtilis strain harbouring the relevant expression
plasmid is streaked on a LB-agar plate with 10 micro g/ml kanamycin
from -80.degree. C. stock, and grown overnight at 37.degree. C. The
colonies are transferred to 100 ml PS-1 media supplemented with 10
micro g/ml chloamphinicol in a 500 ml shaking flask.
2 Composition of PS-1 medium: Pearl sugar 100 g/l Soy Bean Meal 40
g/l Na2HPO4, 12 H2O 10 g/l PluronicTM PE 6100 0.1 g/l CaCO3 5
g/l
[0172] The culture is shaken at 37.degree. C. at 270 rpm for 5
days.
[0173] 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 0.2M 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 0-0.3M 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.
[0174] Stability Determination of Purified Variants
[0175] All stability trials of purified variants are made using the
same set up. The method is as follows: 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.
[0176] 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.
[0177] Specific Activity Determination
[0178] The specific activity is determined using the Phadebas assay
(Pharmacia) as activity/mg enzyme. The manufactures instructions
are followed (see also below under "Assay for .alpha.-amylase
activity).
[0179] Assays for Alpha-Amylase Activity
[0180] 1. Phadebas Assay
[0181] Alpha-amylase activity is determined by a method employing
Phadebas.RTM. tablets as substrate. Phadebas tablets (Phadebas.RTM.
a 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.
[0182] 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 CaCl2, 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.
[0183] 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 colour will be
produced. The colour 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.
[0184] 2. Alternative Method
[0185] 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 .lambda.=405 nm (400-420
nm). Kits containing PNP-G7 substrate and alpha-Glucosidase is
manufactured by Boehringer-Mannheim (cat. No.1054635).
[0186] 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 micro 1
sample to a 96 well microtitre plate and incubating at 25.degree.
C. 200 micro 1 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.
[0187] 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
[0188] 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.
[0189] Error-prone PCR Mutagenesis and Library Construction
[0190] 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' (SEQID 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 MgCl2, 0.3 mM MnCl2, 0.1 mM
dGTP/dATP, 0.5 mM dTTP/dCTP, and 2.5 units Taq polymerase per 100
micro 1 reaction.
[0191] 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., Biotechniques, 23 (1997), 304-310).
[0192] Screening
[0193] 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.
[0194] Results:
[0195] Increased stability at pH 4.5, 5 ppm calcium incubated at
90.degree. C.
3 Name wt LE488 LE489 7.19.1 8.9.1 Mutations - D207V K170Q E132A
D207E D207V D207V E250G N280S H406L L427I Stability.sup.1) - + + +
+ .sup.1)A "+" indicates significant increase in stability relative
to wild type.
[0196] Increased stability at pH 4.5, 5 ppm calcium incubated at
90.degree. C.
4 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 Stabi- -- + + + + + lity 1) 1) A "+" indicates
significant increase in stability relative to wt.
[0197] Increased stability at pH 4.5, 5 ppm calcium incubated at
90.degree. C.
5 Name wt E132-1 D207-7 D207-6 E250-8 Mutations - E132P D207L D207G
E250F Stability.sup.1) - + + + + .sup.1)A "+" indicates significant
increase in stability relative to wt.
Example 2
[0198] 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.
[0199] Site-directed Mutagenesis
[0200] Mutations from LE493 (K176R+D207V+Y402F) were transferred to
LE399 yielding LE495. This was performed by the overlap PCR method
(Kirchhoff and Desrosiers, PCR Methods and Applications, 2 (1993),
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 AA 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 Y402 multi 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.
[0201] 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.
[0202] Results:
[0203] Stabilization of LE399 variant at pH 4.5, 5 ppm calcium
incubated at 9.degree. C.
6 Name LE399 LE495 LE497 Mutations - K176R K176R (backbone) D207V
Y402F Y402F Stability.sup.1) - + + .sup.1)A "+" indicates
significant increase in stability relative to backbone.
[0204]
Sequence CWU 1
1
30 1 1455 DNA Bacillus sp. CDS (1)..(1455) 1 cat cat aat gga aca
aat ggt act atg atg caa tat ttc gaa tgg tat 48 His His Asn Gly Thr
Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr 1 5 10 15 ttg cca aat
gac ggg aat cat tgg aac agg ttg agg gat gac gca gct 96 Leu Pro Asn
Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30 aac
tta aag agt aaa ggg ata aca gct gta tgg atc cca cct gca tgg 144 Asn
Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp 35 40
45 aag ggg act tcc cag aat gat gta ggt tat gga gcc tat gat tta tat
192 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr
50 55 60 gat ctt gga gag ttt aac cag aag ggg acg gtt cgt aca aaa
tat gga 240 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys
Tyr Gly 65 70 75 80 aca cgc aac cag cta cag gct gcg gtg acc tct tta
aaa aat aac ggc 288 Thr Arg Asn Gln Leu Gln Ala Ala Val Thr Ser Leu
Lys Asn Asn Gly 85 90 95 att cag gta tat ggt gat gtc gtc atg aat
cat aaa ggt gga gca gat 336 Ile Gln Val Tyr Gly Asp Val Val Met Asn
His Lys Gly Gly Ala Asp 100 105 110 ggt acg gaa att gta aat gcg gta
gaa gtg aat cgg agc aac cga aac 384 Gly Thr Glu Ile Val Asn Ala Val
Glu Val Asn Arg Ser Asn Arg Asn 115 120 125 cag gaa acc tca gga gag
tat gca ata gaa gcg tgg aca aag ttt gat 432 Gln Glu Thr Ser Gly Glu
Tyr Ala Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 ttt cct gga aga
gga aat aac cat tcc agc ttt aag tgg cgc tgg tat 480 Phe Pro Gly Arg
Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 cat
ttt gat ggg aca gat tgg gat cag tca cgc cag ctt caa aac aaa 528 His
Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys 165 170
175 ata tat aaa ttc agg gga aca ggc aag gcc tgg gac tgg gaa gtc gat
576 Ile Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp
180 185 190 aca gag aat ggc aac tat gac tat ctt atg tat gca gac gtg
gat atg 624 Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val
Asp Met 195 200 205 gat cac cca gaa gta ata cat gaa ctt aga aac tgg
gga gtg tgg tat 672 Asp His Pro Glu Val Ile His Glu Leu Arg Asn Trp
Gly Val Trp Tyr 210 215 220 acg aat aca ctg aac ctt gat gga ttt aga
ata gat gca gtg aaa cat 720 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg
Ile Asp Ala Val Lys His 225 230 235 240 ata aaa tat agc ttt acg aga
gat tgg ctt aca cat gtg cgt aac acc 768 Ile Lys Tyr Ser Phe Thr Arg
Asp Trp Leu Thr His Val Arg Asn Thr 245 250 255 aca ggt aaa cca atg
ttt gca gtg gct gag ttt tgg aaa aat gac ctt 816 Thr Gly Lys Pro Met
Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 ggt gca att
gaa aac tat ttg aat aaa aca agt tgg aat cac tcg gtg 864 Gly Ala Ile
Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285 ttt
gat gtt cct ctc cac tat aat ttg tac aat gca tct aat agc ggt 912 Phe
Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295
300 ggt tat tat gat atg aga aat att tta aat ggt tct gtg gtg caa aaa
960 Gly Tyr Tyr Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys
305 310 315 320 cat cca aca cat gcc gtt act ttt gtt gat aac cat gat
tct cag ccc 1008 His Pro Thr His Ala Val Thr Phe Val Asp Asn His
Asp Ser Gln Pro 325 330 335 ggg gaa gca ttg gaa tcc ttt gtt caa caa
tgg ttt aaa cca ctt gca 1056 Gly Glu Ala Leu Glu Ser Phe Val Gln
Gln Trp Phe Lys Pro Leu Ala 340 345 350 tat gca ttg gtt ctg aca agg
gaa caa ggt tat cct tcc gta ttt tat 1104 Tyr Ala Leu Val Leu Thr
Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365 ggg gat tac tac
ggt atc cca acc cat ggt gtt ccg gct atg aaa tct 1152 Gly Asp Tyr
Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380 aaa
ata gac cct ctt ctg cag gca cgt caa act ttt gcc tat ggt acg 1200
Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala Tyr Gly Thr 385
390 395 400 cag cat gat tac ttt gat cat cat gat att atc ggt tgg aca
aga gag 1248 Gln His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp
Thr Arg Glu 405 410 415 gga aat agc tcc cat cca aat tca ggc ctt gcc
acc att atg tca gat 1296 Gly Asn Ser Ser His Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asp 420 425 430 ggt cca ggt ggt aac aaa tgg atg
tat gtg ggg aaa aat aaa gcg gga 1344 Gly Pro Gly Gly Asn Lys Trp
Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 caa gtt tgg aga gat
att acc gga aat agg aca ggc acc gtc aca att 1392 Gln Val Trp Arg
Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 aat gca
gac gga tgg ggt aat ttc tct gtt aat gga ggg tcc gtt tcg 1440 Asn
Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470
475 480 gtt tgg gtg aag caa 1455 Val Trp Val Lys Gln 485 2 485 PRT
Bacillus sp. 2 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe
Glu Trp Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu
Arg Asp Asp Ala Ala 20 25 30 Asn Leu Lys Ser Lys Gly Ile Thr Ala
Val Trp Ile Pro Pro Ala Trp 35 40 45 Lys Gly Thr Ser Gln Asn Asp
Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn
Gln Leu Gln Ala Ala Val Thr Ser Leu Lys Asn Asn Gly 85 90 95 Ile
Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105
110 Gly Thr Glu Ile Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn
115 120 125 Gln Glu Thr Ser Gly Glu Tyr Ala Ile Glu Ala Trp Thr Lys
Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys
Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Thr Asp Trp Asp Gln
Ser Arg Gln Leu Gln Asn Lys 165 170 175 Ile Tyr Lys Phe Arg Gly Thr
Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn
Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205 Asp His Pro
Glu Val Ile His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr
Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230
235 240 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn
Thr 245 250 255 Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys
Asn Asp Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser
Trp Asn His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu
Tyr Asn Ala Ser Asn Ser Gly 290 295 300 Gly Tyr Tyr Asp Met Arg Asn
Ile Leu Asn Gly Ser Val Val Gln Lys 305 310 315 320 His Pro Thr His
Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Gly Glu
Ala Leu Glu Ser Phe Val Gln Gln Trp Phe Lys Pro Leu Ala 340 345 350
Tyr Ala Leu Val Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355
360 365 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys
Ser 370 375 380 Lys Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Phe Ala
Tyr Gly Thr 385 390 395 400 Gln His Asp Tyr Phe Asp His His Asp Ile
Ile Gly Trp Thr Arg Glu 405 410 415 Gly Asn Ser Ser His Pro Asn Ser
Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 Gly Pro Gly Gly Asn Lys
Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445 Gln Val Trp Arg
Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 450 455 460 Asn Ala
Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475
480 Val Trp Val Lys Gln 485 3 1455 DNA Bacillus sp. CDS (1)..(1455)
3 cat cat aat ggg aca aat ggg acg atg atg caa tac ttt gaa tgg cac
48 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His
1 5 10 15 ttg cct aat gat ggg aat cac tgg aat aga tta aga gat gat
gct agt 96 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp
Ala Ser 20 25 30 aat cta aga aat aga ggt ata acc gct att tgg att
ccg cct gcc tgg 144 Asn Leu Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile
Pro Pro Ala Trp 35 40 45 aaa ggg act tcg caa aat gat gtg ggg tat
gga gcc tat gat ctt tat 192 Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr
Gly Ala Tyr Asp Leu Tyr 50 55 60 gat tta ggg gaa ttt aat caa aag
ggg acg gtt cgt act aag tat ggg 240 Asp Leu Gly Glu Phe Asn Gln Lys
Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80 aca cgt agt caa ttg gag
tct gcc atc cat gct tta aag aat aat ggc 288 Thr Arg Ser Gln Leu Glu
Ser Ala Ile His Ala Leu Lys Asn Asn Gly 85 90 95 gtt caa gtt tat
ggg gat gta gtg atg aac cat aaa gga gga gct gat 336 Val Gln Val Tyr
Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 gct aca
gaa aac gtt ctt gct gtc gag gtg aat cca aat aac cgg aat 384 Ala 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 432
Gln Glu Ile Ser Gly Asp Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp 130
135 140 ttt cca ggg agg ggt aat aca tac tca gac ttt aaa tgg cgt tgg
tat 480 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp
Tyr 145 150 155 160 cat ttc gat ggt gta gat tgg gat caa tca cga caa
ttc caa aat cgt 528 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg Gln
Phe Gln Asn Arg 165 170 175 atc tac aaa ttc cga ggt gat ggt aag gca
tgg gat tgg gaa gta gat 576 Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala
Trp Asp Trp Glu Val Asp 180 185 190 tcg gaa aat gga aat tat gat tat
tta atg tat gca gat gta gat atg 624 Ser 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 672 Asp His Pro Glu Val Val
Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215 220 aca aat aca tta
aat ctt gat gga ttt agg atc gat gcg gtg aag cat 720 Thr Asn Thr Leu
Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240 att
aaa tat agc ttt aca cgt gat tgg ttg acc cat gta aga aac gca 768 Ile
Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala 245 250
255 acg gga aaa gaa atg ttt gct gtt gct gaa ttt tgg aaa aat gat tta
816 Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu
260 265 270 ggt gcc ttg gag aac tat tta aat aaa aca aac tgg aat cat
tct gtc 864 Gly 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 912 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala
Ser Asn Ser Gly 290 295 300 ggc aac tat gac atg gca aaa ctt ctt aat
gga acg gtt gtt caa aag 960 Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn
Gly Thr Val Val Gln Lys 305 310 315 320 cat cca atg cat gcc gta act
ttt gtg gat aat cac gat tct caa cct 1008 His Pro Met His Ala Val
Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 ggg gaa tca tta
gaa tca ttt gta caa gaa tgg ttt aag cca ctt gct 1056 Gly Glu Ser
Leu Glu Ser Phe Val Gln Glu Trp Phe Lys Pro Leu Ala 340 345 350 tat
gcg ctt att tta aca aga gaa caa ggc tat ccc tct gtc ttc tat 1104
Tyr 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 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His Ser Val Pro Ala Met
Lys Ala 370 375 380 aag att gat cca atc tta gag gcg cgt caa aat ttt
gca tat gga aca 1200 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Asn
Phe Ala Tyr Gly Thr 385 390 395 400 caa cat gat tat ttt gac cat cat
aat ata atc gga tgg aca cgt gaa 1248 Gln His Asp Tyr Phe Asp His
His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415 gga aat acc acg cat
ccc aat tca gga ctt gcg act atc atg tcg gat 1296 Gly Asn Thr Thr
His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420 425 430 ggg cca
ggg gga gag aaa tgg atg tac gta ggg caa aat aaa gca ggt 1344 Gly
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
1392 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr Val Thr
Ile 450 455 460 aat gca gat gga tgg gct aat ttt tca gta aat gga gga
tct gtt tcc 1440 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly
Gly Ser Val Ser 465 470 475 480 att tgg gtg aaa cga 1455 Ile Trp
Val Lys Arg 485 4 485 PRT Bacillus sp. 4 His His Asn Gly Thr Asn
Gly Thr Met Met Gln Tyr Phe Glu Trp His 1 5 10 15 Leu Pro Asn Asp
Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30 Asn Leu
Arg Asn Arg Gly Ile Thr Ala Ile Trp Ile Pro Pro Ala Trp 35 40 45
Lys Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50
55 60 Asp Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly 65 70 75 80 Thr Arg Ser Gln Leu Glu Ser Ala Ile His Ala Leu Lys
Asn Asn Gly 85 90 95 Val Gln Val Tyr Gly Asp Val Val Met Asn His
Lys Gly Gly Ala Asp 100 105 110 Ala Thr Glu Asn Val Leu Ala Val Glu
Val Asn Pro Asn Asn Arg Asn 115 120 125 Gln Glu Ile Ser Gly Asp Tyr
Thr Ile Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly
Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe
Asp Gly Val Asp Trp Asp Gln Ser Arg Gln Phe Gln Asn Arg 165 170 175
Ile Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180
185 190 Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp
Met 195 200 205 Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly
Glu Trp Tyr 210 215 220 Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile
Asp Ala Val Lys His 225 230 235 240 Ile Lys Tyr Ser Phe Thr Arg Asp
Trp Leu Thr His Val Arg Asn Ala 245 250 255 Thr Gly Lys Glu Met Phe
Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270 Gly Ala Leu Glu
Asn Tyr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285 Phe Asp
Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300
Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gln Lys 305
310 315 320 His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser
Gln Pro 325 330 335 Gly Glu Ser Leu Glu Ser Phe Val Gln Glu Trp Phe
Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Ile Leu Thr Arg Glu Gln Gly
Tyr Pro Ser Val Phe Tyr 355 360 365 Gly Asp Tyr Tyr Gly Ile Pro
Thr His Ser Val Pro Ala Met Lys Ala 370 375 380 Lys Ile Asp Pro Ile
Leu Glu Ala Arg Gln Asn Phe Ala Tyr Gly Thr 385 390 395 400 Gln His
Asp Tyr Phe Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405 410 415
Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 420
425 430 Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gln Asn Lys Ala
Gly 435 440 445 Gln Val Trp His Asp Ile Thr Gly Asn Lys Pro Gly Thr
Val Thr Ile 450 455 460 Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn
Gly Gly Ser Val Ser 465 470 475 480 Ile Trp Val Lys Arg 485 5 1548
DNA Bacillus stearothermophilus CDS (1)..(1548) 5 gcc gca ccg ttt
aac ggc acc atg atg cag tat ttt gaa tgg tac ttg 48 Ala Ala Pro Phe
Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 ccg gat
gat ggc acg tta tgg acc aaa gtg gcc aat gaa gcc aac aac 96 Pro Asp
Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30
tta tcc agc ctt ggc atc acc gct ctt tgg ctg ccg ccc gct tac aaa 144
Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35
40 45 gga aca agc cgc agc gac gta ggg tac gga gta tac gac ttg tat
gac 192 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr
Asp 50 55 60 ctc ggc gaa ttc aat caa aaa ggg acc gtc cgc aca aaa
tac gga aca 240 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys
Tyr Gly Thr 65 70 75 80 aaa gct caa tat ctt caa gcc att caa gcc gcc
cac gcc gct gga atg 288 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala Ala
His Ala Ala Gly Met 85 90 95 caa gtg tac gcc gat gtc gtg ttc gac
cat aaa ggc ggc gct gac ggc 336 Gln Val Tyr Ala Asp Val Val Phe Asp
His Lys Gly Gly Ala Asp Gly 100 105 110 acg gaa tgg gtg gac gcc gtc
gaa gtc aat ccg tcc gac cgc aac caa 384 Thr Glu Trp Val Asp Ala Val
Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 gaa atc tcg ggc acc
tat caa atc caa gca tgg acg aaa ttt gat ttt 432 Glu Ile Ser Gly Thr
Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 ccc ggg cgg
ggc aac acc tac tcc agc ttt aag tgg cgc tgg tac cat 480 Pro Gly Arg
Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160
ttt gac ggc gtt gat tgg gac gaa agc cga aaa ttg agc cgc att tac 528
Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr 165
170 175 aaa ttc cgc ggc atc ggc aaa gcg tgg gat tgg gaa gta gac acg
gaa 576 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp Thr
Glu 180 185 190 aac gga aac tat gac tac tta atg tat gcc gac ctt gat
atg gat cat 624 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp
Met Asp His 195 200 205 ccc gaa gtc gtg acc gag ctg aaa aac tgg ggg
aaa tgg tat gtc aac 672 Pro Glu Val Val Thr Glu Leu Lys Asn Trp Gly
Lys Trp Tyr Val Asn 210 215 220 aca acg aac att gat ggg ttc cgg ctt
gat gcc gtc aag cat att aag 720 Thr Thr Asn Ile Asp Gly Phe Arg Leu
Asp Ala Val Lys His Ile Lys 225 230 235 240 ttc agt ttt ttt cct gat
tgg ttg tcg tat gtg cgt tct cag act ggc 768 Phe Ser Phe Phe Pro Asp
Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly 245 250 255 aag ccg cta ttt
acc gtc ggg gaa tat tgg agc tat gac atc aac aag 816 Lys Pro Leu Phe
Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 ttg cac
aat tac att acg aaa aca gac gga acg atg tct ttg ttt gat 864 Leu His
Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp 275 280 285
gcc ccg tta cac aac aaa ttt tat acc gct tcc aaa tca ggg ggc gca 912
Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala 290
295 300 ttt gat atg cgc acg tta atg acc aat act ctc atg aaa gat caa
ccg 960 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gln
Pro 305 310 315 320 aca ttg gcc gtc acc ttc gtt gat aat cat gac acc
gaa ccc ggc caa 1008 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp
Thr Glu Pro Gly Gln 325 330 335 gcg ctg cag tca tgg gtc gac cca tgg
ttc aaa ccg ttg gct tac gcc 1056 Ala Leu Gln Ser Trp Val Asp Pro
Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350 ttt att cta act cgg cag
gaa gga tac ccg tgc gtc ttt tat ggt gac 1104 Phe Ile Leu Thr Arg
Gln Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 tat tat ggc
att cca caa tat aac att cct tcg ctg aaa agc aaa atc 1152 Tyr Tyr
Gly Ile Pro Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380
gat ccg ctc ctc atc gcg cgc agg gat tat gct tac gga acg caa cat
1200 Asp Pro Leu Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln
His 385 390 395 400 gat tat ctt gat cac tcc gac atc atc ggg tgg aca
agg gaa ggg ggc 1248 Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp
Thr Arg Glu Gly Gly 405 410 415 act gaa aaa cca gga tcc gga ctg gcc
gca ctg atc acc gat ggg ccg 1296 Thr Glu Lys Pro Gly Ser Gly Leu
Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 gga gga agc aaa tgg atg
tac gtt ggc aaa caa cac gct gga aaa gtg 1344 Gly Gly Ser Lys Trp
Met Tyr Val Gly Lys Gln His Ala Gly Lys Val 435 440 445 ttc tat gac
ctt acc ggc aac cgg agt gac acc gtc acc atc aac agt 1392 Phe Tyr
Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr Ile Asn Ser 450 455 460
gat gga tgg ggg gaa ttc aaa gtc aat ggc ggt tcg gtt tcg gtt tgg
1440 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val
Trp 465 470 475 480 gtt cct aga aaa acg acc gtt tct acc atc gct cgg
ccg atc aca acc 1488 Val Pro Arg Lys Thr Thr Val Ser Thr Ile Ala
Arg Pro Ile Thr Thr 485 490 495 cga ccg tgg act ggt gaa ttc gtc cgt
tgg acc gaa cca cgg ttg gtg 1536 Arg Pro Trp Thr Gly Glu Phe Val
Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 gca tgg cct tga 1548
Ala Trp Pro 515 6 515 PRT Bacillus stearothermophilus 6 Ala Ala Pro
Phe Asn Gly Thr Met Met Gln Tyr Phe Glu Trp Tyr Leu 1 5 10 15 Pro
Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25
30 Leu Ser Ser Leu Gly Ile Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys
35 40 45 Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu
Tyr Asp 50 55 60 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr
Lys Tyr Gly Thr 65 70 75 80 Lys Ala Gln Tyr Leu Gln Ala Ile Gln Ala
Ala His Ala Ala Gly Met 85 90 95 Gln Val Tyr Ala Asp Val Val Phe
Asp His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Trp Val Asp Ala
Val Glu Val Asn Pro Ser Asp Arg Asn Gln 115 120 125 Glu Ile Ser Gly
Thr Tyr Gln Ile Gln Ala Trp Thr Lys Phe Asp Phe 130 135 140 Pro Gly
Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155
160 Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg Ile Tyr
165 170 175 Lys Phe Arg Gly Ile Gly Lys Ala Trp Asp Trp Glu Val Asp
Thr Glu 180 185 190 Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu
Asp Met Asp His 195 200 205 Pro Glu Val Val Thr Glu Leu Lys Asn Trp
Gly Lys Trp Tyr Val Asn 210 215 220 Thr Thr Asn Ile Asp Gly Phe Arg
Leu Asp Ala Val Lys His Ile Lys 225 230 235 240 Phe Ser Phe Phe Pro
Asp Trp Leu Ser Tyr Val Arg Ser Gln Thr Gly 245 250 255 Lys Pro Leu
Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp Ile Asn Lys 260 265 270 Leu
His Asn Tyr Ile Thr Lys Thr Asp Gly Thr Met Ser Leu Phe Asp 275 280
285 Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Ala
290 295 300 Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp
Gln Pro 305 310 315 320 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp
Thr Glu Pro Gly Gln 325 330 335 Ala Leu Gln Ser Trp Val Asp Pro Trp
Phe Lys Pro Leu Ala Tyr Ala 340 345 350 Phe Ile Leu Thr Arg Gln Glu
Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 360 365 Tyr Tyr Gly Ile Pro
Gln Tyr Asn Ile Pro Ser Leu Lys Ser Lys Ile 370 375 380 Asp Pro Leu
Leu Ile Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gln His 385 390 395 400
Asp Tyr Leu Asp His Ser Asp Ile Ile Gly Trp Thr Arg Glu Gly Gly 405
410 415 Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
Pro 420 425 430 Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gln His Ala
Gly Lys Val 435 440 445 Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr
Val Thr Ile Asn Ser 450 455 460 Asp Gly Trp Gly Glu Phe Lys Val Asn
Gly Gly Ser Val Ser Val Trp 465 470 475 480 Val Pro Arg Lys Thr Thr
Val Ser Thr Ile Ala Arg Pro Ile Thr Thr 485 490 495 Arg Pro Trp Thr
Gly Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp
Pro 515 7 1920 DNA Bacillus licheniformis CDS (421)..(1872) 7
cggaagattg gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg
60 gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat
gctgctgaag 120 agattattaa aaagctgaaa gcaaaaggct atcaattggt
aactgtatct cagcttgaag 180 aagtgaagaa gcagagaggc tattgaataa
atgagtagaa gcgccatatc ggcgcttttc 240 ttttggaaga aaatataggg
aaaatggtac ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt
cacattgaaa ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360
cgattgctga cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg
420 gca aat ctt aat ggg acg ctg atg cag tat ttt gaa tgg tac atg ccc
468 Ala Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro
1 5 10 15 aat gac ggc caa cat tgg agg cgt ttg caa aac gac tcg gca
tat ttg 516 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala
Tyr Leu 20 25 30 gct gaa cac ggt att act gcc gtc tgg att ccc ccg
gca tat aag gga 564 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro
Ala Tyr Lys Gly 35 40 45 acg agc caa gcg gat gtg ggc tac ggt gct
tac gac ctt tat gat tta 612 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala
Tyr Asp Leu Tyr Asp Leu 50 55 60 ggg gag ttt cat caa aaa ggg acg
gtt cgg aca aag tac ggc aca aaa 660 Gly Glu Phe His Gln Lys Gly Thr
Val Arg Thr Lys Tyr Gly Thr Lys 65 70 75 80 gga gag ctg caa tct gcg
atc aaa agt ctt cat tcc cgc gac att aac 708 Gly 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 756 Val Tyr Gly Asp
Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 gaa gat
gta acc gcg gtt gaa gtc gat ccc gct gac cgc aac cgc gta 804 Glu Asp
Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125
att tca gga gaa cac cta att aaa gcc tgg aca cat ttt cat ttt ccg 852
Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130
135 140 ggg cgc ggc agc aca tac agc gat ttt aaa tgg cat tgg tac cat
ttt 900 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His
Phe 145 150 155 160 gac gga acc gat tgg gac gag tcc cga aag ctg aac
cgc atc tat aag 948 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn
Arg Ile Tyr Lys 165 170 175 ttt caa gga aag gct tgg gat tgg gaa gtt
tcc aat gaa aac ggc aac 996 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val
Ser Asn Glu Asn Gly Asn 180 185 190 tat gat tat ttg atg tat gcc gac
atc gat tat gac cat cct gat gtc 1044 Tyr Asp Tyr Leu Met Tyr Ala
Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 gca gca gaa att aag
aga tgg ggc act tgg tat gcc aat gaa ctg caa 1092 Ala Ala Glu Ile
Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220 ttg gac
ggt ttc cgt ctt gat gct gtc aaa cac att aaa ttt tct ttt 1140 Leu
Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230
235 240 ttg cgg gat tgg gtt aat cat gtc agg gaa aaa acg ggg aag gaa
atg 1188 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys
Glu Met 245 250 255 ttt acg gta gct gaa tat tgg cag aat gac ttg ggc
gcg ctg gaa aac 1236 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu
Gly Ala Leu Glu Asn 260 265 270 tat ttg aac aaa aca aat ttt aat cat
tca gtg ttt gac gtg ccg ctt 1284 Tyr Leu Asn Lys Thr Asn Phe Asn
His Ser Val Phe Asp Val Pro Leu 275 280 285 cat tat cag ttc cat gct
gca tcg aca cag gga ggc ggc tat gat atg 1332 His Tyr Gln Phe His
Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300 agg aaa ttg
ctg aac ggt acg gtc gtt tcc aag cat ccg ttg aaa tcg 1380 Arg Lys
Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315
320 gtt aca ttt gtc gat aac cat gat aca cag ccg ggg caa tcg ctt gag
1428 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu
Glu 325 330 335 tcg act gtc caa aca tgg ttt aag ccg ctt gct tac gct
ttt att ctc 1476 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr
Ala Phe Ile Leu 340 345 350 aca agg gaa tct gga tac cct cag gtt ttc
tac ggg gat atg tac ggg 1524 Thr Arg Glu Ser Gly Tyr Pro Gln Val
Phe Tyr Gly Asp Met Tyr Gly 355 360 365 acg aaa gga gac tcc cag cgc
gaa att cct gcc ttg aaa cac aaa att 1572 Thr Lys Gly Asp Ser Gln
Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 gaa ccg atc tta
aaa gcg aga aaa cag tat gcg tac gga gca cag cat 1620 Glu Pro Ile
Leu Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400
gat tat ttc gac cac cat gac att gtc ggc tgg aca agg gaa ggc gac
1668 Asp Tyr Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly
Asp 405 410 415 agc tcg gtt gca aat tca ggt ttg gcg gca tta ata aca
gac gga ccc 1716 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile
Thr Asp Gly Pro 420 425 430 ggt ggg gca aag cga atg tat gtc ggc cgg
caa aac gcc ggt gag aca 1764 Gly Gly Ala Lys Arg Met Tyr Val Gly
Arg Gln Asn Ala Gly Glu Thr 435 440 445 tgg cat gac att acc gga aac
cgt tcg gag ccg gtt gtc atc aat tcg 1812 Trp His Asp Ile Thr Gly
Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460 gaa ggc tgg gga
gag ttt cac gta aac ggc ggg tcg gtt tca att tat 1860 Glu Gly Trp
Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480
gtt caa aga tag aagagcagag aggacggatt tcctgaagga aatccgtttt 1912
Val Gln Arg tttatttt 1920 8 483 PRT Bacillus licheniformis 8 Ala
Asn Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10
15 Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu
20 25 30 Ala 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 60 Gly Glu Phe His Gln Lys Gly Thr Val Arg
Thr Lys Tyr Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile Lys
Ser Leu His Ser Arg Asp Ile Asn 85 90
95 Val Tyr Gly Asp Val Val Ile Asn His Lys Gly Gly Ala Asp Ala Thr
100 105 110 Glu Asp Val Thr Ala Val Glu Val Asp Pro Ala Asp Arg Asn
Arg Val 115 120 125 Ile Ser Gly Glu His Leu Ile Lys Ala Trp Thr His
Phe His Phe Pro 130 135 140 Gly Arg Gly Ser Thr Tyr Ser Asp Phe Lys
Trp His Trp Tyr His Phe 145 150 155 160 Asp Gly Thr Asp Trp Asp Glu
Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165 170 175 Phe Gln Gly Lys Ala
Trp Asp Trp Glu Val Ser Asn Glu Asn Gly Asn 180 185 190 Tyr Asp Tyr
Leu Met Tyr Ala Asp Ile Asp Tyr Asp His Pro Asp Val 195 200 205 Ala
Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215
220 Leu Asp Gly Phe Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe
225 230 235 240 Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly
Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu
Gly Ala Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr Asn Phe Asn His
Ser Val Phe Asp Val Pro Leu 275 280 285 His Tyr Gln Phe His Ala Ala
Ser Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg Lys Leu Leu Asn
Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 Val Thr
Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330 335
Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile Leu 340
345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly Asp Met Tyr
Gly 355 360 365 Thr Lys Gly Asp Ser Gln Arg Glu Ile Pro Ala Leu Lys
His Lys Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg Lys Gln Tyr Ala
Tyr Gly Ala Gln His 385 390 395 400 Asp Tyr Phe Asp His His Asp Ile
Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn Ser
Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ala Lys
Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 Trp His
Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455 460
Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr 465
470 475 480 Val Gln Arg 9 2084 DNA Bacillus amyloliquefaciens CDS
(343)..(1794) 9 gccccgcaca tacgaaaaga ctggctgaaa acattgagcc
tttgatgact gatgatttgg 60 ctgaagaagt ggatcgattg tttgagaaaa
gaagaagacc ataaaaatac cttgtctgtc 120 atcagacagg gtatttttta
tgctgtccag actgtccgct gtgtaaaaat aaggaataaa 180 ggggggttgt
tattatttta ctgatatgta aaatataatt tgtataagaa aatgagaggg 240
agaggaaaca tgattcaaaa acgaaagcgg acagtttcgt tcagacttgt gcttatgtgc
300 acgctgttat ttgtcagttt gccgattaca aaaacatcag cc gta aat ggc acg
354 Val Asn Gly Thr 1 ctg atg cag tat ttt gaa tgg tat acg ccg aac
gac ggc cag cat tgg 402 Leu Met Gln Tyr Phe Glu Trp Tyr Thr Pro Asn
Asp Gly Gln His Trp 5 10 15 20 aaa cga ttg cag aat gat gcg gaa cat
tta tcg gat atc gga atc act 450 Lys Arg Leu Gln Asn Asp Ala Glu His
Leu Ser Asp Ile Gly Ile Thr 25 30 35 gcc gtc tgg att cct ccc gca
tac aaa gga ttg agc caa tcc gat aac 498 Ala Val Trp Ile Pro Pro Ala
Tyr Lys Gly Leu Ser Gln Ser Asp Asn 40 45 50 gga tac gga cct tat
gat ttg tat gat tta gga gaa ttc cag caa aaa 546 Gly Tyr Gly Pro Tyr
Asp Leu Tyr Asp Leu Gly Glu Phe Gln Gln Lys 55 60 65 ggg acg gtc
aga acg aaa tac ggc aca aaa tca gag ctt caa gat gcg 594 Gly Thr Val
Arg Thr Lys Tyr Gly Thr Lys Ser Glu Leu Gln Asp Ala 70 75 80 atc
ggc tca ctg cat tcc cgg aac gtc caa gta tac gga gat gtg gtt 642 Ile
Gly Ser Leu His Ser Arg Asn Val Gln Val Tyr Gly Asp Val Val 85 90
95 100 ttg aat cat aag gct ggt gct gat gca aca gaa gat gta act gcc
gtc 690 Leu Asn His Lys Ala Gly Ala Asp Ala Thr Glu Asp Val Thr Ala
Val 105 110 115 gaa gtc aat ccg gcc aat aga aat cag gaa act tcg gag
gaa tat caa 738 Glu Val Asn Pro Ala Asn Arg Asn Gln Glu Thr Ser Glu
Glu Tyr Gln 120 125 130 atc aaa gcg tgg acg gat ttt cgt ttt ccg ggc
cgt gga aac acg tac 786 Ile Lys Ala Trp Thr Asp Phe Arg Phe Pro Gly
Arg Gly Asn Thr Tyr 135 140 145 agt gat ttt aaa tgg cat tgg tat cat
ttc gac gga gcg gac tgg gat 834 Ser Asp Phe Lys Trp His Trp Tyr His
Phe Asp Gly Ala Asp Trp Asp 150 155 160 gaa tcc cgg aag atc agc cgc
atc ttt aag ttt cgt ggg gaa gga aaa 882 Glu Ser Arg Lys Ile Ser Arg
Ile Phe Lys Phe Arg Gly Glu Gly Lys 165 170 175 180 gcg tgg gat tgg
gaa gta tca agt gaa aac ggc aac tat gac tat tta 930 Ala Trp Asp Trp
Glu Val Ser Ser Glu Asn Gly Asn Tyr Asp Tyr Leu 185 190 195 atg tat
gct gat gtt gac tac gac cac cct gat gtc gtg gca gag aca 978 Met Tyr
Ala Asp Val Asp Tyr Asp His Pro Asp Val Val Ala Glu Thr 200 205 210
aaa aaa tgg ggt atc tgg tat gcg aat gaa ctg tca tta gac ggc ttc
1026 Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu Leu Ser Leu Asp Gly
Phe 215 220 225 cgt att gat gcc gcc aaa cat att aaa ttt tca ttt ctg
cgt gat tgg 1074 Arg Ile Asp Ala Ala Lys His Ile Lys Phe Ser Phe
Leu Arg Asp Trp 230 235 240 gtt cag gcg gtc aga cag gcg acg gga aaa
gaa atg ttt acg gtt gcg 1122 Val Gln Ala Val Arg Gln Ala Thr Gly
Lys Glu Met Phe Thr Val Ala 245 250 255 260 gag tat tgg cag aat aat
gcc ggg aaa ctc gaa aac tac ttg aat aaa 1170 Glu Tyr Trp Gln Asn
Asn Ala Gly Lys Leu Glu Asn Tyr Leu Asn Lys 265 270 275 aca agc ttt
aat caa tcc gtg ttt gat gtt ccg ctt cat ttc aat tta 1218 Thr Ser
Phe Asn Gln Ser Val Phe Asp Val Pro Leu His Phe Asn Leu 280 285 290
cag gcg gct tcc tca caa gga ggc gga tat gat atg agg cgt ttg ctg
1266 Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met Arg Arg Leu
Leu 295 300 305 gac ggt acc gtt gtg tcc agg cat ccg gaa aag gcg gtt
aca ttt gtt 1314 Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala
Val Thr Phe Val 310 315 320 gaa aat cat gac aca cag ccg gga cag tca
ttg gaa tcg aca gtc caa 1362 Glu Asn His Asp Thr Gln Pro Gly Gln
Ser Leu Glu Ser Thr Val Gln 325 330 335 340 act tgg ttt aaa ccg ctt
gca tac gcc ttt att ttg aca aga gaa tcc 1410 Thr Trp Phe Lys Pro
Leu Ala Tyr Ala Phe Ile Leu Thr Arg Glu Ser 345 350 355 ggt tat cct
cag gtg ttc tat ggg gat atg tac ggg aca aaa ggg aca 1458 Gly Tyr
Pro Gln Val Phe Tyr Gly Asp Met Tyr Gly Thr Lys Gly Thr 360 365 370
tcg cca aag gaa att ccc tca ctg aaa gat aat ata gag ccg att tta
1506 Ser Pro Lys Glu Ile Pro Ser Leu Lys Asp Asn Ile Glu Pro Ile
Leu 375 380 385 aaa gcg cgt aag gag tac gca tac ggg ccc cag cac gat
tat att gac 1554 Lys Ala Arg Lys Glu Tyr Ala Tyr Gly Pro Gln His
Asp Tyr Ile Asp 390 395 400 cac ccg gat gtg atc gga tgg acg agg gaa
ggt gac agc tcc gcc gcc 1602 His Pro Asp Val Ile Gly Trp Thr Arg
Glu Gly Asp Ser Ser Ala Ala 405 410 415 420 aaa tca ggt ttg gcc gct
tta atc acg gac gga ccc ggc gga tca aag 1650 Lys Ser Gly Leu Ala
Ala Leu Ile Thr Asp Gly Pro Gly Gly Ser Lys 425 430 435 cgg atg tat
gcc ggc ctg aaa aat gcc ggc gag aca tgg tat gac ata 1698 Arg Met
Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr Trp Tyr Asp Ile 440 445 450
acg ggc aac cgt tca gat act gta aaa atc gga tct gac ggc tgg gga
1746 Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly Ser Asp Gly Trp
Gly 455 460 465 gag ttt cat gta aac gat ggg tcc gtc tcc att tat gtt
cag aaa taa 1794 Glu Phe His Val Asn Asp Gly Ser Val Ser Ile Tyr
Val Gln Lys 470 475 480 ggtaataaaa aaacacctcc aagctgagtg cgggtatcag
cttggaggtg cgtttatttt 1854 ttcagccgta tgacaaggtc ggcatcaggt
gtgacaaata cggtatgctg gctgtcatag 1914 gtgacaaatc cgggttttgc
gccgtttggc tttttcacat gtctgatttt tgtataatca 1974 acaggcacgg
agccggaatc tttcgccttg gaaaaataag cggcgatcgt agctgcttcc 2034
aatatggatt gttcatcggg atcgctgctt ttaatcacaa cgtgggatcc 2084 10 483
PRT Bacillus amyloliquefaciens 10 Val Asn Gly Thr Leu Met Gln Tyr
Phe Glu Trp Tyr Thr Pro Asn Asp 1 5 10 15 Gly Gln His Trp Lys Arg
Leu Gln Asn Asp Ala Glu His Leu Ser Asp 20 25 30 Ile Gly Ile Thr
Ala Val Trp Ile Pro Pro Ala Tyr Lys Gly Leu Ser 35 40 45 Gln Ser
Asp Asn Gly Tyr Gly Pro Tyr Asp Leu Tyr Asp Leu Gly Glu 50 55 60
Phe Gln Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr Lys Ser Glu 65
70 75 80 Leu Gln Asp Ala Ile Gly Ser Leu His Ser Arg Asn Val Gln
Val Tyr 85 90 95 Gly Asp Val Val Leu Asn His Lys Ala Gly Ala Asp
Ala Thr Glu Asp 100 105 110 Val Thr Ala Val Glu Val Asn Pro Ala Asn
Arg Asn Gln Glu Thr Ser 115 120 125 Glu Glu Tyr Gln Ile Lys Ala Trp
Thr Asp Phe Arg Phe Pro Gly Arg 130 135 140 Gly Asn Thr Tyr Ser Asp
Phe Lys Trp His Trp Tyr His Phe Asp Gly 145 150 155 160 Ala Asp Trp
Asp Glu Ser Arg Lys Ile Ser Arg Ile Phe Lys Phe Arg 165 170 175 Gly
Glu Gly Lys Ala Trp Asp Trp Glu Val Ser Ser Glu Asn Gly Asn 180 185
190 Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Tyr Asp His Pro Asp Val
195 200 205 Val Ala Glu Thr Lys Lys Trp Gly Ile Trp Tyr Ala Asn Glu
Leu Ser 210 215 220 Leu Asp Gly Phe Arg Ile Asp Ala Ala Lys His Ile
Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Gln Ala Val Arg
Gln Ala Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala Glu Tyr Trp
Gln Asn Asn Ala Gly Lys Leu Glu Asn 260 265 270 Tyr Leu Asn Lys Thr
Ser Phe Asn Gln Ser Val Phe Asp Val Pro Leu 275 280 285 His Phe Asn
Leu Gln Ala Ala Ser Ser Gln Gly Gly Gly Tyr Asp Met 290 295 300 Arg
Arg Leu Leu Asp Gly Thr Val Val Ser Arg His Pro Glu Lys Ala 305 310
315 320 Val Thr Phe Val Glu Asn His Asp Thr Gln Pro Gly Gln Ser Leu
Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala
Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr
Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Thr Ser Pro Lys Glu Ile
Pro Ser Leu Lys Asp Asn Ile 370 375 380 Glu Pro Ile Leu Lys Ala Arg
Lys Glu Tyr Ala Tyr Gly Pro Gln His 385 390 395 400 Asp Tyr Ile Asp
His Pro Asp Val Ile Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser
Ala Ala Lys Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430
Gly Gly Ser Lys Arg Met Tyr Ala Gly Leu Lys Asn Ala Gly Glu Thr 435
440 445 Trp Tyr Asp Ile Thr Gly Asn Arg Ser Asp Thr Val Lys Ile Gly
Ser 450 455 460 Asp Gly Trp Gly Glu Phe His Val Asn Asp Gly Ser Val
Ser Ile Tyr 465 470 475 480 Val Gln Lys 11 1458 DNA Bacillus sp.
CDS (1)..(1458) 11 cac cat aat ggt acg aac ggc aca atg atg cag tac
ttt gaa tgg tat 48 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr
Phe Glu Trp Tyr 1 5 10 15 cta cca aat gac gga aac cat tgg aat aga
tta agg tct gat gca agt 96 Leu Pro Asn Asp Gly Asn His Trp Asn Arg
Leu Arg Ser Asp Ala Ser 20 25 30 aac cta aaa gat aaa ggg atc tca
gcg gtt tgg att cct cct gca tgg 144 Asn Leu Lys Asp Lys Gly Ile Ser
Ala Val Trp Ile Pro Pro Ala Trp 35 40 45 aag ggt gcc tct caa aat
gat gtg ggg tat ggt gct tat gat ctg tat 192 Lys Gly Ala Ser Gln Asn
Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 gat tta gga gaa
ttc aat caa aaa gga acc att cgt aca aaa tat gga 240 Asp Leu Gly Glu
Phe Asn Gln Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 acg cgc
aat cag tta caa gct gca gtt aac gcc ttg aaa agt aat gga 288 Thr Arg
Asn Gln Leu Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95
att caa gtg tat ggc gat gtt gta atg aat cat aaa ggg gga gca gac 336
Ile Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100
105 110 gct acc gaa atg gtt agg gca gtt gaa gta aac ccg aat aat aga
aat 384 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg
Asn 115 120 125 caa gaa gtg tcc ggt gaa tat aca att gag gct tgg aca
aag ttt gac 432 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr
Lys Phe Asp 130 135 140 ttt cca gga cga ggt aat act cat tca aac ttc
aaa tgg aga tgg tat 480 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe
Lys Trp Arg Trp Tyr 145 150 155 160 cac ttt gat gga gta gat tgg gat
cag tca cgt aag ctg aac aat cga 528 His Phe Asp Gly Val Asp Trp Asp
Gln Ser Arg Lys Leu Asn Asn Arg 165 170 175 att tat aaa ttt aga ggt
gat gga aaa ggg tgg gat tgg gaa gtc gat 576 Ile Tyr Lys Phe Arg Gly
Asp Gly Lys Gly Trp Asp Trp Glu Val Asp 180 185 190 aca gaa aac ggt
aac tat gat tac cta atg tat gca gat att gac atg 624 Thr Glu Asn Gly
Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 gat cac
cca gag gta gtg aat gag cta aga aat tgg ggt gtt tgg tat 672 Asp His
Pro Glu Val Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220
acg aat aca tta ggc ctt gat ggt ttt aga ata gat gca gta aaa cat 720
Thr Asn Thr Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225
230 235 240 ata aaa tac agc ttt act cgt gat tgg att aat cat gtt aga
agt gca 768 Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg
Ser Ala 245 250 255 act ggc aaa aat atg ttt gcg gtt gcg gaa ttt tgg
aaa aat gat tta 816 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp
Lys Asn Asp Leu 260 265 270 ggt gct att gaa aac tat tta aac aaa aca
aac tgg aac cat tca gtc 864 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr
Asn Trp Asn His Ser Val 275 280 285 ttt gat gtt ccg ctg cac tat aac
ctc tat aat gct tca aaa agc gga 912 Phe Asp Val Pro Leu His Tyr Asn
Leu Tyr Asn Ala Ser Lys Ser Gly 290 295 300 ggg aat tat gat atg agg
caa ata ttt aat ggt aca gtc gtg caa aga 960 Gly Asn Tyr Asp Met Arg
Gln Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 cat cca atg
cat gct gtt aca ttt gtt gat aat cat gat tcg caa cct 1008 His Pro
Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335
gaa gaa gct tta gag tct ttt gtt gaa gaa tgg ttc aaa cca tta gcg
1056 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu
Ala 340 345 350 tat gct ttg aca tta aca cgt gaa caa ggc tac cct tct
gta ttt tat 1104 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro
Ser Val Phe Tyr 355 360 365 gga gat tat tat ggc att cca acg cat ggt
gta cca gcg atg aaa tcg 1152 Gly Asp Tyr Tyr Gly Ile Pro Thr His
Gly Val Pro Ala Met Lys Ser 370 375 380 aaa att gac ccg att cta gaa
gcg cgt caa aag tat gca tat gga aga 1200 Lys Ile Asp Pro Ile Leu
Glu Ala Arg Gln Lys Tyr Ala Tyr Gly Arg 385 390 395 400 caa aat gac
tac tta gac cat cat aat atc atc ggt tgg aca cgt gaa 1248 Gln Asn
Asp Tyr Leu Asp His His Asn Ile Ile Gly Trp Thr Arg Glu 405
410 415 ggg aat aca gca cac ccc aac tcc ggt tta gct act atc atg tcc
gat 1296 Gly Asn Thr Ala His Pro Asn Ser Gly Leu Ala Thr Ile Met
Ser Asp 420 425 430 ggg gca gga gga aat aag tgg atg ttt gtt ggg cgt
aat aaa gct ggt 1344 Gly Ala Gly Gly Asn Lys Trp Met Phe Val Gly
Arg Asn Lys Ala Gly 435 440 445 caa gtt tgg acc gat atc act gga aat
cgt gca ggt act gtt acg att 1392 Gln Val Trp Thr Asp Ile Thr Gly
Asn Arg Ala Gly Thr Val Thr Ile 450 455 460 aat gct gat gga tgg ggt
aat ttt tct gta aat gga gga tca gtt tct 1440 Asn Ala Asp Gly Trp
Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 att tgg
gta aac aaa taa 1458 Ile Trp Val Asn Lys 485 12 485 PRT Bacillus
sp. 12 His His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp
Tyr 1 5 10 15 Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Ser
Asp Ala Ser 20 25 30 Asn Leu Lys Asp Lys Gly Ile Ser Ala Val Trp
Ile Pro Pro Ala Trp 35 40 45 Lys Gly Ala Ser Gln Asn Asp Val Gly
Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60 Asp Leu Gly Glu Phe Asn Gln
Lys Gly Thr Ile Arg Thr Lys Tyr Gly 65 70 75 80 Thr Arg Asn Gln Leu
Gln Ala Ala Val Asn Ala Leu Lys Ser Asn Gly 85 90 95 Ile Gln Val
Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110 Ala
Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120
125 Gln Glu Val Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp
130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg
Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp Trp Asp Gln Ser Arg
Lys Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe Arg Gly Asp Gly Lys
Gly Trp Asp Trp Glu Val Asp 180 185 190 Thr Glu Asn Gly Asn Tyr Asp
Tyr Leu Met Tyr Ala Asp Ile Asp Met 195 200 205 Asp His Pro Glu Val
Val Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220 Thr Asn Thr
Leu Gly Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His 225 230 235 240
Ile Lys Tyr Ser Phe Thr Arg Asp Trp Ile Asn His Val Arg Ser Ala 245
250 255 Thr Gly Lys Asn Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp
Leu 260 265 270 Gly Ala Ile Glu Asn Tyr Leu Asn Lys Thr Asn Trp Asn
His Ser Val 275 280 285 Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn
Ala Ser Lys Ser Gly 290 295 300 Gly Asn Tyr Asp Met Arg Gln Ile Phe
Asn Gly Thr Val Val Gln Arg 305 310 315 320 His Pro Met His Ala Val
Thr Phe Val Asp Asn His Asp Ser Gln Pro 325 330 335 Glu Glu Ala Leu
Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala
Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr 355 360 365
Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ala Met Lys Ser 370
375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln Lys Tyr Ala Tyr Gly
Arg 385 390 395 400 Gln Asn Asp Tyr Leu Asp His His Asn Ile Ile Gly
Trp Thr Arg Glu 405 410 415 Gly Asn Thr Ala His Pro Asn Ser Gly Leu
Ala Thr Ile Met Ser Asp 420 425 430 Gly Ala Gly Gly Asn Lys Trp Met
Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln Val Trp Thr Asp Ile
Thr Gly Asn Arg Ala Gly Thr Val Thr Ile 450 455 460 Asn Ala Asp Gly
Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480 Ile
Trp Val Asn Lys 485 13 197 PRT Bacillus sp 707 13 Phe Asp Val Pro
Leu His Tyr Asn Leu Tyr Asn Ala Ser Lys Ser Gly 1 5 10 15 Gly Asn
Tyr Asp Met Arg Asn Ile Phe Asn Gly Thr Val Val Gln Arg 20 25 30
His Pro Ser His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro 35
40 45 Glu Glu Ala Leu Glu Ser Phe Val Glu Glu Trp Phe Lys Pro Leu
Ala 50 55 60 Tyr Ala Leu Thr Leu Thr Arg Glu Gln Gly Tyr Pro Ser
Val Phe Tyr 65 70 75 80 Gly Asp Tyr Tyr Gly Ile Pro Thr His Gly Val
Pro Ala Met Arg Ser 85 90 95 Lys Ile Asp Pro Ile Leu Glu Ala Arg
Gln Lys Tyr Ala Tyr Gly Lys 100 105 110 Gln Asn Asp Tyr Leu Asp His
His Asn Ile Ile Gly Trp Thr Arg Glu 115 120 125 Gly Asn Thr Ala His
Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp 130 135 140 Gly Ala Gly
Gly Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 145 150 155 160
Gln Val Trp Ser Asp Ile Thr Gly Asn Arg Thr Gly Thr Val Thr Ile 165
170 175 Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val
Ser 180 185 190 Ile Trp Val Asn Lys 195 14 24 DNA Artificial
Sequence Primer 22149 14 cgattgctga cgctgttatt tgcg 24 15 19 DNA
Artificial Sequence Primer 24814 15 gatcacccgc gataccgtc 19 16 31
DNA Artificial Sequence Primer # 24 16 gaatgtatgt cggccggcaa
aacgccggtg a 31 17 30 DNA Artificial Sequence Primer # 27 17
gccgccgctg ctgcagaatg aggcagcaag 30 18 48 DNA Artificial Sequence
Primer # 312 18 cccgaaagct gaaccgcatc tataggtttc aagggaagac
ttgggatt 48 19 23 DNA Artificial Sequence Primer 290 19 aggatggtca
taatcaaagt cgg 23 20 52 DNA Artificial Sequence Primer #313 20
ccgactttga ttatgaccat cctgttgtcg tagcagagat taagagatgg gg 52 21 45
DNA Artificial Sequence Primer # 314 21 cgacaatgtc atggtggtcg
aaaaaatcat gctgtgctcc gtacg 45 22 23 DNA Artificial Sequence Primer
#296 22 tttcgaccac catgacattg tcg 23 23 24 DNA Artificial Sequence
Primer #305 23 tatagatgcg gttcagcttt cggg 24 24 1650 DNA Bacillus
sp. 24 cttgaatcat tatttaaagc tggttatgat atatgtaagc gttatcatta
aaaggaggta 60 tttgatgaaa agatgggtag tagcaatgct ggcagtgtta
tttttatttc cttcggtagt 120 agttgcagat ggcttgaatg gaacgatgat
gcagtattat gagtggcatc tagagaatga 180 tgggcaacac tggaatcggt
tgcatgatga tgccgaagct ttaagtaatg cgggtattac 240 agctatttgg
atacccccag cctacaaagg aaatagtcag gctgatgttg ggtatggtgc 300
atacgacctt tatgatttag gggagtttaa tcaaaaaggt accgttcgaa cgaaatacgg
360 gacaaaggct cagcttgagc gagctatagg gtccctaaag tcgaatgata
tcaatgttta 420 tggggatgtc gtaatgaatc ataaattagg agctgatttc
acggaggcag tgcaagctgt 480 tcaagtaaat ccttcgaacc gttggcagga
tatttcaggt gtctacacga ttgatgcatg 540 gacgggattt gactttccag
ggcgcaacaa tgcctattcc gattttaaat ggagatggtt 600 ccattttaat
ggcgttgact gggatcaacg ctatcaagaa aaccatcttt ttcgctttgc 660
aaatacgaac tggaactggc gagtggatga agagaatggt aattatgact atttattagg
720 atcgaacatt gactttagcc acccagaggt tcaagaggaa ttaaaggatt
gggggagctg 780 gtttacggat gagctagatt tagatgggta tcgattggat
gctattaagc atattccatt 840 ctggtatacg tcagattggg ttaggcatca
gcgaagtgaa gcagaccaag atttatttgt 900 cgtaggggag tattggaagg
atgacgtagg tgctctcgaa ttttatttag atgaaatgaa 960 ttgggagatg
tctctattcg atgttccgct caattataat ttttaccggg cttcaaagca 1020
aggcggaagc tatgatatgc gtaatatttt acgaggatct ttagtagaag cacatccgat
1080 tcatgcagtt acgtttgttg ataatcatga tactcagcca ggagagtcat
tagaatcatg 1140 ggtcgctgat tggtttaagc cacttgctta tgcgacaatc
ttgacgcgtg aaggtggtta 1200 tccaaatgta ttttacggtg actactatgg
gattcctaac gataacattt cagctaagaa 1260 ggatatgatt gatgagttgc
ttgatgcacg tcaaaattac gcatatggca cacaacatga 1320 ctattttgat
cattgggata tcgttggatg gacaagagaa ggtacatcct cacgtcctaa 1380
ttcgggtctt gctactatta tgtccaatgg tcctggagga tcaaaatgga tgtacgtagg
1440 acagcaacat gcaggacaaa cgtggacaga tttaactggc aatcacgcgg
cgtcggttac 1500 gattaatggt gatggctggg gcgaattctt tacaaatgga
ggatctgtat ccgtgtatgt 1560 gaaccaataa taaaaagcct tgagaaggga
ttcctcccta actcaaggct ttctttatgt 1620 cgtttagctc aacgcttcta
cgaagcttta 1650 25 501 PRT Bacillus sp. 25 Met Lys Arg Trp Val Val
Ala Met Leu Ala Val Leu Phe Leu Phe Pro 1 5 10 15 Ser Val Val Val
Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr 20 25 30 Glu Trp
His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 35 40 45
Asp Ala Glu Ala Leu Ser Asn Ala Gly Ile Thr Ala Ile Trp Ile Pro 50
55 60 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala
Tyr 65 70 75 80 Asp 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 100 105 110 Ser Asn Asp Ile Asn Val Tyr Gly Asp
Val Val Met Asn His Lys Leu 115 120 125 Gly Ala Asp Phe Thr Glu Ala
Val Gln Ala Val Gln Val Asn Pro Ser 130 135 140 Asn Arg Trp Gln Asp
Ile Ser Gly Val Tyr Thr Ile Asp Ala Trp Thr 145 150 155 160 Gly 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
185 190 Asn His Leu Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val
Asp 195 200 205 Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn
Ile Asp Phe 210 215 220 Ser His Pro Glu Val Gln Glu Glu Leu Lys Asp
Trp Gly Ser Trp Phe 225 230 235 240 Thr 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 260 265 270 Ala Asp Gln Asp
Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val 275 280 285 Gly Ala
Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 290 295 300
Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Lys Gln Gly 305
310 315 320 Gly 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 340 345 350 Gly Glu Ser Leu Glu Ser Trp Val Ala Asp
Trp Phe Lys Pro Leu Ala 355 360 365 Tyr Ala Thr Ile Leu Thr Arg Glu
Gly Gly Tyr Pro Asn Val Phe Tyr 370 375 380 Gly Asp Tyr Tyr Gly Ile
Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 385 390 395 400 Met 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
430 Gly Thr Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn
435 440 445 Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Gln Gln His
Ala Gly 450 455 460 Gln Thr Trp Thr Asp Leu Thr Gly Asn His Ala Ala
Ser Val Thr Ile 465 470 475 480 Asn Gly Asp Gly Trp Gly Glu Phe Phe
Thr Asn Gly Gly Ser Val Ser 485 490 495 Val Tyr Val Asn Gln 500 26
1745 DNA Bacillus sp. CDS (190)..(1692) 26 aactaagtaa catcgattca
ggataaaagt atgcgaaacg atgcgcaaaa ctgcgcaact 60 actagcactc
ttcagggact aaaccacctt ttttccaaaa atgacatcat ataaacaaat 120
ttgtctacca atcactattt aaagctgttt atgatatatg taagcgttat cattaaaagg
180 aggtatttg atg aga aga tgg gta gta gca atg ttg gca gtg tta ttt
tta 231 Met Arg Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu -20
-15 -10 ttt cct tcg gta gta gtt gca gat gga ttg aac ggt acg atg atg
cag 279 Phe Pro Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met
Gln -5 -1 1 5 tat tat gag tgg cat ttg gaa aac gac ggg cag cat tgg
aat cgg ttg 327 Tyr Tyr Glu Trp His Leu Glu Asn Asp Gly Gln His Trp
Asn Arg Leu 10 15 20 25 cac gat gat gcc gca gct ttg agt gat gct ggt
att aca gct att tgg 375 His Asp Asp Ala Ala Ala Leu Ser Asp Ala Gly
Ile Thr Ala Ile Trp 30 35 40 att ccg cca gcc tac aaa ggt aat agt
cag gcg gat gtt ggg tac ggt 423 Ile Pro Pro Ala Tyr Lys Gly Asn Ser
Gln Ala Asp Val Gly Tyr Gly 45 50 55 gca tac gat ctt tat gat tta
gga gag ttc aat caa aag ggt act gtt 471 Ala Tyr Asp Leu Tyr Asp Leu
Gly Glu Phe Asn Gln Lys Gly Thr Val 60 65 70 cga acg aaa tac gga
act aag gca cag ctt gaa cga gct att ggg tcc 519 Arg Thr Lys Tyr Gly
Thr Lys Ala Gln Leu Glu Arg Ala Ile Gly Ser 75 80 85 ctt aaa tct
aat gat atc aat gta tac gga gat gtc gtg atg aat cat 567 Leu Lys Ser
Asn Asp Ile Asn Val Tyr Gly Asp Val Val Met Asn His 90 95 100 105
aaa atg gga gct gat ttt acg gag gca gtg caa gct gtt caa gta aat 615
Lys Met Gly Ala Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn 110
115 120 cca acg aat cgt tgg cag gat att tca ggt gcc tac acg att gat
gcg 663 Pro Thr Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp
Ala 125 130 135 tgg acg ggt ttc gac ttt tca ggg cgt aac aac gcc tat
tca gat ttt 711 Trp Thr Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr
Ser Asp Phe 140 145 150 aag tgg aga tgg ttc cat ttt aat ggt gtt gac
tgg gat cag cgc tat 759 Lys Trp Arg Trp Phe His Phe Asn Gly Val Asp
Trp Asp Gln Arg Tyr 155 160 165 caa gaa aat cat att ttc cgc ttt gca
aat acg aac tgg aac tgg cga 807 Gln Glu Asn His Ile Phe Arg Phe Ala
Asn Thr Asn Trp Asn Trp Arg 170 175 180 185 gtg gat gaa gag aac ggt
aat tat gat tac ctg tta gga tcg aat atc 855 Val Asp Glu Glu Asn Gly
Asn Tyr Asp Tyr Leu Leu Gly Ser Asn Ile 190 195 200 gac ttt agt cat
cca gaa gta caa gat gag ttg aag gat tgg ggt agc 903 Asp Phe Ser His
Pro Glu Val Gln Asp Glu Leu Lys Asp Trp Gly Ser 205 210 215 tgg ttt
acc gat gag tta gat ttg gat ggt tat cgt tta gat gct att 951 Trp Phe
Thr Asp Glu Leu Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile 220 225 230
aaa cat att cca ttc tgg tat aca tct gat tgg gtt cgg cat cag cgc 999
Lys His Ile Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg 235
240 245 aac gaa gca gat caa gat tta ttt gtc gta ggg gaa tat tgg aag
gat 1047 Asn Glu Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp
Lys Asp 250 255 260 265 gac gta ggt gct ctc gaa ttt tat tta gat gaa
atg aat tgg gag atg 1095 Asp Val Gly Ala Leu Glu Phe Tyr Leu Asp
Glu Met Asn Trp Glu Met 270 275 280 tct cta ttc gat gtt cca ctt aat
tat aat ttt tac cgg gct tca caa 1143 Ser Leu Phe Asp Val Pro Leu
Asn Tyr Asn Phe Tyr Arg Ala Ser Gln 285 290 295 caa ggt gga agc tat
gat atg cgt aat att tta cga gga tct tta gta 1191 Gln Gly Gly Ser
Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val 300 305 310 gaa gcg
cat ccg atg cat gca gtt acg ttt gtt gat aat cat gat act 1239 Glu
Ala His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Thr 315 320
325 cag cca ggg gag tca tta gag tca tgg gtt gct gat tgg ttt aag cca
1287 Gln Pro Gly Glu Ser Leu Glu Ser Trp Val Ala Asp Trp Phe Lys
Pro 330 335 340 345 ctt gct tat gcg aca att ttg acg cgt gaa ggt ggt
tat cca aat gta 1335 Leu Ala Tyr Ala Thr Ile Leu Thr Arg Glu Gly
Gly Tyr Pro Asn Val 350 355 360 ttt tac ggt gat tac tat ggg att cct
aac gat aac att tca gct aaa 1383 Phe Tyr Gly Asp Tyr Tyr Gly Ile
Pro Asn Asp Asn Ile Ser Ala Lys 365 370 375 aaa gat atg att gat gag
ctg ctt gat gca cgt caa aat tac gca tat 1431 Lys Asp Met Ile Asp
Glu Leu Leu Asp Ala Arg
Gln Asn Tyr Ala Tyr 380 385 390 ggc acg cag cat gac tat ttt gat cat
tgg gat gtt gta gga tgg act 1479 Gly Thr Gln His Asp Tyr Phe Asp
His Trp Asp Val Val Gly Trp Thr 395 400 405 agg gaa gga tct tcc tcc
aga cct aat tca ggc ctt gcg act att atg 1527 Arg Glu Gly Ser Ser
Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met 410 415 420 425 tcg aat
gga cct ggt ggt tcc aag tgg atg tat gta gga cgt cag aat 1575 Ser
Asn Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn 430 435
440 gca gga caa aca tgg aca gat tta act ggt aat aac gga gcg tcc gtt
1623 Ala Gly Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala Ser
Val 445 450 455 aca att aat ggc gat gga tgg ggc gaa ttc ttt acg aat
gga gga tct 1671 Thr Ile Asn Gly Asp Gly Trp Gly Glu Phe Phe Thr
Asn Gly Gly Ser 460 465 470 gta tcc gtg tac gtg aac caa taacaaaaag
ccttgagaag ggattcctcc 1722 Val Ser Val Tyr Val Asn Gln 475 480
ctaactcaag gctttcttta tgt 1745 27 501 PRT Bacillus sp. 27 Met Arg
Arg Trp Val Val Ala Met Leu Ala Val Leu Phe Leu Phe Pro -20 -15 -10
Ser Val Val Val Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr -5
-1 1 5 10 Glu Trp His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu
His Asp 15 20 25 Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala
Ile Trp Ile Pro 30 35 40 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp
Val Gly Tyr Gly Ala Tyr 45 50 55 Asp Leu Tyr Asp Leu Gly Glu Phe
Asn Gln Lys Gly Thr Val Arg Thr 60 65 70 75 Lys Tyr Gly Thr Lys Ala
Gln Leu Glu Arg Ala Ile Gly Ser Leu Lys 80 85 90 Ser Asn Asp Ile
Asn Val Tyr Gly Asp Val Val Met Asn His Lys Met 95 100 105 Gly Ala
Asp Phe Thr Glu Ala Val Gln Ala Val Gln Val Asn Pro Thr 110 115 120
Asn Arg Trp Gln Asp Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr 125
130 135 Gly Phe Asp Phe Ser Gly Arg Asn Asn Ala Tyr Ser Asp Phe Lys
Trp 140 145 150 155 Arg Trp Phe His Phe Asn Gly Val Asp Trp Asp Gln
Arg Tyr Gln Glu 160 165 170 Asn His Ile Phe Arg Phe Ala Asn Thr Asn
Trp Asn Trp Arg Val Asp 175 180 185 Glu Glu Asn Gly Asn Tyr Asp Tyr
Leu Leu Gly Ser Asn Ile Asp Phe 190 195 200 Ser His Pro Glu Val Gln
Asp Glu Leu Lys Asp Trp Gly Ser Trp Phe 205 210 215 Thr Asp Glu Leu
Asp Leu Asp Gly Tyr Arg Leu Asp Ala Ile Lys His 220 225 230 235 Ile
Pro Phe Trp Tyr Thr Ser Asp Trp Val Arg His Gln Arg Asn Glu 240 245
250 Ala Asp Gln Asp Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val
255 260 265 Gly Ala Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met
Ser Leu 270 275 280 Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala
Ser Gln Gln Gly 285 290 295 Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg
Gly Ser Leu Val Glu Ala 300 305 310 315 His Pro Met His Ala Val Thr
Phe Val Asp Asn His Asp Thr Gln Pro 320 325 330 Gly Glu Ser Leu Glu
Ser Trp Val Ala Asp Trp Phe Lys Pro Leu Ala 335 340 345 Tyr Ala Thr
Ile Leu Thr Arg Glu Gly Gly Tyr Pro Asn Val Phe Tyr 350 355 360 Gly
Asp Tyr Tyr Gly Ile Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 365 370
375 Met Ile Asp Glu Leu Leu Asp Ala Arg Gln Asn Tyr Ala Tyr Gly Thr
380 385 390 395 Gln His Asp Tyr Phe Asp His Trp Asp Val Val Gly Trp
Thr Arg Glu 400 405 410 Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala
Thr Ile Met Ser Asn 415 420 425 Gly Pro Gly Gly Ser Lys Trp Met Tyr
Val Gly Arg Gln Asn Ala Gly 430 435 440 Gln Thr Trp Thr Asp Leu Thr
Gly Asn Asn Gly Ala Ser Val Thr Ile 445 450 455 Asn Gly Asp Gly Trp
Gly Glu Phe Phe Thr Asn Gly Gly Ser Val Ser 460 465 470 475 Val Tyr
Val Asn Gln 480 28 501 PRT Bacillus sp 28 Met Arg Arg Trp Val Val
Ala Met Leu Ala Val Leu Phe Leu Phe Pro 1 5 10 15 Ser Val Val Val
Ala Asp Gly Leu Asn Gly Thr Met Met Gln Tyr Tyr 20 25 30 Glu Trp
His Leu Glu Asn Asp Gly Gln His Trp Asn Arg Leu His Asp 35 40 45
Asp Ala Ala Ala Leu Ser Asp Ala Gly Ile Thr Ala Ile Trp Ile Pro 50
55 60 Pro Ala Tyr Lys Gly Asn Ser Gln Ala Asp Val Gly Tyr Gly Ala
Tyr 65 70 75 80 Asp 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 100 105 110 Ser Asn Asp Ile Asn Val Tyr Gly Asp
Val Val Met Asn His Lys Met 115 120 125 Gly Ala Asp Phe Thr Glu Ala
Val Gln Ala Val Gln Val Asn Pro Thr 130 135 140 Asn Arg Trp Gln Asp
Ile Ser Gly Ala Tyr Thr Ile Asp Ala Trp Thr 145 150 155 160 Gly Phe
Asp Phe Ser 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
185 190 Asn His Ile Phe Arg Phe Ala Asn Thr Asn Trp Asn Trp Arg Val
Asp 195 200 205 Glu Glu Asn Gly Asn Tyr Asp Tyr Leu Leu Gly Ser Asn
Ile Asp Phe 210 215 220 Ser His Pro Glu Val Gln Asp Glu Leu Lys Asp
Trp Gly Ser Trp Phe 225 230 235 240 Thr 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 Asn Glu 260 265 270 Ala Asp Gln Asp
Leu Phe Val Val Gly Glu Tyr Trp Lys Asp Asp Val 275 280 285 Gly Ala
Leu Glu Phe Tyr Leu Asp Glu Met Asn Trp Glu Met Ser Leu 290 295 300
Phe Asp Val Pro Leu Asn Tyr Asn Phe Tyr Arg Ala Ser Gln Gln Gly 305
310 315 320 Gly Ser Tyr Asp Met Arg Asn Ile Leu Arg Gly Ser Leu Val
Glu Ala 325 330 335 His Pro Met His Ala Val Thr Phe Val Asp Asn His
Asp Thr Gln Pro 340 345 350 Gly Glu Ser Leu Glu Ser Trp Val Ala Asp
Trp Phe Lys Pro Leu Ala 355 360 365 Tyr Ala Thr Ile Leu Thr Arg Glu
Gly Gly Tyr Pro Asn Val Phe Tyr 370 375 380 Gly Asp Tyr Tyr Gly Ile
Pro Asn Asp Asn Ile Ser Ala Lys Lys Asp 385 390 395 400 Met 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 Val Val Gly Trp Thr Arg Glu 420 425
430 Gly Ser Ser Ser Arg Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asn
435 440 445 Gly Pro Gly Gly Ser Lys Trp Met Tyr Val Gly Arg Gln Asn
Ala Gly 450 455 460 Gln Thr Trp Thr Asp Leu Thr Gly Asn Asn Gly Ala
Ser Val Thr Ile 465 470 475 480 Asn Gly Asp Gly Trp Gly Glu Phe Phe
Thr Asn Gly Gly Ser Val Ser 485 490 495 Val Tyr Val Asn Gln 500 29
1920 DNA Bacillus licheniformis CDS (421)..(1872) 29 cggaagattg
gaagtacaaa aataagcaaa agattgtcaa tcatgtcatg agccatgcgg 60
gagacggaaa aatcgtctta atgcacgata tttatgcaac gttcgcagat gctgctgaag
120 agattattaa aaagctgaaa gcaaaaggct atcaattggt aactgtatct
cagcttgaag 180 aagtgaagaa gcagagaggc tattgaataa atgagtagaa
gcgccatatc ggcgcttttc 240 ttttggaaga aaatataggg aaaatggtac
ttgttaaaaa ttcggaatat ttatacaaca 300 tcatatgttt cacattgaaa
ggggaggaga atcatgaaac aacaaaaacg gctttacgcc 360 cgattgctga
cgctgttatt tgcgctcatc ttcttgctgc ctcattctgc agcagcggcg 420 gca aat
ctt aat ggg acg ctg atg cag tat ttt gaa tgg tac atg ccc 468 Ala Asn
Leu Asn Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15
aat gac ggc caa cat tgg agg cgt ttg caa aac gac tcg gca tat ttg 516
Asn Asp Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20
25 30 gct gaa cac ggt att act gcc gtc tgg att ccc ccg gca tat aag
gga 564 Ala Glu His Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Tyr Lys
Gly 35 40 45 acg agc caa gcg gat gtg ggc tac ggt gct tac gac ctt
tat gat tta 612 Thr Ser Gln Ala Asp Val Gly Tyr Gly Ala Tyr Asp Leu
Tyr Asp Leu 50 55 60 ggg gag ttt cat caa aaa ggg acg gtt cgg aca
aag tac ggc aca aaa 660 Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr
Lys Tyr Gly Thr Lys 65 70 75 80 gga gag ctg caa tct gcg atc aaa agt
ctt cat tcc cgc gac att aac 708 Gly 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 756 Val Tyr Gly Asp Val Val Ile
Asn His Lys Gly Gly Ala Asp Ala Thr 100 105 110 gaa gat gta acc gcg
gtt gaa gtc gat ccc gct gac cgc aac cgc gta 804 Glu Asp Val Thr Ala
Val Glu Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 att tca gga
gaa cac cta att aaa gcc tgg aca cat ttt cat ttt ccg 852 Ile Ser Gly
Glu His Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 ggg
cgc ggc agc aca tac agc gat ttt aaa tgg cat tgg tac cat ttt 900 Gly
Arg Gly Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150
155 160 gac gga acc gat tgg gac gag tcc cga aag ctg aac cgc atc tat
aag 948 Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr
Lys 165 170 175 ttt caa gga aag gct tgg gat tgg gaa gtt tcc aat gaa
aac ggc aac 996 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu
Asn Gly Asn 180 185 190 tat gat tat ttg atg tat gcc gac atc gat tat
gac cat cct gat gtc 1044 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp
Tyr Asp His Pro Asp Val 195 200 205 gca gca gaa att aag aga tgg ggc
act tgg tat gcc aat gaa ctg caa 1092 Ala Ala Glu Ile Lys Arg Trp
Gly Thr Trp Tyr Ala Asn Glu Leu Gln 210 215 220 ttg gac ggt ttc cgt
ctt gat gct gtc aaa cac att aaa ttt tct ttt 1140 Leu Asp Gly Phe
Arg Leu Asp Ala Val Lys His Ile Lys Phe Ser Phe 225 230 235 240 ttg
cgg gat tgg gtt aat cat gtc agg gaa aaa acg ggg aag gaa atg 1188
Leu Arg Asp Trp Val Asn His Val Arg Glu Lys Thr Gly Lys Glu Met 245
250 255 ttt acg gta gct gaa tat tgg cag aat gac ttg ggc gcg ctg gaa
aac 1236 Phe Thr Val Ala Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu
Glu Asn 260 265 270 tat ttg aac aaa aca aat ttt aat cat tca gtg ttt
gac gtg ccg ctt 1284 Tyr Leu Asn Lys Thr Asn Phe Asn His Ser Val
Phe Asp Val Pro Leu 275 280 285 cat tat cag ttc cat gct gca tcg aca
cag gga ggc ggc tat gat atg 1332 His Tyr Gln Phe His Ala Ala Ser
Thr Gln Gly Gly Gly Tyr Asp Met 290 295 300 agg aaa ttg ctg aac ggt
acg gtc gtt tcc aag cat ccg ttg aaa tcg 1380 Arg Lys Leu Leu Asn
Gly Thr Val Val Ser Lys His Pro Leu Lys Ser 305 310 315 320 gtt aca
ttt gtc gat aac cat gat aca cag ccg ggg caa tcg ctt gag 1428 Val
Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly Gln Ser Leu Glu 325 330
335 tcg act gtc caa aca tgg ttt aag ccg ctt gct tac gct ttt att ctc
1476 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu Ala Tyr Ala Phe Ile
Leu 340 345 350 aca agg gaa tct gga tac cct cag gtt ttc tac ggg gat
atg tac ggg 1524 Thr Arg Glu Ser Gly Tyr Pro Gln Val Phe Tyr Gly
Asp Met Tyr Gly 355 360 365 acg aaa gga gac tcc cag cgc gaa att cct
gcc ttg aaa cac aaa att 1572 Thr Lys Gly Asp Ser Gln Arg Glu Ile
Pro Ala Leu Lys His Lys Ile 370 375 380 gaa ccg atc tta aaa gcg aga
aaa cag tat gcg tac gga gca cag cat 1620 Glu Pro Ile Leu Lys Ala
Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 gat tat ttc
gac cac cat gac att gtc ggc tgg aca agg gaa ggc gac 1668 Asp Tyr
Phe Asp His His Asp Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415
agc tcg gtt gca aat tca ggt ttg gcg gca tta ata aca gac gga ccc
1716 Ser Ser Val Ala Asn Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly
Pro 420 425 430 ggt ggg gca aag cga atg tat gtc ggc cgg caa aac gcc
ggt gag aca 1764 Gly Gly Ala Lys Arg Met Tyr Val Gly Arg Gln Asn
Ala Gly Glu Thr 435 440 445 tgg cat gac att acc gga aac cgt tcg gag
ccg gtt gtc atc aat tcg 1812 Trp His Asp Ile Thr Gly Asn Arg Ser
Glu Pro Val Val Ile Asn Ser 450 455 460 gaa ggc tgg gga gag ttt cac
gta aac ggc ggg tcg gtt tca att tat 1860 Glu Gly Trp Gly Glu Phe
His Val Asn Gly Gly Ser Val Ser Ile Tyr 465 470 475 480 gtt caa aga
tag aagagcagag aggacggatt tcctgaagga aatccgtttt 1912 Val Gln Arg
tttatttt 1920 30 483 PRT Bacillus licheniformis 30 Ala Asn Leu Asn
Gly Thr Leu Met Gln Tyr Phe Glu Trp Tyr Met Pro 1 5 10 15 Asn Asp
Gly Gln His Trp Arg Arg Leu Gln Asn Asp Ser Ala Tyr Leu 20 25 30
Ala 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 60 Gly Glu Phe His Gln Lys Gly Thr Val Arg Thr Lys Tyr
Gly Thr Lys 65 70 75 80 Gly Glu Leu Gln Ser Ala Ile Lys Ser Leu His
Ser Arg Asp Ile Asn 85 90 95 Val Tyr Gly Asp Val Val Ile Asn His
Lys Gly Gly Ala Asp Ala Thr 100 105 110 Glu Asp Val Thr Ala Val Glu
Val Asp Pro Ala Asp Arg Asn Arg Val 115 120 125 Ile Ser Gly Glu His
Leu Ile Lys Ala Trp Thr His Phe His Phe Pro 130 135 140 Gly Arg Gly
Ser Thr Tyr Ser Asp Phe Lys Trp His Trp Tyr His Phe 145 150 155 160
Asp Gly Thr Asp Trp Asp Glu Ser Arg Lys Leu Asn Arg Ile Tyr Lys 165
170 175 Phe Gln Gly Lys Ala Trp Asp Trp Glu Val Ser Asn Glu Asn Gly
Asn 180 185 190 Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Tyr Asp His
Pro Asp Val 195 200 205 Ala Ala Glu Ile Lys Arg Trp Gly Thr Trp Tyr
Ala Asn Glu Leu Gln 210 215 220 Leu Asp Gly Phe Arg Leu Asp Ala Val
Lys His Ile Lys Phe Ser Phe 225 230 235 240 Leu Arg Asp Trp Val Asn
His Val Arg Glu Lys Thr Gly Lys Glu Met 245 250 255 Phe Thr Val Ala
Glu Tyr Trp Gln Asn Asp Leu Gly Ala Leu Glu Asn 260 265 270 Tyr Leu
Asn Lys Thr Asn Phe Asn His Ser Val Phe Asp Val Pro Leu 275 280 285
His Tyr Gln Phe His Ala Ala Ser Thr Gln Gly Gly Gly Tyr Asp Met 290
295 300 Arg Lys Leu Leu Asn Gly Thr Val Val Ser Lys His Pro Leu Lys
Ser 305 310 315 320 Val Thr Phe Val Asp Asn His Asp Thr Gln Pro Gly
Gln Ser Leu Glu 325 330 335 Ser Thr Val Gln Thr Trp Phe Lys Pro Leu
Ala Tyr Ala Phe Ile Leu 340 345 350 Thr Arg Glu Ser Gly Tyr Pro Gln
Val Phe Tyr Gly Asp Met Tyr Gly 355 360 365 Thr Lys Gly Asp Ser Gln
Arg Glu Ile Pro Ala Leu Lys His Lys Ile 370 375 380 Glu Pro Ile Leu
Lys Ala Arg Lys Gln Tyr Ala Tyr Gly Ala Gln His 385 390 395 400 Asp
Tyr Phe Asp His His Asp
Ile Val Gly Trp Thr Arg Glu Gly Asp 405 410 415 Ser Ser Val Ala Asn
Ser Gly Leu Ala Ala Leu Ile Thr Asp Gly Pro 420 425 430 Gly Gly Ala
Lys Arg Met Tyr Val Gly Arg Gln Asn Ala Gly Glu Thr 435 440 445 Trp
His Asp Ile Thr Gly Asn Arg Ser Glu Pro Val Val Ile Asn Ser 450 455
460 Glu Gly Trp Gly Glu Phe His Val Asn Gly Gly Ser Val Ser Ile Tyr
465 470 475 480 Val Gln Arg
* * * * *