U.S. patent application number 13/785190 was filed with the patent office on 2014-01-16 for alpha-amylase mutants.
This patent application is currently assigned to NOVOZYMES A/S. The applicant listed for this patent is NOVOZYMES A/S. Invention is credited to Carsten Andersen, Torben Vedel Borchert, Soren Kjaerulff, Bjarne Nielsen, Torben Lauesgaard Nissen, Allan Svendsen.
Application Number | 20140017715 13/785190 |
Document ID | / |
Family ID | 55701615 |
Filed Date | 2014-01-16 |
United States Patent
Application |
20140017715 |
Kind Code |
A1 |
Borchert; Torben Vedel ; et
al. |
January 16, 2014 |
Alpha-amylase Mutants
Abstract
The invention relates to a variant of a parent Termamyl-like
.alpha.-amylase, which exhibits an alteration in at least one of
the following properties relative to said parent .alpha.-amylase:
i) improved pH stability at a pH from 8 to 10.5; and/or ii)
improved Ca.sup.2+ stability at pH 8 to 10.5, and/or iii) increased
specific activity at temperatures from 10 to 60.degree. C.
Inventors: |
Borchert; Torben Vedel;
(Copenhagen O, DK) ; Svendsen; Allan; (Birkeroed,
DK) ; Andersen; Carsten; (Vaerloese, DK) ;
Nielsen; Bjarne; (Virum, DK) ; Nissen; Torben
Lauesgaard; (Frederiksberg, DK) ; Kjaerulff;
Soren; (Vanloese, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
NOVOZYMES A/S
Bagsvaerd
DK
|
Family ID: |
55701615 |
Appl. No.: |
13/785190 |
Filed: |
March 5, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11953532 |
Dec 10, 2007 |
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13785190 |
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10980923 |
Nov 4, 2004 |
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11953532 |
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10665667 |
Sep 19, 2003 |
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10980923 |
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09769864 |
Jan 25, 2001 |
6673589 |
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10665667 |
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09183412 |
Oct 30, 1998 |
6204232 |
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09769864 |
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60093234 |
Jul 17, 1998 |
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60064662 |
Nov 6, 1997 |
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Current U.S.
Class: |
435/22 ; 435/187;
435/202; 435/252.31; 435/254.11; 435/320.1; 510/226; 510/235;
510/320; 510/392; 536/23.2 |
Current CPC
Class: |
C12Y 302/01001 20130101;
C11D 3/386 20130101; C11D 3/38636 20130101; C12N 9/2417 20130101;
C07K 14/32 20130101; C11D 3/38609 20130101 |
Class at
Publication: |
435/22 ; 435/202;
536/23.2; 435/320.1; 435/252.31; 435/254.11; 435/187; 510/392;
510/226; 510/235; 510/320 |
International
Class: |
C12N 9/28 20060101
C12N009/28 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 30, 1997 |
DK |
PA 1240/97 |
Jul 14, 1998 |
DK |
PA 1998 00936 |
Claims
1. A variant of a parent Termamyl-like .alpha.-amylase, which
variant has .alpha.-amylase activity, said variant comprises one or
more mutations corresponding to the following mutations in the
amino acid sequence shown in SEQ ID NO: 2: T141, K142, F143, D144,
F145, P146, G147, R148, G149, Q174, R181, G182, D183, G184, K185,
A186, W189, 5193, N195 H107, K108, G109,D166, W167, D168, Q169,
5170, R171, Q172, F173, F267, W268, K269, N270, D271, L272, G273,
A274, L275, K311, E346, K385, G456, N457, K458,P459, G460, T461,
V462, T463.
2. The variant according to claim 1, which variant has one or more
of the following substitutions or deletions:
T141A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
K142A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
F143A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
D144A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F145A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
P146A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G147A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R148A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G149A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R181*,A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G182*,A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D183*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G184*,A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W189A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
S193A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
H107A,D,R,N,C,E,Q,G,I,L,K,M,F,P,S,T,W,Y,V;
K108A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G109A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D166A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W167A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
D168A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q169A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
S170A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
R171A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q172A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F173A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
Q174*,A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F267A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
W268A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D271A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L272A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
G273A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
A274D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L275A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
K311A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
E346A,D,R,N,C,Q,G,H,I,K,L,M,F,P,S,T,W,Y,V;
K385A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G456A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
N457A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G460A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
V462A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;
T463A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.
3. The variant according to claim 2, wherein the variant has one or
more of the following substitutions or deletions: K142R; S193P;
N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R; K458R; P459T;
T461P; Q174*; R181Q,N,S; G182T,S,N; D183*; G184*;
K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R;
W189T,S,N,Q.
4. The variant according to claim 1, wherein the variant has a
deletion in position D183+G184, and further one or more of the
following substitutions or deletions: K142R; S193P; N195F; K269R,Q;
N270Y,R,D; K311R; E346Q; K385R; K458R; P459T; T461P; Q174*;
R181Q,N,S; G182T,S,N; D183*; G184*;
K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R;
W189T,S,N,Q.
5. The variant according to claim 1, wherein the variants exhibits
an alteration in at least one of the following properties relative
to the parent .alpha.-amylase: i) improved pH stability at a pH
from 8 to 10.5; and/or ii) improved Ca.sup.2+ stability at pH 8 to
10.5, and/or iii) increased specific activity at temperatures from
10 to 60.degree. C., preferably 20-50.degree. C., especially
30-40.degree. C.
6. The variant according to claim 1, exhibiting improved stability
at pH 8 to 10.5, having mutations in one or more of the position(s)
corresponding to the following positions (using SEQ ID NO: 2
numbering): T141, K142, F143, D144, F145, P146, G147, R148, G149,
R181, A186, 5193, N195, K269, N270, K311, K458, P459, T461.
7. The variant according to claim 6, which variant has one or more
of the following substitutions:
T141A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
K142A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
F143A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
D144A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F145A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
P146A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G147A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R148A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G149A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
K181A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
A186D,R,N,C,E,Q,G,H,I,L,P,K,M,F,S,T,W,Y,V;
S193A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K311A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.
8. The variant according to claim 7, wherein the variant has one or
more of the following substitutions: K142R, R181S, A186T, S193P,
N195F, K269R, N270Y, K311R, K458R, P459T and T461P.
9. The variant according to claim 1, exhibiting improved Ca.sup.2+
stability at pH 8 to 10.5, having mutations in one or more of the
following positions (using the SEQ ID NO: 2 numbering): R181, G182,
D183, G184, K185, A186, W189, N195, N270, E346, K385, K458,
P459.
10. The variant according to claim 9, which variant has one or more
of the following substitutions or deletions:
R181*,A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G182*,A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D183*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G184*,A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W189A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
N270A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
E346A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K385A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
K458A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,R,D,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V.
11. The variant according to claim 10, wherein the variant has one
or more of the following substitutions or deletions: R181Q,N;
G182T,S,N; D183*; G184*; K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V;
A186T,S,N,I,V; W189T,S,N,Q; N195F; N270R,D; E346Q; K385R; K458R;
P459T.
12. A variant according to claim 1, wherein the parent
Termamyl-like .alpha.-amylase is selected from: the Bacillus strain
NCIB 12512 .alpha.-amylase having the sequence shown in SEQ ID NO:
1; the B. amyloliquefaciens .alpha.-amylase having the sequence
shown in SEQ ID NO: 5; the B. licheniformis .alpha.-amylase having
the sequence shown in SEQ ID NO: 4.
13. The variant according to claim 1, exhibiting increased specific
activity at a temperatures from 10 to 60.degree. C., preferably
20-50.degree. C., especially 30-40.degree. C., having mutation(s)
in one or more of the following positions (using the SEQ ID NO: 2
numbering): H107, K108, G109, D166, W167, D168, Q169, 5170, R171,
Q172, F173, Q174, D183, G184, N195, F267, W268, K269,N270, D271,
L272, G273, A274, L275, G456, N457, K458, P459, G460, T461, V462,
T463.
14. The variant according to claim 13, which variant has one or
more of the following substitutions:
H107A,D,R,N,C,E,Q,G,I,L,K,M,F,P,S,T,W,Y,V;
K108A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G109A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D166A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W167A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
D168A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q169A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
S170A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
R171A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q172A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F173A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
Q174*,A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D183*,A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
G184*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F267A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
W268A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D271A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L272A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
G273A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
A274D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L275A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
G456A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
N457A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G460A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
V462A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;
T463A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.
15. The variant according to claim 14, wherein the variant has one
or more of the following substitutions or deletions: Q174*, D183*,
G184*, N195F, K269S.
16. The variant according to claim 13, wherein the parent
Termamyl-like .alpha.-amylase is the B. licheniformis
.alpha.-amylase having the sequence shown in SEQ ID NO: 4.
17. A DNA construct comprising a DNA sequence encoding an
.alpha.-amylase variant according to claim 1.
18. A recombinant expression vector which carries a DNA construct
according to claim 17.
19. A cell which is transformed with a DNA construct according to
claim 17 or a vector according to claim 18.
20. A cell according to claim 19, which is a microorganism.
21. A cell according to claim 20, which is a bacterium or a
fungus.
22. The cell according to claim 21, which is a Gram positive
bacterium such as Bacillus subtilis, Bacillus licheniformis,
Bacillus lentos, Bacillus brevis, Bacillus stearothermophilus,
Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus
coagulans, Bacillus circulans, Bacillus lautus or Bacillus
thuringiensis.
23. A detergent additive comprising an .alpha.-amylase variant
according to claim 1, optionally in the form of a non-dusting
granulate, stabilized liquid or protected enzyme.
24. A detergent additive according to claim 23 which contains
0.02-200 mg of enzyme protein/g of the additive.
25. A detergent additive according to claim 23, which additionally
comprises another enzyme such as a protease, a lipase, a
peroxidase, another amylolytic enzyme and/or a cellulase.
26. A detergent composition comprising an .alpha.-amylase variant
according to claim 1.
27. A detergent composition according to claim 26 which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, another amylolytic enzyme and/or a cellulase.
28. A manual or automatic dishwashing detergent composition
comprising an .alpha.-amylase variant according to claim 1.
29. A dishwashing detergent composition according to claim 28 which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, another amylolytic enzyme and/or a cellulase.
30. A manual or automatic laundry washing composition comprising an
.alpha.-amylase variant according to claim 1.
31. A laundry washing composition according to claim 30, which
additionally comprises another enzyme such as a protease, a lipase,
a peroxidase, an amylolytic enzyme and/or a cellulase.
32. Method for providing .alpha.-amylases with 1) altered pH
optimum, and/or 2) altered temperature optimum, and/or 3) improved
stability, comprising the following steps: i) identifying (a)
target position(s) and/or region(s) for mutation of the
.alpha.-amylase by comparing the molecular dynamics of two or more
.alpha.-amylase's 3D structures having substantially different pH,
temperature and/or stability profiles, ii) substituting, adding
and/or deleting one or more amino acids in the identified
position(s) and/or region(s).
33. The method according to claim 32, wherein a medium temperature
.alpha.-amylase is compared with a high temperature
.alpha.-amylase.
34. The method according to claim 32, wherein a low temperature
.alpha.-amylase is compared with a medium or high temperature
.alpha.-amylase.
35. The method according to claim 32, wherein the .alpha.-amylases
are at least 70%, preferably 80%, up to 90%, such as up to 95%,
especially 95% homologous.
36. The method according to claim 35, wherein the .alpha.-amylases
compared are Termamyl-like .alpha.-amylases.
37. The method according to claim 27, wherein the .alpha.-amylases
compared are any of the .alpha.-amylases shown in SEQ ID NO: 1 to
SEQ ID NO: 8.
38. The method according to claim 32, wherein the stability profile
of the .alpha.-amylases compared are the Ca.sup.2+ dependency
profile.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/953,532 filed Dec. 10, 2007 (pending) which is a divisional
of U.S. application Ser. No. 10/980,923, filed Nov. 4, 2004 (now
abandoned), which is a continuation of U.S. application Ser. No.
10/665,667, filed Sep. 19, 2003 (now abandoned), which is a
divisional of U.S. application Ser. No. 09/769,864, filed on Jan.
25, 2001 (now U.S. Pat. No. 6,673,589), which is a divisional of
U.S. application Ser. No. 09/183,412, filed on Oct. 30, 1998(now
U.S. Pat. No. 6,204,232), and claims priority under 35 U.S.C. 119
of Danish application no. 1240/97, filed on Oct. 30, 1997, Danish
application no. PA 1998 00936, filed on Jul. 14, 1998, U.S.
provisional application No. 60/064,662, filed on Nov. 6, 1997 and
U.S. provisional application No. 60/093,234, filed on Jul. 17,
1998, 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 with higher activity at
medium temperatures and/or high pH.
BACKGROUND OF THE INVENTION
[0003] .alpha.-Amylases (.alpha.-1,4-glucan-4-glucanohydrolases, EC
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.
[0004] There is a very extensive body of patent and scientific
literature relating to this industrially very important class of
enzymes. A number of .alpha.-amylases such as Termamyl-like
.alpha.-amylases variants are known from e.g. WO 90/11352, WO
95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
[0005] Among more recent disclosures relating to .alpha.-amylases,
WO 96/23874 provides three-dimensional, X-ray crystal structural
data for a Termamyl-like .alpha.-amylase which consists of the 300
N-terminal amino acid residues of the B. amyloliquefaciens
.alpha.-amylase (BAN.TM.) and amino acids 301-483 of the C-terminal
end of the B. licheniformis .alpha.-amylase comprising the amino
acid sequence (the latter being available commercially under the
tradename Termamyl.TM.), and which is thus closely related to the
industrially important Bacillus .alpha.-amylases (which in the
present context are embraced within the meaning of the term
"Termamyl-like .alpha.-amylases", and which include, inter alia,
the B. licheniformis, B. amyloliquefaciens (BAN.TM.) and B.
stearothermophilus (BSG.TM.) .alpha.-amylases). WO 96/23874 further
describes methodology for designing, on the basis of an analysis of
the structure of a parent Termamyl-like .alpha.-amylase, variants
of the parent Termamyl-like .alpha.-amylase which exhibit altered
properties relative to the parent.
BRIEF DISCLOSURE OF THE INVENTION
[0006] The present invention relates to novel .alpha.-amylolytic
variants (mutants) of a Termamyl-like .alpha.-amylase which exhibit
improved wash performance (relative to the parent .alpha.-amaylase)
at high pH and at a medium temperature.
[0007] The term "medium temperature" means in the context of the
invention a temperature from 10.degree. C. to 60.degree. C.,
preferably 20.degree. C. to 50.degree. C., especially 30-40.degree.
C.
[0008] The term "high pH" means the alkaline pH which today are
used for washing, more specifically from about pH 8 to 10.5.
[0009] In the context of the invention a "low temperature
.alpha.-amylase" means an .alpha.-amylase which has an relative
optimum activity in the temperature range from 0-30.degree. C.
[0010] In the context of the invention a "medium temperature
.alpha.-amylase" means an .alpha.-amylase which has an optimum
activity in the temperature range from 30-60.degree. C. For
instance, SP690 and SP722 .alpha.-amylases, respectively, are
"medium temperature .alpha.-amylases.
[0011] In the context of the invention a "high temperature
.alpha.-amylase" is an .alpha.-amylase having the optimum activity
in the temperature range from 60-110.degree. C. For instance,
Termamyl is a "high temperature .alpha.-amylase.
[0012] Alterations in properties which may be achieved in variants
(mutants) of the invention are alterations in: the stability of the
Termamyl-like .alpha.-amylase at a pH from 8 to 10.5, and/or the
Ca.sup.2+ stability at pH 8 to 10.5, and/or the specific activity
at temperatures from 10 to 60.degree. C., preferably 20-50.degree.
C., especially 30-40.degree. C.
[0013] It should be noted that the relative temperature optimum
often is dependent on the specific pH used. In other words the
relative temperature optimum determined at, e.g., pH 8 may be
substantially different from the relative temperature optimum
determined at, e.g., pH 10.
The Temperature's Influence on the Enzymatic Activity
[0014] The dynamics in the active site and surroundings are
dependent on the temperature and the amino acid composition and of
strong importance for the relative temperature optimum of an
enzyme. By comparing the dynamics of medium and high temperature
.alpha.-amylases, regions of importance for the function of high
temperature .alpha.-amylases at medium temperatures can be
determined. The temperature activity profile of the SP722
.alpha.-amaylase (SEQ ID NO: 2) and the B. licheniformis
.alpha.-amylase (available from Novo Nordisk as Termamyl.RTM.) (SEQ
ID NO: 4) are shown in FIG. 2.
[0015] The relative temperature optimum of SP722 in absolute
activities are shown to be higher at medium range temperatures
(30-60.degree. C.) than the homologous B. licheniformis
.alpha.-amylase, which have an optimum activity around
60-100.degree. C. The profiles are mainly dependent on the
temperature stability and the dynamics of the active site residues
and their surroundings. Further, the activity profiles are
dependent on the pH used and the pKa of the active site
residues.
[0016] In the first aspect the invention relates to a variant of a
parent Termamyl-like .alpha.-amylase, which variant has
.alpha.-amylase activity, said variant comprises one or more
mutations corresponding to the following mutations in the amino
acid sequence shown in SEQ ID NO: 2:
T141, K142, F143, D144, F145, P146, G147, R148, G149, Q174, R181,
G182, D183, G184, K185, A186, W189, 5193, N195, H107, K108, G109,
D166, W167, D168, Q169, 5170, R171, Q172, F173, F267, W268, K269,
N270, D271, L272, G273, A274, L275, K311, E346, K385, G456, N457,
K458, P459, G460, T461, V462, T463.
[0017] A variant of the invention have one or more of the following
substitutions or deletions:
T141A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
K142A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
F143A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
D144A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F145A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
P146A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G147A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R148A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G149A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R181*,A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G182*,A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D183*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G184*,A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W189A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
S193A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
H107A,D,R,N,C,E,Q,G,I,L,K,M,F,P,S,T,W,Y,V;
K108A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G109A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D166A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W167A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
D168A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q169A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
S170A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
R171A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q172A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F173A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
Q174*,A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F267A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
W268A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D271A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L272A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
G273A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
A274D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L275A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
K311A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
E346A,D,R,N,C,Q,G,H,I,K,L,M,F,P,S,T,W,Y,V;
K385A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G456A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
N457A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G460A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
V462A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;
T463A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.
[0018] Preferred are variants having one or more of the following
substitutions or deletions:
K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R;
K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; D183*; G184*;
K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R;
W189T,S,N,Q.
[0019] Especially preferred are variants having a deletion in
positions D183 and G184 and further one or more of the following
substitutions or deletions:
K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q; K385R;
K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N;
K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R;
W189T,S,N,Q.
[0020] The variants of the invention mentioned above exhibits an
alteration in at least one of the following properties relative to
the parent .alpha.-amylase:
i) improved pH stability at a pH from 8 to 10.5; and/or ii)
improved Ca.sup.2+ stability at pH 8 to 10.5, and/or iii) increased
specific activity at temperatures from 10 to 60.degree. C.,
preferably 20-50.degree. C., especially 30-40.degree. C. Further,
details will be described below.
[0021] The invention further relates to DNA constructs encoding
variants of the invention; to methods for preparing variants of the
invention; and to the use of variants of the invention, alone or in
combination with other enzymes, in various industrial products or
processes, e.g., in detergents or for starch liquefaction.
[0022] In a final aspect the invention relates to a method of
providing .alpha.-amylases with altered pH optimum, and/or altered
temperature optimum, and/or improved stability.
Nomenclature
[0023] 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:
Original amino acid(s):position(s):substituted amino acid(s)
[0024] According to this nomenclature, for instance the
substitution of alanine for asparagine in position 30 is shown
as:
[0025] Ala30Asn or A30N
a deletion of alanine in the same position is shown as:
[0026] Ala30* or A30*
and insertion of an additional amino acid residue, such as lysine,
is shown as:
[0027] Ala30AlaLys or A30AK
[0028] 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).
[0029] 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:
[0030] *36Asp or *36D
for insertion of an aspartic acid in position 36 Multiple mutations
are separated by plus signs, i.e.:
[0031] Ala30Asp+Glu34Ser or A30N+E34S
representing mutations in positions 30 and 34 substituting alanine
and glutamic acid for asparagine and serine, respectively.
[0032] When one or more alternative amino acid residues may be
inserted in a given position it is indicated as
[0033] A30N,E or
[0034] A30N or A30E
[0035] Furthermore, when a position suitable for modification is
identified herein without any specific modification being
suggested, it is to be understood that any amino acid residue may
be substituted for the amino acid residue present in the position.
Thus, for instance, when a modification of an alanine in position
30 is mentioned, but not specified, it is to be understood that the
alanine may be deleted or substituted for any other amino acid,
i.e., any one of:
R,N,D,A,C,Q,E,G,H,I,L,K,M,F,P,S,T,W,Y,V.
BRIEF DESCRIPTION OF THE DRAWING
[0036] FIGS. 1 (a)-(c) is an alignment of the amino acid sequences
of six parent Termamyl-like .alpha.-amylases. The numbers on the
extreme left designate the respective amino acid sequences as
follows:
[0037] 1: SEQ ID NO: 2
[0038] 2: Kaoamyl
[0039] 3: SEQ ID NO: 1
[0040] 4: SEQ ID NO: 5
[0041] 5: SEQ ID NO: 4
[0042] 6: SEQ ID NO: 3.
[0043] FIG. 2 shows the temperature activity profile of SP722 (SEQ
ID NO: 2) (at pH 9) and B. licheniformis .alpha.-amylase (SEQ ID
NO: 4) (at pH 7.3).
[0044] FIG. 3 shows the temperature profile for SP690 (SEQ ID NO:
1), SP722 (SEQ ID NO: 2), B. licheniformis .alpha.-amylase (SEQ ID
NO: 4) at pH 10.
[0045] FIGS. 4(a)-(b) is an alignment of the amino acid sequences
of five .alpha.-amylases. The numbers on the extreme left designate
the respective amino acid sequences as follows:
[0046] 1: amyp_mouse
[0047] 2: amyp_rat
[0048] 3: amyp_pig porcine pancreatic alpha-amylase (PPA)
[0049] 4: amyp_human
[0050] 5: amy_altha A. haloplanctis alpha-amylase (AHA)
DETAILED DISCLOSURE OF THE INVENTION
The Termamyl-Like .alpha.-Amylase
[0051] It is well known that a number of .alpha.-amylases produced
by Bacillus spp. are highly homologous on the amino acid level. For
instance, the B. licheniformis .alpha.-amylase comprising the amino
acid sequence shown in SEQ ID NO: 4 (commercially available as
Termamyl.TM.) has been found to be about 89% homologous with the B.
amyloliquefaciens .alpha.-amylase comprising the amino acid
sequence shown in SEQ ID NO: 5 and about 79% homologous with the B.
stearothermophilus .alpha.-amylase comprising the amino acid
sequence shown in SEQ ID NO: 3. Further homologous .alpha.-amylases
include an .alpha.-amylase derived from a strain of the Bacillus
sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which
are described in detail in WO 95/26397, and the .alpha.-amylase
described by Tsukamoto et al., Biochemical and Biophysical Research
Communications, 151 (1988), pp. 25-31, (see SEQ ID NO: 6).
[0052] 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 B.
licheniformis .alpha.-amylases are comprised in the products
Optitherm.TM. and Takatherm.TM. (available from 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).
[0053] 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".
[0054] Accordingly, in the present context, the term "Termamyl-like
.alpha.-amylase" is intended to indicate an .alpha.-amylase which,
at the amino acid level, exhibits a substantial homology to
Termamyl.TM., i.e., the B. licheniformis .alpha.-amylase having the
amino acid sequence shown in SEQ ID NO:4 herein. In other words,
all the following .alpha.-amylases which has the amino acid
sequences shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8 herein, or
the amino acid sequence shown in SEQ ID NO: 1 of WO 95/26397 (the
same as the amino acid sequence shown as SEQ ID NO: 7 herein) or in
SEQ ID NO: 2 of WO 95/26397 (the same as the amino acid sequence
shown as SEQ ID NO: 8 herein) or in Tsukamoto et al., 1988, (which
amino acid sequence is shown in SEQ ID NO: 6 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%,
e.g., at least 85%, at least 90% or at least 95% homology with at
least one of said amino acid sequences shown in SEQ ID NOS: 1-8
and/or ii) displays immunological cross-reactivity with an antibody
raised against at least one of said .alpha.-amylases, and/or iii)
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: 9, 10, 11, or 12 of the present application (which
encoding sequences encode the amino acid sequences shown in SEQ ID
NOS: 1, 2, 3, 4 and 5 herein, respectively), from SEQ ID NO: 4 of
WO 95/26397 (which DNA sequence, together with the stop codon TAA,
is shown in SEQ ID NO: 13 herein and encodes the amino acid
sequence shown in SEQ ID NO: 8 herein) and from SEQ ID NO: 5 of WO
95/26397 (shown in SEQ ID NO: 14 herein), respectively.
[0055] In connection with property i), the "homology" may be
determined by use of any conventional algorithm, preferably by use
of the GAP programme from the GCG package version 7.3 (June 1993)
using default values for GAP penalties, which is a GAP creation
penalty of 3.0 and GAP extension penalty of 0.1, (Genetic Computer
Group (1991) Programme Manual for the GCG Package, version 7, 575
Science Drive, Madison, Wis., USA 53711).
[0056] A structural alignment between Termamyl (SEQ ID NO: 4) and a
Termamyl-like .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, A E, PROTEIN SCIENCE Vol. 7, No. 1 pp. 142-149 (1998).
[0057] Property ii) of the .alpha.-amylase, i.e., the immunological
cross reactivity, may be assayed using an antibody raised against,
or reactive with, at least one epitope of the relevant
Termamyl-like .alpha.-amylase. The antibody, which may either be
monoclonal or polyclonal, may be produced by methods known in the
art, e.g., as described by Hudson et al., Practical Immunology,
Third edition (1989), Blackwell Scientific Publications. The
immunological cross-reactivity may be determined using assays known
in the art, examples of which are Western Blotting or radial
immunodiffusion assay, e.g., as described by Hudson et al., 1989.
In this respect, immunological cross-reactivity between the
.alpha.-amylases having the amino acid sequences SEQ ID NOS: 1, 2,
3, 4, 5, 6, 7, or 8, respectively, has been found.
[0058] The oligonucleotide probe used in the characterisation of
the Termamyl-like .alpha.-amylase in accordance with property iii)
above may suitably be prepared on the basis of the full or partial
nucleotide or amino acid sequence of the .alpha.-amylase in
question.
[0059] 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.
[0060] In the present context, "derived from" is intended not only
to indicate an .alpha.-amylase produced or producible by a strain
of the organism in question, but also an .alpha.-amylase encoded by
a DNA sequence isolated from such strain and produced in a host
organism transformed with said DNA sequence. Finally, the term is
intended to indicate an .alpha.-amylase which is encoded by a DNA
sequence of synthetic and/or cDNA origin and which has the
identifying characteristics of the .alpha.-amylase in question. The
term is also intended to indicate that the parent .alpha.-amylase
may be a variant of a naturally occurring .alpha.-amylase, i.e. a
variant which is the result of a modification (insertion,
substitution, deletion) of one or more amino acid residues of the
naturally occurring .alpha.-amylase.
Parent Hybrid .alpha.-Amylases
[0061] The parent .alpha.-amylase (i.e., backbone .alpha.-amylase)
may 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.
[0062] The parent hybrid .alpha.-amylase may be one which on the
basis of amino acid homology and/or immunological cross-reactivity
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.
[0063] 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, e.g., be any of
those specific Termamyl-like .alpha.-amylase referred to
herein.
[0064] 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: 5 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: 4, 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: 4, 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: 5; 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:
3 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: 4.
[0065] 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). Increased activity was achieved by substituting one or
more of the following positions of the above hybrid .alpha.-amylase
(BAN:1-300/Termamyl:301-483): Q360, F290, and N102. Particularly
interesting substitutions are one or more of the following
substitutions: Q360E,D; F290A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T;
N102D,E;
[0066] The corresponding positions in the SP722 .alpha.-amylase
shown in SEQ ID NO: 2 are one or more of: 5365, Y295, N106.
Corresponding substitutions of particular interest in said
.alpha.-amylase shown in SEQ ID NO: 2 are one or more of: S365D,E;
Y295 A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; and N106D,E.
[0067] The corresponding positions in the SP690 .alpha.-amylase
shown in SEQ ID NO: 1 are one or more of: 5365, Y295, N106. The
corresponding substitutions of particular interest are one or more
of: S365D,E; Y295 A,C,D,E,G,H,I,K,L,M,N,P,Q,R,S,T; N106D,E.
[0068] The above mentioned non-Termamyl-like .alpha.-amylase may,
e.g., be a fungal .alpha.-amylase, a mammalian or a plant
.alpha.-amylase or a bacterial .alpha.-amylase (different from a
Termamyl-like .alpha.-amylase). Specific examples of such
.alpha.-amylases include the Aspergillus oryzae TAKA
.alpha.-amylase, the A. niger acid .alpha.-amylase, the Bacillus
subtilis .alpha.-amylase, the porcine pancreatic .alpha.-amylase
and a barley .alpha.-amylase. All of these .alpha.-amylases have
elucidated structures which are markedly different from the
structure of a typical Termamyl-like .alpha.-amylase as referred to
herein.
[0069] The fungal .alpha.-amylases mentioned above, i.e., derived
from A. niger and A. oryzae, are highly homologous on the amino
acid level and generally considered to belong to the same family of
.alpha.-amylases. The fungal .alpha.-amylase derived from
Aspergillus oryzae is commercially available under the tradename
Fungamyl.TM.
[0070] Furthermore, when a particular variant of a Termamyl-like
.alpha.-amylase (variant of the invention) is referred to--in a
conventional manner--by reference to modification (e.g., deletion
or substitution) of specific amino acid residues in the amino acid
sequence of a specific Termamyl-like .alpha.-amylase, it is to be
understood that variants of another Termamyl-like .alpha.-amylase
modified in the equivalent position(s) (as determined from the best
possible amino acid sequence alignment between the respective amino
acid sequences) are encompassed thereby.
[0071] In a preferred embodiment of the invention the
.alpha.-amylase backbone is derived from B. licheniformis (as the
parent Termamyl-like .alpha.-amylase), 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: 4.
Altered Properties of Variants of the Invention
[0072] The following discusses the relationship between mutations
which are present in variants of the invention, and desirable
alterations in properties (relative to those a parent Termamyl-like
.alpha.-amylase) which may result therefrom.
Improved Stability at pH 8-10.5
[0073] In the context of the present invention, mutations
(including amino acid substitutions) of importance with respect to
achieving improved stability at high pH (i.e., pH 8-10.5) include
mutations corresponding to mutations in one or more of the
following positions in SP722 .alpha.-amylase (having the amino acid
sequence shown in SEQ ID NO: 2): T141, K142, F143, D144, F145,
P146, G147, R148, G149, R181, A186, 5193, N195, K269, N270, K311,
K458, P459, T461.
[0074] The variant of the invention have one or more of the
following substitutions (using the SEQ ID NO: 2 numbering):
T141A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
K142A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
F143A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
D144A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F145A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
P146A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G147A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
R148A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G149A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
K181A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
A186D,R,N,C,E,Q,G,H,I,L,P,K,M,F,S,T,W,Y,V;
S193A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K311A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.
[0075] Preferred high pH stability variants include one or more of
the following substitutions in the SP722 .alpha.-amylase (having
the amino acid sequence shown in SEQ ID NO: 2):
K142R, R181S, A186T, S193P, N195F, K269R, N270Y, K311R, K458R,
P459T and T461P.
[0076] In specific embodiments the Bacillus strain NCIB 12512
.alpha.-amylase having the sequence shown in SEQ ID NO: 1, or the
B. stearothermophilus .alpha.-amylase having the sequence shown in
SEQ ID NO: 3, or the B. licheniformis .alpha.-amylase having the
sequence shown in SEQ ID NO: 4, or the B. amyloliquefaciens
.alpha.-amylase having the sequence shown in SEQ ID NO: 5 is used
as the backbone, i.e., parent Termamyl-like .alpha.-amylase, for
these mutations.
[0077] As can been seen from the alignment in FIG. 1 the B.
stearothermophilus .alpha.-amylase already has a Tyrosine at
position corresponding to N270 in SP722. Further, the Bacillus
strain NCIB 12512 .alpha.-amylase, the B. stearothermophilus
.alpha.-amylase, the B. licheniformis .alpha.-amylase and the B.
amyloliquefaciens .alpha.-amylase already have Arginine at position
corresponding to K458 in SP722. Furthermore, the B. licheniformis
.alpha.-amylase already has a Proline at position corresponding to
T461 in SP722. Therefore, for said .alpha.-amylases these
substitutions are not relevant.
[0078] .alpha.-amylase variants with improved stability at high pH
can be constructed by making substitutions in the regions found
using the molecular dynamics simulation mentioned in Example 2. The
simulation depicts the region(s) that has a higher flexibility or
mobility at high pH (i.e., pH 8-10.5) when compared to medium
pH.
[0079] By using the structure of any bacterial alpha-amylase with
homology (as defined below) to the Termamyl-like .alpha.-amylase
(BA2), of which the 3D structure is disclosed in Appendix 1 of WO
96/23874 (from Novo Nordisk), it is possible to modelbuild the
structure of such alpha-amylase and to subject it to molecular
dynamics simulations. The homology of said bacterial
.alpha.-amylase may be at least 60%, preferably be more than 70%,
more preferably more than 80%, most preferably more than 90%
homologous to the above mentioned Termamyl-like .alpha.-amylase
(BA2), measured using the UWGCG GAP program from the GCG package
version 7.3 (June 1993) using default values for GAP penalties
[Genetic Computer Group (1991) Programme Manual for the GCG
Package, version 7, 575 Science Drive, Madison, Wis., USA 53711].
Substitution of the unfavorable residue for another would be
applicable.
Improved Ca.sup.2+ Stability at pH 8-10.5
[0080] Improved Ca.sup.2+ stability means the stability of the
enzyme under Ca.sup.2+ depletion has been improved. In the context
of the present invention, mutations (including amino acid
substitutions) of importance with respect to achieving improved
Ca.sup.2+ stability at high pH include mutation or deletion in one
or more positions corresponding to the following positions in the
SP722 .alpha.-amylase having the amino acid sequence shown in SEQ
ID NO: 2: R181, G182, D183, G184, K185, A186, W189, N195, N270,
E346, K385, K458, P459.
[0081] A variant of the invention have one or more of the following
substitutions or deletions:
R181*,A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G182*,A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D183*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
G184*,A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W189A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
N270A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
E346A,R,D,N,C,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K385A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
K458A,R,D,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,R,D,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V.
[0082] Preferred are variants having one or more of the following
substitutions or deletions:
R181Q,N; G182T,S,N; D183*; G184*;
K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V;
W189T,S,N,Q; N195F, N270R,D; E346Q; K385R; K458R; P459T.
[0083] In specific embodiments the Bacillus strain NCIB 12512
.alpha.-amylase having the sequence shown in SEQ ID NO: 1, or the
B. amyloliquefaciens .alpha.-amylase having the sequence shown in
SEQ ID NO: 5, or the B. licheniformis .alpha.-amylase having the
sequence shown in SEQ ID NO: 4 are used as the backbone for these
mutations.
[0084] As can been seen from the alignment in FIG. 1 the B.
licheniformis .alpha.-amylase does not have the positions
corresponding to D183 and G184 in SP722. Therefore for said
.alpha.-amylases these deletions are not relevant.
[0085] In a preferred embodiment the variant is the Bacillus strain
NCIB 12512 .alpha.-amylase with deletions in D183 and G184 and
further one of the following substitutions: R181Q,N and/or
G182T,S,N and/or D183*; G184* and/or
K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V and/or A186T,S,N,I,V
and/or W189T,S,N,Q and/or N195F and/or N270R,D and/or E346Q and/or
K385R and/or K458R and/or P459T.
Increased Specific Activity at Medium Temperature
[0086] In a further aspect of the present invention, important
mutations with respect to obtaining variants exhibiting increased
specific activity at temperatures from 10-60.degree. C., preferably
20-50.degree. C., especially 30-40.degree. C., include mutations
corresponding to one or more of the following positions in the
SP722 .alpha.-amylase having the amino acid sequence shown in SEQ
ID NO: 2:
H107, K108, G109, D166, W167, D168, Q169, 5170, R171, Q172, F173,
Q174, D183, G184, N195, F267, W268, K269, N270, D271, L272, G273,
A274, L275, G456, N457, K458, P459, G460, T461, V462, T463.
[0087] The variant of the invention have one or more of the
following substitutions:
H107A,D,R,N,C,E,Q,G,I,L,K,M,F,P,S,T,W,Y,V;
K108A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
G109A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
D166A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
W167A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
D168A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q169A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
S170A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,T,W,Y,V;
R171A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
Q172A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F173A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
Q174*,A,D,R,N,C,E,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D183*,A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
G184*,A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
N195A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
F267A,D,R,N,C,E,Q,G,H,I,L,K,M,P,S,T,W,Y,V;
W268A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,Y,V;
K269A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
N270A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
D271A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L272A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
G273A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
A274D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
L275A,D,R,N,C,E,Q,G,H,I,K,M,F,P,S,T,W,Y,V;
G456A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
N457A,D,R,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,V;
K458A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,V;
P459A,D,R,N,C,E,Q,G,H,I,L,K,M,F,S,T,W,Y,V;
G460A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,V;
T461A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V;
V462A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y;
T463A,D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,W,Y,V.
[0088] Preferred variants has one or more of the following
substitutions or deletions: Q174*, D183*, G184*, K269S.
[0089] In a specific embodiment the B. licheniformis
.alpha.-amylase having the sequence shown in SEQ ID NO: 4 is used
as the backbone for these mutations.
General Mutations in Variants of the Invention: Increased Specific
Activity at Medium Temperatures
[0090] The particularly interesting amino acid substitution are
those that increase the mobility around the active site of the
enzyme. This is accomplished by changes that disrupt stabilizing
interaction in the vicinity of the active site, i.e., within
preferably 10 .ANG. or 8 .ANG. or 6 .ANG. or 4 .ANG. from any of
the residues constituting the active site.
[0091] Examples are mutations that reduce the size of side chains,
such as
Ala to Gly,
Val to Ala or Gly,
Ile or Leu to Val, Ala, or Gly
Thr to Ser
[0092] Such mutations are expected to cause increased flexibility
in the active site region either by the introduction of cavities or
by the structural rearrangements that fill the space left by the
mutation.
[0093] It may be preferred that a variant of the invention
comprises one or more modifications in addition to those outlined
above. Thus, it may be advantageous that one or more Proline
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.
[0094] Analogously, it may be preferred that one or more Cysteine
residues present among the amino acid residues with which the
parent .alpha.-amylase is modified is/are replaced with a
non-Cysteine residue such as Serine, Alanine, Threonine, Glycine,
Valine or Leucine.
[0095] 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: 4 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: 4 by an Arg.
[0096] It will be understood that the present invention encompasses
variants incorporating two or more of the above outlined
modifications.
[0097] Furthermore, it may be advantageous to introduce
point-mutations in any of the variants described herein.
.alpha.-Amylase Variants Having Increased Mobility Around the
Active Site:
[0098] The mobility of .alpha.-amylase variants of the invention
may be increased by replacing one or more amino acid residue at one
or more positions close to the substrate site. These positions are
(using the SP722 .alpha.-amylase (SEQ ID NO: 2) numbering): V56,
K108, D168, Q169, Q172, L201, K269, L272, L275, K446, P459.
[0099] Therefore, in an aspect the invention relates to variants
being mutated in one or more of the above mentioned positions.
[0100] Preferred substitutions are one or more of the
following:
V56A,G,S,T;
K108A,D,E,Q,G,H,I,L,M,N,S,T,V;
D168A,G,I,V,N,S,T;
Q169A,D,G,H,I,L,M,N,S,T,V;
Q172A,D,G,H,I,L,M,N,S,T,V;
L201A,G,I,V,S,T;
K269A,D,E,Q,G,H,I,L,M,N,S,T,V;
L272A,G,I,V,S,T;
L275A,G,I,V,S,T;
Y295A,D,E,Q,G,H,I,L,M,N,F,S,T,V;
K446A,D,E,Q,G,H,I,L,M,N,S,T,V;
P459A,G,I,L,S,T,V.
[0101] In specific embodiments of the invention the Bacillus strain
NCIB 12512 .alpha.-amylase having the sequence shown in SEQ ID NO:
1, or the B. stearothermophilus .alpha.-amylase having the sequence
shown in SEQ ID NO: 3, or the B. licheniformis .alpha.-amylase
having the sequence shown in SEQ ID NO: 4, or the B.
amyloliquefaciens .alpha.-amylase having the sequence shown in SEQ
ID NO: 5 are used as the backbone for these mutations.
[0102] As can been seen from the alignment in FIG. 1 the B.
licheniformis .alpha.-amylase and the B. amyloliquefaciens
.alpha.-amylase have a Glutamine at position corresponding to K269
in SP722. Further, the B. stearothermophilus .alpha.-amylase has a
Serine at position corresponding to K269 in SP722. Therefore, for
said .alpha.-amylases these substitutions are not relevant.
[0103] Furthermore, as can been seen from the alignment in FIG. 1
the B. amyloliquefaciens .alpha.-amylase has an Alanine at position
corresponding to L272 in SP722, and the B. stearothermophilus
.alpha.-amylase has a Isoleucine at the position corresponding to
L272 in SP722. Therefore, for said .alpha.-amylases these
substitutions are not relevant.
[0104] As can been seen from the alignment in FIG. 1, the Bacillus
strain 12512 .alpha.-amylase has a Isoleucine at position
corresponding to L275 in SP722. Therefore for said .alpha.-amylase
this substitution is not relevant.
[0105] As can been seen from the alignment in FIG. 1 the B.
amyloliquefaciens .alpha.-amylase has a Phenylalanine at position
corresponding to Y295 in SP722. Further, the B. stearothermophilus
.alpha.-amylase has an Asparagine at position corresponding to Y295
in SP722. Therefore, for said .alpha.-amylases these substitutions
are not relevant.
[0106] As can been seen from the alignment in FIG. 1 the B.
licheniformis .alpha.-amylase and the B. amyloliquefaciens
.alpha.-amylase have a Asparagine at position corresponding to K446
in SP722. Further, the B. stearothermophilus .alpha.-amylase has a
Histidine at position corresponding to K446 in SP722. Therefore,
for said .alpha.-amylases these substitutions are not relevant.
[0107] As can been seen from the alignment in FIG. 1 the B.
licheniformis .alpha.-amylase, the B. amyloliquefaciens
.alpha.-amylase and the B. stearothermophilus .alpha.-amylase have
a Serine at position corresponding to P459 in SP722. Further, the
Bacillus strain 12512 .alpha.-amylase has a Threonine at position
corresponding to P459 in SP722. Therefore, for said
.alpha.-amylases these substitutions are not relevant.
Stabilization of Enzymes Having High Activity at Medium
Temperatures
[0108] In a further embodiment the invention relates to improving
the stability of low temperature .alpha.-amylases (e.g, Alteromonas
haloplanctis (Feller et al., (1994), Eur. J. Biochem 222:441-447),
and medium temperature .alpha.-amylases (e.g., SP722 and SP690)
possessing medium temperature activity, i.e., commonly known as
psychrophilic enzymes and mesophilic enzymes. The stability can for
this particular enzyme class be understood either as
thermostability or the stability at Calcium depletion
conditions.
[0109] Typically, enzymes displaying the high activity at medium
temperatures also display severe problems under conditions that
stress the enzyme, such as temperature or Calcium depletion.
[0110] Consequently, the objective is to provide enzymes that at
the same time display the desired high activity at medium
temperatures without loosing their activity under slightly stressed
conditions.
[0111] The activity of the stabilized variant measured at medium
temperatures should preferably be between 100% or more and 50%, and
more preferably between 100% or more and 70%, and most preferably
between 100% or more and 85% of the original activity at that
specific temperature before stabilization of the enzyme and the
resulting enzyme should withstand longer incubation at stressed
condition than the wild type enzyme.
[0112] Contemplated enzymes include .alpha.-amylases of, e.g.,
bacterial or fungal origin.
[0113] An example of such a low temperature .alpha.-amylase is the
one isolated from Alteromonas haloplanctis (Feller et al., (1994),
Eur. J. Biochem 222:441-447). The crystal structure of this
alpha-amylase has been solved (Aghajari et al., (1998), Protein
Science 7:564-572).
[0114] The A. haloplanctis alpha-amylase (5 in alignment shown in
FIG. 4) has a homology of approximately 66% to porcine pancreatic
alpha-amylase (PPA) (3 in the alignment shown in FIG. 4). The PPA
3D structure is known, and can be obtained from Brookhaven database
under the name 10SE or 1DHK. Based on the homology to other more
stable alpha amylases, stabilization of "the low temperature highly
active enzyme" from Alteromonas haloplanctis alpha-amylase, can be
obtained and at the same time retaining the desired high activity
at medium temperatures.
[0115] FIG. 4 shown a multiple sequence alignments of five
.alpha.-amylases, including the AHA and the PPA .alpha.-amylase.
Specific mutations giving increased stability in Alteromonas
haloplanctis alpha-amylase:
T66P, Q69P, R155P, Q177R, A205P, A232P, L243R, V295P, S315R.
Methods for Preparing .alpha.-Amylase Variants
[0116] Several methods for introducing mutations into genes are
known in the art. After a brief discussion of the cloning of
.alpha.-amylase-encoding DNA sequences, methods for generating
mutations at specific sites within the .alpha.-amylase-encoding
sequence will be discussed.
Cloning a DNA Sequence Encoding an .alpha.-Amylase Cloning a DNA
Sequence Encoding an .alpha.-Amylase Cloning a DNA Sequence
Encoding an .alpha.-Amylase Cloning a DNA Sequence Encoding an
.alpha.-Amylase Cloning a DNA Sequence Encoding an .alpha.-Amylase
Cloning a DNA Sequence Encoding an .alpha.-Amylase Cloning a DNA
Sequence Encoding an .alpha.-Amylase Cloning a DNA Sequence
Encoding an .alpha.-Amylase Cloning a DNA Sequence Encoding an
.alpha.-Amylase
[0117] 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.
[0118] 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.
[0119] 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 (1981) or the method described by Matthes et al. (1984).
In the phosphoroamidite method, oligonucleotides are synthesized,
e.g., in an automatic DNA synthesizer, purified, annealed, ligated
and cloned in appropriate vectors.
[0120] 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. (1988).
Expression of .alpha.-Amylase Variants
[0121] 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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).
[0129] 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.
[0130] 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.
[0131] Examples of suitable bacteria are Gram positive bacteria
such as Bacillus subtilis, Bacillus licheniformis, Bacillus lentos,
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.
[0132] 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.
[0133] 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.
[0134] 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).
[0135] The .alpha.-amylase variant secreted from the host cells may
conveniently be recovered from the culture medium by well-known
procedures, including separating the cells from the medium by
centrifugation or filtration, and precipitating proteinaceous
components of the medium by means of a salt such as ammonium
sulphate, followed by the use of chromatographic procedures such as
ion exchange chromatography, affinity chromatography, or the
like.
INDUSTRIAL APPLICATIONS
[0136] The .alpha.-amylase variants of this invention possesses
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.
[0137] Numerous variants are particularly useful in the production
of sweeteners and ethanol from starch, and/or for textile desizing.
Conditions for conventional starch-conversion processes, including
starch liquefaction and/or saccharification processes, are
described in, e.g., U.S. Pat. No. 3,912,590 and in EP patent
publications Nos. 252,730 and 63,909.
Detergent Compositions
[0138] 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.
[0139] Detergent compositions comprising a variant of the invention
may additionally comprise one or more other enzymes, such as a
lipase, cutinase, protease, cellulase, peroxidase or laccase,
and/or another .alpha.-amylase.
[0140] .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
0.00001-1 mg (calculated as pure, active enzyme protein) of
.alpha.-amylase per liter of wash/dishwash liquor using
conventional dosing levels of detergent.
[0141] The invention also relates to a method of providing
.alpha.-amylases with 1) altered pH optimum, and/or 2) altered
temperature optimum, and/or 3) improved stability, comprising the
following steps:
i) identifying (a) target position(s) and/or region(s) for mutation
of the .alpha.-amylase by comparing the molecular dynamics of two
or more .alpha.-amylase 3D structures having substantially
different pH, temperature and/or stability profiles, ii)
substituting, adding and/or deleting one or more amino acids in the
identified position(s) and/or region(s).
[0142] In embodiment of the invention a medium temperature
.alpha.-amylase is compared with a high temperature
.alpha.-amylase. In another embodiment a low temperature
.alpha.-amylase is compared with either a medium or a high
temperature .alpha.-amylase.
[0143] The .alpha.-amylases compared should preferably be at least
70%, preferably 80%, up to 90%, such as up to 95%, especially 95%
homologous with each other.
[0144] The .alpha.-amylases compared may be Termamyl-like
.alpha.-amylases as defined above. In specific embodiment the
.alpha.-amylases compared are the .alpha.-amylases shown in SEQ ID
NO: 1 to SEQ ID NO: 8.
[0145] In another embodiment the stability profile of the
.alpha.-amylases in question compared are the Ca.sup.2+ dependency
profile.
Materials and Methods
Enzymes:
[0146] SP722: (SEQ ID NO: 2, available from Novo Nordisk)
Termamyl.TM. (SEQ ID NO: 4, available from Novo Nordisk) SP690:
(SEQ ID NO: 1, available from Novo Nordisk) Bacillus subtilis
SHA273: see WO 95/10603
Plasmids
[0147] pJE1 contains the gene encoding a variant of SP722
.alpha.-amylase (SEQ ID NO: 2): viz. deletion of 6 nucleotides
corresponding to amino acids D183-G184 in the mature protein.
Transcription of the JE1 gene is directed from the amyL promoter.
The plasmid further more contains the origin of replication and
cat-gene conferring resistance towards kanamycin obtained from
plasmid pUB110 (Gryczan, T J et al. (1978), J. Bact.
134:318-329).
Methods:
[0148] Construction of Library Vector pDorK101
[0149] The E. coli/Bacillus shuttle vector pDorK101 (described
below) can be used to introduce mutations without expression of
.alpha.-amylase in E. coli and then be modified in such way that
the .alpha.-amylase is active in Bacillus. The vector was
constructed as follows: The JE1 encoding gene (SP722 with the
deletion of D183-G184) was inactivated in pJE1 by gene interruption
in the PstI site in the 5' coding region of the SEQ ID NO: 2: SP722
by a 1.2 kb fragment containing an E. coli origin of replication.
This fragment was PCR amplified from the pUC19 (GenBank Accession
#:X02514) using the forward primer: 5'-gacctgcagtcaggcaacta-3' and
the reverse primer: 5'-tagagtcgacctgcaggcat-3'. The PCR amplicon
and the pJE1 vector were digested with PstI at 37.degree. C. for
hours. The pJE1 vector fragment and the PCR fragment were ligated
at room temperature. for 1 hour and transformed in E. coli by
electrotransformation. The resulting vector is designated
pDorK101.
Filter Screening Assays
[0150] The assay can be used to screening of Termamyl-like
.alpha.-amylase variants having an improved stability at high pH
compared to the parent enzyme and Termamyl-like .alpha.-amylase
variants having an improved stability at high pH and medium
temperatures compared to the parent enzyme depending of the
screening temperature setting
High pH Filter Assay
[0151] 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 .mu.g/ml kanamycin at
37.degree. C. for at least 21 hours. The cellulose acetate layer is
located on the TY agar plate.
[0152] 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
glycin-NaOH buffer, pH 8.6-10.6 and incubated at room temperature
(can be altered from 10.degree.-60.degree. C.) for 15 min. The
cellulose acetate filters with colonies are stored on the TY-plates
at room temperature until use. After incubation, residual activity
is detected on plates containing 1% agarose, 0.2% starch in
glycin-NaOH buffer, pH 8.6-10.6. The assay plates with
nitrocellulose filters are marked the same way as the filter
sandwich and incubated for 2 hours. at room temperature. 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 rescreened twice under the same conditions as
the first screen.
Low Calcium Filter Assay
[0153] The Bacillus library 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 a relevant antibiotic,
e.g., kanamycin or chloramphenicol, at 37.degree. C. for at least
21 hours. The cellulose acetate layer is located on the TY agar
plate.
[0154] 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
carbonate/bicarbonate buffer pH 8.5-10 and with different EDTA
concentrations (0.001 mM-100 mM). The filters are incubated at room
temperature for 1 hour. The cellulose acetate filters with colonies
are stored on the TY-plates at room temperature until use. After
incubation, residual activity is detected on plates containing 1%
agarose, 0.2% starch in carbonate/bicarbonate buffer pH 8.5-10. The
assay plates with nitrocellulose filters are marked the same way as
the filter sandwich and incubated for 2 hours. at room temperature.
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 rescreened twice under the same
conditions as the first screen.
Method to Obtaining the Regions of Interest:
[0155] There are three known 3D structures of bacterial
.alpha.-amylases. Two of B. licheniformis .alpha.-amylase,
Brookhaven database 1BPL (Machius et al. (1995), J. Mol. Biol. 246,
p. 545-559) and 1VJS (Song et al. (1996), Enzymes for Carbohydrate
163 Engineering (Prog. Biotechnol. V 12). These two structures are
lacking an important piece of the structure from the so-called
B-domain, in the area around the two Calcium ions and one Sodium
ion binding sites. We have therefore used a 3D structure of an
.alpha.-amylase BA2 (WO 96/23874 which are a hybrid between BAN.TM.
(SEQ ID NO. 5) and B. licheniformis .alpha.-amylase (SEQ ID NO. 4).
On basis of the structure a model of B. licheniformis alpha amylase
and the SP722 .alpha.-amylase has been build.
Fermentation and Purification of .alpha.-Amylase Variants
[0156] Fermentation and purification may be performed by methods
well known in the art.
Stability Determination
[0157] All stability trials are made using the same set up. The
method are:
The enzyme is incubated under the relevant conditions (1-4).
[0158] 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 (0.1M 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.
[0159] The activity measured before incubation (0 minutes) is used
as reference (100%). The decline in percent is calculated as a
function of the incubation time. The table shows the residual
activity after, e.g., 30 minutes of incubation.
Specific Activity Determination
[0160] 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).
Assays for .alpha.-Amylase Activity
1. Phadebas Assay
[0161] .alpha.-amylase activity is determined by a method employing
Phadebas.RTM. tablets as substrate. Phadebas tablets (Phadebas.RTM.
Amylase Test, supplied by Pharmacia Diagnostic) contain a
cross-linked insoluble blue-colored starch polymer which has been
mixed with bovine serum albumin and a buffer substance and
tabletted.
[0162] For every single measurement one tablet is suspended in a
tube containing 5 ml 50 mM Britton-Robinson buffer (50 mM acetic
acid, 50 mM phosphoric acid, 50 mM boric acid, 0.1 mM CaCl.sub.2,
pH adjusted to the value of interest with NaOH). The test is
performed in a water bath at the temperature of interest. The
.alpha.-amylase to be tested is diluted in x ml of 50 mM
Britton-Robinson buffer. 1 ml of this .alpha.-amylase solution is
added to the 5 ml 50 mM Britton-Robinson buffer. The starch is
hydrolyzed by the .alpha.-amylase giving soluble blue fragments.
The absorbance of the resulting blue solution, measured
spectrophotometrically at 620 nm, is a function of the
.alpha.-amylase activity.
[0163] 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.
2. Alternative Method
[0164] .alpha.-amylase activity is determined by a method employing
the PNP-G7 substrate. PNP-G7 which is a abbreviation for
p-nitrophenyl-.alpha.,D-maltoheptaoside is a blocked
oligosaccharide which can be cleaved by an endo-amylase. Following
the cleavage, the .alpha.-Glucosidase included in the kit digest
the substrate to liberate a free PNP molecule which has a yellow
colour and thus can be measured by visible spectophometry at
.lamda.=405 nm. (400-420 nm.). Kits containing PNP-G7 substrate and
.alpha.-Glucosidase is manufactured by Boehringer-Mannheim (cat.
No. 1054635).
[0165] To prepare the substrate one bottle of substrate (BM
1442309) is added to 5 ml buffer (BM1442309). To prepare the
.alpha.-Glucosidase one bottle of .alpha.-Glucosidase (BM 1462309)
is added to 45 ml buffer (BM1442309). The working solution is made
by mixing 5 ml .alpha.-Glucosidase solution with 0.5 ml
substrate.
[0166] The assay is performed by transforming 20 .mu.l enzyme
solution to a 96 well microtitre plate and incubating at 25.degree.
C. 200 .mu.l working solution, 25.degree. C. is added. The solution
is mixed and pre-incubated 1 minute and absorption is measured
every 15 sec. over 3 minutes at OD 405 nm.
[0167] The slope of the time dependent absorption-curve is directly
proportional to the specific activity (activity per mg enzyme) of
the .alpha.-amylase in question under the given set of
conditions.
General Method for Random Mutagenesis by Use of the DOPE
Program
[0168] The random mutagenesis may be carried out by the following
steps:
1. Select regions of interest for modification in the parent enzyme
2. Decide on mutation sites and non-mutated sites in the selected
region 3. Decide on which kind of mutations should be carried out,
e.g. with respect to the desired stability and/or performance of
the variant to be constructed 4. Select structurally reasonable
mutations. 5. Adjust the residues selected by step 3 with regard to
step 4. 6. Analyze by use of a suitable dope algorithm the
nucleotide distribution. 7. If necessary, adjust the wanted
residues to genetic code realism (e.g., taking into account
constraints resulting from the genetic code (e.g. in order to avoid
introduction of stop codons)) (the skilled person will be aware
that some codon combinations cannot be used in practice and will
need to be adapted) 8. Make primers 9. Perform random mutagenesis
by use of the primers 10. Select resulting .alpha.-amylase variants
by screening for the desired improved properties.
[0169] Suitable dope algorithms for use in step 6 are well known in
the art. One algorithm is described by Tomandl, D. et al., Journal
of Computer-Aided Molecular Design, 11 (1997), pp. 29-38). Another
algorithm, DOPE, is described in the following:
The Dope Program
[0170] The "DOPE" program is a computer algorithm useful to
optimize the nucleotide composition of a codon triplet in such a
way that it encodes an amino acid distribution which resembles most
the wanted amino acid distribution. In order to assess which of the
possible distributions is the most similar to the wanted amino acid
distribution, a scoring function is needed. In the "Dope" program
the following function was found to be suited:
s .ident. i = 1 N ( x i y i ( 1 - x i ) 1 - y i y i y i ( 1 - y i )
1 - y i ) w i , ##EQU00001##
where the x.sub.i's are the obtained amounts of amino acids and
groups of amino acids as calculated by the program, y.sub.i's are
the wanted amounts of amino acids and groups of amino acids as
defined by the user of the program (e.g. specify which of the 20
amino acids or stop codons are wanted to be introduced, e.g. with a
certain percentage (e.g. 90% Ala, 3% Ile, 7% Val), and w.sub.i's
are assigned weight factors as defined by the user of the program
(e.g., depending on the importance of having a specific amino acid
residue inserted into the position in question). N is 21 plus the
number of amino acid groups as defined by the user of the program.
For purposes of this function 0.sup.0 is defined as being 1.
[0171] A Monte-Carlo algorithm (one example being the one described
by Valleau, J. P. & Whittington, S. G. (1977) A guide to Monte
Carlo for statistical mechanics: 1 Highways. In "Stastistical
Mechanics, Part A" Equlibrium Techniqeues ed. B. J. Berne, New
York: Plenum) is used for finding the maximum value of this
function. In each iteration the following steps are performed:
[0172] 1. A new random nucleotide composition is chosen for each
base, where the absolute difference between the current and the new
composition is smaller than or equal to d for each of the four
nucleotides G,A,T,C in all three positions of the codon (see below
for definition of d). [0173] 2. The scores of the new composition
and the current composition are compared by the use of the function
s as described above. If the new score is higher or equal to the
score of the current composition, the new composition is kept and
the current composition is changed to the new one. If the new score
is smaller, the probability of keeping the new composition is
exp(1000(new_score-current_score)).
[0174] A cycle normally consists of 1000 iterations as described
above in which d is decreasing linearly from 1 to 0. One hundred or
more cycles are performed in an optimization process. The
nucleotide composition resulting in the highest score is finally
presented.
EXAMPLES
Example 1
Example on Homology Building of Termamyl.TM.
[0175] The overall homology of the B. licheniformis .alpha.-amylase
(in the following referred to as Termamyl.TM.) to other
Termamyl-like .alpha.-amylases is high and the percent similarity
is extremely high. The similarity calculated for Termamyl.TM. to
BSG (the B. stearothermophilus .alpha.-amylase having SEQ ID NO:
3), and BAN (the B. amyloliquefaciens .alpha.-amylase having SEQ ID
NO: 5) using the University of Wisconsin Genetics Computer Group's
program GCG gave 89% and 78%, respectively. TERM has a deletion of
2 residues between residue G180 and K181 compared to BAN.TM. and
BSG. BSG has a deletion of 3 residues between G371 and 1372 in
comparison with BAN.TM. and Termamyl.TM.. Further BSG has a
C-terminal extension of more than 20 residues compared to BAN.TM.
and Termamyl.TM.. BAN.TM. has 2 residues less and Termamyl has one
residue less in the N-terminal compared to BSG.
[0176] The structure of the B. licheniformis (Termamyl.TM.) and of
the B. amyloliquefaciens .alpha.-amylase (BAN.TM.), respectively,
was model built on the structure disclosed in Appendix 1 of WO
96/23974. The structure of other Termamyl-like .alpha.-amylases
(e.g. those disclosed herein) may be built analogously.
[0177] In comparison with the .alpha.-amylase used for elucidating
the present structure, Termamyl.TM. differs in that it lacks two
residues around 178-182. In order to compensate for this in the
model structure, the HOMOLOGY program from BIOSYM was used to
substitute the residues in equivalent positions in the structure
(not only structurally conserved regions) except for the deletion
point. A peptide bond was established between G179(G177) and
K180(K180) in Termamy.TM. (BAN.TM.). The close structural
relationship between the solved structure and the model structure
(and thus the validity of the latter) is indicated by the presence
of only very few atoms found to be too close together in the
model.
[0178] To this very rough structure of Termamyl.TM. was then added
all waters (605) and ions (4 Calcium and 1 Sodium) from the solved
structure (See Appendix 1 of WO 96/23874) at the same coordinates
as for said solved structure using the INSIGHT program. This could
be done with only few overlaps--in other words with a very nice
fit. This model structure were then minimized using 200 steps of
Steepest descent and 600 steps of Conjugated gradient (see Brooks
et al 1983, J. Computational Chemistry 4, p. 187-217). The
minimized structure was then subjected to molecular dynamics, 5 ps
heating followed by up to 200 ps equilibration but more than 35 ps.
The dynamics as run with the Verlet algorithm and the equilibration
temperature 300K were kept using the Behrendsen coupling to a water
bath (Berendsen et. al., 1984, J. Chemical Physics 81, p.
3684-3690). Rotations and translations were removed every pico
second.
Example 2
Method of Extracting Important Regions for Identifying
.alpha.-Amylase Variants with Improved pH Stability and Altered
Temperature Activity
[0179] The X-ray structure and/or the model build structure of the
enzyme of interest, here SP722 and Termamyl.TM., are subjected to
molecular dynamics simulations. The molecular dynamics simulation
are made using the CHARMM (from Molecular simulations (MSI))
program or other suited program like, e.g., DISCOVER (from MSI).
The molecular dynamic analysis is made in vacuum, or more preferred
including crystal waters, or with the enzyme embedded in water,
e.g., a water sphere or a water box. The simulation are run for 300
pico seconds (ps) or more, e.g., 300-1200 ps. The isotropic
fluctuations are extracted for the CA carbons of the structures and
compared between the structures. Where the sequence has deletions
and/or insertions the isotropic fluctuations from the other
structure are inserted thus giving 0 as difference in isotropic
fluctuation. For explanation of isotropic fluctuations see the
CHARMM manual (obtainable from MSI).
[0180] The molecular dynamics simulation can be done using standard
charges on the chargeable amino acids. This is Asp and Glu are
negatively charged and Lys and Arg are positively charged. This
condition resembles the medium pH of approximately 7. To analyze a
higher or lower pH, titration of the molecule can be done to obtain
the altered pKa's of the standard titrateable residues normally
within pH 2-10; Lys, Arg, Asp, Glu, Tyr and His. Also Ser, Thr and
Cys are titrateable but are not taking into account here. Here the
altered charges due to the pH has been described as both Asp and
Glu are negative at high pH, and both Arg and Lys are uncharged.
This imitates a pH around 10 to 11 where the titration of Lys and
Arg starts, as the normal pKa of these residues are around
9-11.
[0181] 1. The approach used for extracting important regions for
identifying .alpha.-amylase variants with high pH stability:
[0182] The important regions for constructing variants with
improved pH stability are the regions which at the extreme pH
display the highest mobility, i.e., regions having the highest
isotropic fluctuations.
[0183] Such regions are identified by carrying out two molecular
dynamics simulations: i) a high pH run at which the basic amino
acids, Lys and Arg, are seen as neutral (i.e. not protonated) and
the acidic amino acids, Asp and Glu, have the charge (-1) and ii) a
neutral pH run with the basic amino acids, Lys and Arg, having the
net charge of (+1) and the acidic amino acids having a charge of
(-1).
[0184] The two run are compared and regions displaying the
relatively higher mobility at high pH compared to neutral pH
analysis were identified.
[0185] Introduction of residues improving general stability, e.g.,
hydrogen bonding, making the region more rigid (by mutations such
as Proline substitutions or replacement of Glycine residues), or
improving the charges or their interaction, improves the high pH
stability of the enzyme.
[0186] 2. The approach used for extracting regions for identifying
.alpha.-amylase variants with increased activity at medium
temperatures:
[0187] The important regions for constructing variants with
increased activity at medium temperature was found as the
difference between the isotropic fluctuations in SP722 and
Termamyl, i.e., SP722 minus Termamyl CA isotrophic fluctuations,
The regions with the highest mobility in the isotrophic
fluctuations were selected. These regions and there residues were
expected to increase the activity at medium temperatures. The
activity of an alpha-amylase is only expressed if the correct
mobility of certain residues are present. If the mobility of the
residues is too low the activity is decreased or abandoned.
Example 3
Construction, by Localized Random, Doped Mutagenesis, of
Termamyl-Like .alpha.-Amylase Variants Having an Improved Ca2+
Stability at Medium Temperatures Compared to the Parent Enzyme
[0188] To improve the stability at low calcium concentration of
.alpha.-amylases random mutagenesis in pre-selected region was
performed.
Region: Residue:
SAI: R181--W189
[0189] The DOPE software (see Materials and Methods) was used to
determine spiked codons for each suggested change in the SA1 region
minimizing the amount of stop codons (see table 1). The exact
distribution of nucleotides was calculated in the three positions
of the codon to give the suggested population of amino acid
changes. The doped regions were doped specifically in the indicated
positions to have a high chance of getting the desired residues,
but still allow other possibilities.
TABLE-US-00001 TABLE 1 Distribution of amino acid residues for each
position R181: 72% R, 2% N, 7% Q, 4% H, 4% K, 11% S G182: 73% G,
13% A, 12% S, 2% T K185: 95% K, 5% R A186: 50% A, 4% N, 6% D, 1% E,
1% G, 1% K, 5% S, 31% T W187: 100% W D188: 100% D W189: 92% W, 8%
S
[0190] The resulting doped oligonucleotide strand is shown in table
2 as sense strand: with the wild type nucleotide and amino acid
sequences and the distribution of nucleotides for each doped
position.
TABLE-US-00002 TABLE 2 Position 181 182 185 186 187 188 189 Amino
acid seq. Arg Gly Lys Ala Thr Asp Thr Wt nuc. seq. cga ggt aaa gct
tgg gat tgg Forward primer (SEQ ID NO: 15): FSA: 5'-caa aat cgt atc
tac aaa ttc 123 456 a7g 8910 tgg gat t11g gaa gta gat tcg gaa
aat-3' Distribution of nucleotides for each doped Position 1: 35%
A, 65% C 2: 83% G, 17% A 3: 63% G, 37% T 4: 86% G, 14% A 5: 85% G,
15% C 6: 50% T, 50% C 7: 95% A, 5% G 8: 58% G, 37% A, 5% T 9: 86%
C, 13% A, 1% G 10: 83% T, 17% G 11: 92% G, 8% C Reverse primer (SEQ
ID NO: 16): RSA: 5'-gaa ttt gta gat acg att ttg-3'
Random Mutagenesis
[0191] The spiked oligonucleotides apparent from Table 2 (which by
a common term is designated FSA) and reverse primers RSA for the
SA1 region and specific SEQ ID NO: 2: SP722 primers covering the
SacII and the DraIII sites are used to generate
PCR-library-fragments by the overlap extension method (Horton et
al., Gene, 77 (1989), pp. 61-68) with an overlap of 21 base pairs.
Plasmid pJE1 is template for the Polymerase Chain Reaction. The PCR
fragments are cloned in the E. coli/Bacillus shuttle vector
pDork101 (see Materials and Methods) enabling mutagenesis in E.
coli and immediate expression in Bacillus subtilis preventing
lethal accumulation of amylases in E. coli. After establishing the
cloned PCR fragments in E. coli, a modified pUC19 fragment is
digested out of the plasmid and the promoter and the mutated
Termamyl gene is physically connected and expression can take place
in Bacillus.
Screening
[0192] The library may be screened in the low calcium filter assays
described in the "Material and Methods" section above.
Example 4
Construction of Variants of Amylase SEQ ID NO: 1 (SP690)
[0193] The gene encoding the amylase from SEQ ID NO: 1 is located
in a plasmid pTVB106 described in WO96/23873. The amylase is
expressed from the amyL promoter in this construct in Bacillus
subtilis.
[0194] A variant of the protein is
delta(T183-G184)+Y243F+Q391E+K444Q. Construction of this variant is
described in WO96/23873.
[0195] Construction of delta(T183-G184)+N195F by the mega-primer
method as described by Sarkar and Sommer, (1990), BioTechniques 8:
404-407.
[0196] Gene specific primer B1 (SEQ ID NO: 17) and mutagenic primer
101458 (SEQ ID NO: 19) were used to amplify by PCR an approximately
645 bp DNA fragment from a pTVB106-like plasmid (with the
delta(T183-G184) mutations in the gene encoding the amylase from
SEQ ID NO: 1).
[0197] The 645 bp fragment was purified from an agarose gel and
used as a mega-primer together with primer Y2 (SEQ ID NO: 18) in a
second PCR carried out on the same template.
[0198] The resulting approximately 1080 bp fragment was digested
with restriction enzymes BstEII and AflIII and the resulting
approximately 510 bp DNA fragment was purified and ligated with the
pTVB106-like plasmid (with the delta(T183-G184) mutations in the
gene encoding the amylase from SEQ ID NO: 1) digested with the same
enzymes. Competent Bacillus subtilis SHA273 (amylase and protease
low) cells were transformed with the ligation and Chlorampenicol
resistant transformants and was checked by DNA sequencing to verify
the presence of the correct mutations on the plasmid.
TABLE-US-00003 primer B1: (SEQ ID NO: 17) 5' CGA TTG CTG ACG CTG
TTA TTT GCG 3' primer Y2: (SEQ ID NO: 18) 5' CTT GTT CCC TTG TCA
GAA CCA ATG 3' primer 101458 (SEQ ID NO: 19): 5' GT CAT AGT TGC CGA
AAT CTG TAT CGA CTT C 3'
[0199] The construction of variant: delta(T183-G184)+K185R+A186T
was carried out in a similar way except that mutagenic primer
101638 was used.
TABLE-US-00004 primer 101638: (SEQ ID NO: 20) 5' CC CAG TCC CAC GTA
CGT CCC CTG AAT TTA TAT ATT TTG 3'
[0200] Variants: delta(T183-G184)+A186T, delta(T183-G184)+A1861,
delta(T183-G184)+A186S, delta(T183-G184)+A186N are constructed by a
similar method except that pTVB106-like plasmid (carrying variant
delta(T183-G184)+K185R+A186T) is used as template and as the vector
for the cloning purpose. The mutagenic oligonucleotide (Oligo 1)
is:
TABLE-US-00005 (SEQ ID NO: 21) 5' CC CAG TCC CAG NTCTTT CCC CTG AAT
TTA TAT ATT TTG 3'
[0201] N represents a mixture of the four bases: A, C, G, and T
used in the synthesis of the mutagenicoli-gonucleotide. Sequencing
of transformants identifies the correct codon for amino acid
position 186 in the mature amylase.
[0202] Variant: delta(T183-G184)+K185R+A186T+N195F is constructed
as follows:
PCR is carried out with primer x2 (SEQ ID NO: 22) and primer 101458
(SEQ ID NO: 19) on pTVB106-like plasmid (with mutations
delta(T183-G184)+K185R+A186T). The resulting DNA fragment is used
as a mega-primer together with primer Y2 (SEQ ID NO: 18) in a PCR
on pTVB106-like plasmid (with mutations delta(T183-G184)+N195). The
product of the second PCR is digested with restriction
endonucleases Acc65I and AflIII and cloned into pTVB106 like
plasmid (delta(T183-G184)+N195F) digested with the same
enzymes.
TABLE-US-00006 primer x2: (SEQ ID NO: 22) 5' GCG TGG ACA AAG TTT
GAT TTT CCT G 3'
[0203] Variant:
delta(T183-G184)+K185R+A186T+N195F+Y243F+Q391E+K444Q is constructed
as follows:
[0204] PCR is carried out with primer x2 and primer 101458 on
pTVB106-like plasmid (with mutations delta(T183-G184)+K185R+A186T).
The resulting DNA fragment is used as a mega-primer together with
primer Y2 in a PCR on pTVB106 like plasmid (with mutations
delta(T183-G184)+Y243F+Q391E+K444Q). The product of the second PCR
is digested with restriction endonucleases Acc65I and AflIII and
cloned into pTVB106 like plasmid
(delta(T183-G184)+Y243F+Q391E+K444Q) digested with the same
enzymes.
Example 5
Construction of Site-Directed .alpha.-Amylase Variants in the
Parent SP722 .alpha.-Amylase (SEQ ID NO: 2)
[0205] Construction of variants of amylase SEQ ID NO: 2 (SP722) is
carried out as described below.
[0206] The gene encoding the amylase from SEQ ID NO: 2 is located
in a plasmid pTVB112 described in WO 96/23873. The amylase is
expressed from the amyL promoter in this construct in Bacillus
subtilis.
[0207] Construction of delta(D183-G184)+V56I by the mega-primer
method as described by Sarkar and Sommer, 1990 (BioTechniques 8:
404-407).
[0208] Gene specific primer DA03 and mutagenic primer DA07 are used
to amplify by PCR an approximately 820 bp DNA fragment from a
pTVB112-like plasmid (with the delta(D183-G184) mutations in the
gene encoding the .alpha.-amylase shown in SEQ ID NO: 2.
[0209] The 820 bp fragment is purified from an agarose gel and used
as a mega-primer together with primer DA01 in a second PCR carried
out on the same template.
[0210] The resulting approximately 920 bp fragment is digested with
restriction enzymes NgoM I and Aat II and the resulting
approximately 170 bp DNA fragment is purified and ligated with the
pTVB112-like plasmid (with the delta(D183-G184) mutations in the
gene encoding the amylase shown in SEQ ID NO: 2) digested with the
same enzymes. Competent Bacillus subtilis SHA273 (amylase and
protease low) cells are transformed with the ligation and
Chlorampenicol resistant transformants are checked by DNA
sequencing to verify the presence of the correct mutations on the
plasmid.
TABLE-US-00007 primer DA01: (SEQ ID NO: 23) 5'
CCTAATGATGGGAATCACTGG 3' primer DA03: (SEQ ID NO: 24) 5'
GCATTGGATGCTTTTGAACAACCG 3' primer DA07 (SEQ ID NO: 25): 5'
CGCAAAATGATATCGGGTATGGAGCC 3'
Variants: delta(D183-G184)+K108L, delta(D183-G184)+K108Q,
delta(D183-G184)+K108E, delta(D183-G184)+K108V, were constructed by
the mega-primer method as described by Sarkar and Sommer, 1990
(BioTechniques 8: 404-407):
[0211] PCR is carried out with primer DA03 and mutagenesis primer
DA20 on pTVB112-like plasmid (with mutations delta(D183-G184)). The
resulting DNA fragment is used as a mega-primer together with
primer DA01 in a PCR on pTVB112-like plasmid (with mutations
delta(D183-G184)). The approximately 920 bp product of the second
PCR is digested with restriction endonucleases Aat II and Mlu I and
cloned into pTVB112-like plasmid (delta(D183-G184)) digested with
the same enzymes.
TABLE-US-00008 primer DA20 (SQ ID NO: 26): 5'
GTGATGAACCACSWAGGTGGAGCTGATGC 3'
[0212] S represents a mixture of the two bases: C and G used in the
synthesis of the mutagenic oligonucleotide and W represents a
mixture of the two bases: A and T used in the synthesis of the
mutagenic oligonucleotide.
[0213] Sequencing of transformants identifies the correct codon for
amino acid position 108 in the mature amylase.
[0214] Construction of the variants: delta(D183-G184)+D168A,
delta(D183-G184)+D1681, delta(D183-G184)+D168V,
delta(D183-G184)+D168T is carried out in a similar way except that
mutagenic primer DA14 is used.
TABLE-US-00009 primer DA14 (SEQ ID NO: 27): 5'
GATGGTGTATGGRYCAATCACGACAATTCC 3'
[0215] R represents a mixture of the two bases: A and G used in the
synthesis of the mutagenic oligonucleotide and Y represents a
mixture of the two bases: C and T used in the synthesis of the
mutagenic oligonucleotide.
[0216] Sequencing of transformants identifies the correct codon for
amino acid position 168 in the mature amylase.
[0217] Construction of the variant: delta(D183-G184)+Q169N is
carried out in a similar way except that mutagenic primer DA15 is
used.
TABLE-US-00010 primer DA15 (SEQ ID NO: 28): 5'
GGTGTATGGGATAACTCACGACAATTCC 3'
[0218] Construction of the variant: delta(D183-G184)+Q169L is
carried out in a similar way except that mutagenic primer DA16 is
used.
TABLE-US-00011 primer DA16 (SEQ ID NO: 29): 5'
GGTGTATGGGATCTCTCACGACAATTCC 3'
[0219] Construction of the variant: delta(D183-G184)+Q172N is
carried out in a similar way except that mutagenic primer DA17 is
used.
TABLE-US-00012 primer DA17 (SEQ ID NO: 30): 5'
GGGATCAATCACGAAATTTCCAAAATCGTATC 3'
[0220] Construction of the variant: delta(D183-G184)+Q172L is
carried out in a similar way except that mutagenic primer DA18 is
used.
TABLE-US-00013 primer DA18 (SEQ ID NO: 31): 5'
GGGATCAATCACGACTCTTCCAAAATCGTATC 3'
[0221] Construction of the variant: delta(D183-G184)+L2011 is
carried out in a similar way except that mutagenic primer DA06 is
used.
TABLE-US-00014 primer DA06 (SEQ ID NO: 32): 5'
GGAAATTATGATTATATCATGTATGCAGATGTAG 3'
[0222] Construction of the variant: delta(D183-G184)+K269S is
carried out in a similar way except that mutagenic primer DA09 is
used.
TABLE-US-00015 primer DA09 (SEQ ID NO: 33): 5'
GCTGAATTTTGGTCGAATGATTTAGGTGCC 3'
[0223] Construction of the variant: delta(D183-G184)+K269Q is
carried out in a similar way except that mutagenic primer DA11 is
used.
TABLE-US-00016 primer DA11 (SEQ ID NO: 34): 5'
GCTGAATTTTGGTCGAATGATTTAGGTGCC 3'
[0224] Construction of the variant: delta(D183-G184)+N270Y is
carried out in a similar way except that mutagenic primer DA21 is
used.
TABLE-US-00017 primer DA21 (SEQ ID NO: 35): 5'
GAATTTTGGAAGTACGATTTAGGTCGG 3'
[0225] Construction of the variants: delta(D183-G184)+L272A,
delta(D183-G184)+L272I, delta(D183-G184)+L272V,
delta(D183-G184)+L272T is carried out in a similar way except that
mutagenic primer DA12 is used.
TABLE-US-00018 primer DA12 (SEQ ID NO: 36): 5'
GGAAAAACGATRYCGGTGCCTTGGAGAAC 3'
R represents a mixture of the two bases: A and G used in the
synthesis of the mutagenic oligonucleotide and Y represents a
mixture of the two bases: C and T used in the synthesis of the
mutagenic oligonucleotide. Sequencing of transformants identifies
the correct codon for amino acid position 272 in the mature
amylase.
[0226] Construction of the variants: delta(D183-G184)+L275A,
delta(D183-G184)+L275I, delta(D183-G184)+L275V,
delta(D183-G184)+L275T is carried out in a similar way except that
mutagenic primer DA13 is used.
TABLE-US-00019 primer DA13 (SEQ ID NO: 37): 5'
GATTTAGGTGCCTRYCAGAACTATTTA 3'
R represents a mixture of the two bases: A and G used in the
synthesis of the mutagenic oligonucleotide and Y represents a
mixture of the two bases: C and T used in the synthesis of the
mutagenic oligonucleotide. Sequencing of transformants identifies
the correct codon for amino acid position 275 in the mature
amylase.
[0227] Construction of the variant: delta(D183-G184)+Y295E is
carried out in a similar way except that mutagenic primer DA08 is
used.
TABLE-US-00020 primer DA08 (SEQ ID NO: 38): 5'
CCCCCTTCATGAGAATCTTTATAACG 3'
[0228] Construction of delta(D183-G184)+K446Q by the mega-primer
method as described by Sarkar and Sommer, 1990 (BioTechniques 8:
404-407):
[0229] Gene specific primer DA04, annealing 214-231 by downstream
relative to the STOP-codon and mutagenic primer DA10 were used to
amplify by PCR an approximately 350 bp DNA fragment from a
pTVB112-like plasmid (with the delta(D183-G184) mutations in the
gene encoding the amylase depicted in SEQ ID NO: 2).
[0230] The resulting DNA fragment is used as a mega-primer together
with primer DA05 in a PCR on pTVB112 like plasmid (with mutations
delta(D183-G184)). The app. 460 bp product of the second PCR is
digested with restriction endonucleases SnaB I and Not I and cloned
into pTVB112 like plasmid (delta(D183-G184)) digested with the same
enzymes.
TABLE-US-00021 primer DA04 (SEQ ID NO: 39): 5'
GAATCCGAACCTCATTACACATTCG 3' primer DA05 (SEQ ID NO: 40): 5'
CGGATGGACTCGAGAAGGAAATACCACG 3' primer DA10 (SEQ ID NO: 41): 5'
CGTAGGGCAAAATCAGGCCGGTCAAGTTTGG 3'
[0231] Construction of the variants: delta(D183-G184)+K458R is
carried out in a similar way except that mutagenic primer DA22 is
used.
TABLE-US-00022 primer DA22 (SEQ ID NO: 42): 5'
CATAACTGGAAATCGCCCGGGAACAGTTACG 3'
[0232] Construction of the variants: delta(D183-G184)+P459S and
delta(D183-G184)+P459T is carried out in a similar way except that
mutagenic primer DA19 is used.
TABLE-US-00023 primer DA19 (SEQ ID NO: 43): 5'
CTGGAAATAAAWCCGGAACAGTTACG 3'
W represents a mixture of the two bases: A and T used in the
synthesis of the mutagenic oligonucleotide. Sequencing of
transformants identifies the correct codon for amino acid position
459 in the mature amylase.
[0233] Construction of the variants: delta(D183-G184)+T461P is
carried out in a similar way except that mutagenic primer DA23 is
used.
TABLE-US-00024 primer DA23 (SEQ ID NO: 44): 5'
GGAAATAAACCAGGACCCGTTACGATCAATGC 3'
[0234] Construction of the variant: delta(D183-G184)+K142R is
carried out in a similar way except that mutagenic primer DA32 is
used.
TABLE-US-00025 Primer DA32 (SEQ ID NO: 45): 5'
GAGGCTTGGACTAGGTTTGATTTTCCAG 3'
[0235] Construction of the variant: delta(D183-G184)+K269R is
carried out in a similar way except that mutagenic primer DA31 is
used.
TABLE-US-00026 Primer DA31 (SEQ ID NO: 46): 5'
GCTGAATTTTGGCGCAATGATTTAGGTGCC 3'
Example 6
Construction of Site-Directed .alpha.-Amylase Variants in the
Parent Termamyl .alpha.-Amylase (SEQ ID NO: 4)
[0236] The amyL gene, encoding the Termamyl .alpha.-amylase is
located in plasmid pDN1528 described in WO 95/10603 (Novo Nordisk).
Variants with substitutions N265R and N265D, respectively, of said
parent .alpha.-amylase are constructed by methods described in WO
97/41213 or by the "megaprimer" approach described above.
Mutagenic Oligonucleotides are:
TABLE-US-00027 [0237] Primer b11 for the N265R substitution: (SEQ
ID NO: 56) 5' PCC AGC GCG CCT AGG TCA CGC TGC CAA TAT TCA G Primer
b12 for the N265D substitution: (SEQ ID NO: 57) 5' PCC AGC GCG CCT
AGG TCA TCC TGC CAA TAT TCA G P represents a phosphate group.
Example 7
Determination of pH Stability at Alkaline pH of Variants of the
Parent .alpha.-Amylase Having the Amino Acid Sequence Shown in SEQ
ID NO:2
[0238] In this series of analysis purified enzyme samples were
used. The measurements were made using solutions of the respective
variants in 100 mM CAPS buffer adjusted to pH 10.5. The solutions
were incubated at 75.degree. C.
[0239] After incubation for 20 and 30 min the residual activity was
measured using the PNP-G7 assay (described in the "Materials and
Methods" section above). The residual activity in the samples was
measured using Britton Robinson buffer pH 7.3. The decline in
residual activity was measured relative to a corresponding
reference solution of the same enzyme at 0 minutes, which has not
been incubated at high pH and 75.degree. C.
[0240] The percentage of the initial activity as a function is
shown in the table below for the parent enzyme (SEQ ID NO: 2) and
for the variants in question.
TABLE-US-00028 Residual activity Residual activity Variant after 20
min after 30 min .DELTA. (D183-G184) + M323L 56% 44% .DELTA.
(D183-G184) + M323L + 67% 55% R181S .DELTA. (D183-G184) + M323L +
62% 50% A186T
In an other series of analysis culture supernatants were used. The
measurements were made using solutions of the respective variants
in 100 mM CAPS buffer adjusted to pH 10.5. The solutions were
incubated at 80.degree. C.
[0241] After incubation for 30 minutes the residual activity was
measured using the Phadebas assay (described in the "Materials and
Method" section above. The residual activity in the samples was
measured using Britton Robinson buffer pH 7.3. The decline in
residual activity was measured relative to a corresponding
reference solution of the same enzyme at 0 minutes, which has not
been incubated at high pH and 80.degree. C.
[0242] The percentage of the initial activity as a function is
shown in the table below for the parent enzyme (SEQ ID NO: 2) and
for the variants in question.
TABLE-US-00029 Variant Residual activity after 30 min .DELTA.
(D183-G184) 4% .DELTA. (D183-G184) + P459T 25% .DELTA. (D183-G184)
+ K458R 31% .DELTA. (D183-G184) + K311R 10%
Example 8
Determination of Calcium Stability at Alkaline pH of Variants of
the Parent .alpha.-Amylase Having the Amino Acid Sequence Shown in
SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4
A: Calcium Stability of Variants of the Sequence in SEQ ID NO:
1
[0243] The measurement were made using solutions of the respective
variants in 100 mM CAPS buffer adjusted to pH 10.5 to which
polyphosphate was added (at time t=0) to give a final concentration
of 2400 ppm. The solutions were incubated at 50.degree. C.
[0244] After incubation for 20 and 30 minutes the residual activity
was measured using the PNP-G7 assay (described above). The residual
activity in the samples was measured using Britton Robinson buffer
pH 7.3. The decline in residual activity was measured relative to a
corresponding reference solution of the same enzyme at 0 minutes,
which has not been incubated at high pH and 50.degree. C.
[0245] The percentage of the initial activity as a function is
shown in the table below for the parent enzyme (SEQ ID NO: 1) and
for the variants in question.
TABLE-US-00030 Residual activity Residual activity Variant after 20
min after 30 min .DELTA. (T183-G184) 32% 19% .DELTA. (T183-G184) +
A186T 36% 23% .DELTA. (T183-G184) + K185R + A186T 45% 29% .DELTA.
(T183-G184) + A186I 35% 20% .DELTA. (T183-G184) + N195F 44% n.d.
n.d. = Not determinated
B: Calcium Stability of Variants of the Sequence in SEQ ID NO:
2
[0246] In this series of analysis purified samples of enzymes were
used. The measurement were made using solutions of the respective
variants in 100 mM CAPS buffer adjusted to pH 10.5 to which
polyphosphate was added (at time t=0) to give a final concentration
of 2400 ppm. The solutions were incubated at 50.degree. C.
[0247] After incubation for 20 and 30 minutes the residual activity
was measured using the PNP-G7 assay (described above). The residual
activity in the samples was measured using Britton Robinson buffer
pH 7.3. The decline in residual activity was measured relative to a
corresponding reference solution of the same enzyme at 0 minutes,
which has not been incubated at high pH and 50.degree. C.
[0248] The percentage of the initial activity as a function is
shown in the table below for the parent enzyme (SEQ ID NO: 2) and
for the variants in question.
TABLE-US-00031 Residual activity Residual activity Variant after 20
min after 30 min .DELTA. (D183-G184) + M323L 21% 13% .DELTA.
(D183-G184) + M323L + 32% 19% R181S .DELTA. (D183-G184) + M323L +
28% 17% A186T .DELTA. (D183-G184) + M323L + 30% 18% A186R .DELTA.
(D183-G184) 30% 20% .DELTA. (D183-G184) + N195F 55% 44%
[0249] In this series of analysis culture supernatants were used.
The measurement were made using solutions of the respective
variants in 100 mM CAPS buffer adjusted to pH 10.5 to which
polyphosphate was added (at time t=0) to give a final concentration
of 2400 ppm. The solutions were incubated at 50.degree. C.
[0250] After incubation for 30 minutes the residual activity was
measured using the Phadebas assay as described above. The residual
activity in the samples was measured using Britton Robinson buffer
pH 7.3. The decline in residual activity was measured relative to a
corresponding reference solution of the same enzyme at 0 minutes,
which has not been incubated at high pH and 50.degree. C.
[0251] The percentage of the initial activity as a function is
shown in the table below for the parent enzyme (SEQ ID NO: 2) and
for the variants in question.
TABLE-US-00032 Variant Residual activity after 30 min .DELTA.
(D183-G184) 0% .DELTA. (D183-G184) + P459T 19% .DELTA. (D183-G184)
+ K458R 18% .DELTA. (D183-G184) + T461P 13% .DELTA. (D183-G184) +
E346Q + K385R 4%
C: Calcium Stability of Variants of the Sequence in SEQ ID NO:
4
[0252] The measurement were made using solutions of the respective
variants in 100 mM CAPS buffer adjusted to pH 10.5 to which
polyphosphate was added (at time t=0) to give a final concentration
of 2400 ppm. The solutions were incubated at 60.degree. C. for 20
minutes.
[0253] After incubation for 20 minutes the residual activity was
measured using the PNP-G7 assay (described above). The residual
activity in the samples was measured using Britton Robinson buffer
pH 7.3. The decline in residual activity was measured relative to a
corresponding reference solution of the same enzyme at 0 minutes,
which has not been incubated at high pH and 60.degree. C.
[0254] The percentage of the initial activity as a function is
shown in the table below for the parent enzyme (SEQ ID NO: 4) and
for the variants in question.
TABLE-US-00033 Residual activity after Variant 20 min Termamyl (SEQ
ID NO: 4) 17% N265R 28% N265D 25%
Example 9
Activity Measurement at Medium Temperature of .alpha.-Amylases
Having the Amino Acid Sequence Shown in SEQ ID NO: 1
A: .alpha.-Amylase Activity of Variants of the Sequence in SEQ ID
NO:1
[0255] The measurement were made using solutions of the respective
variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and
using the Phadebas assay described above. The activity in the
samples was measured at 37.degree. C. using 50 mM Britton Robinson
buffer pH 7.3 and at 25.degree. C. using 50 mM CAPS buffer pH
10.5.
[0256] The temperature dependent activity and the percentage of the
activity at 25.degree. C. relative to the activity at 37.degree. C.
is shown in the table below for the parent enzyme (SEQ ID NO: 1)
and for the variants in question.
TABLE-US-00034 NU/mg NU/mg NU (25.degree. C.)/ Variant 25.degree.
C. 37.degree. C. NU (37.degree. C.) SP690 1440 35000 4.1% .DELTA.
(T183-G184) 2900 40000 7.3% .DELTA. (T183-G184) + K269S 1860 12000
15.5% .DELTA. (Q174) 3830 38000 7.9%
[0257] Another measurement was made using solutions of the
respective variants in 50 mM Britton Robinson buffer adjusted to pH
7.3 and using the Phadebas assay described above. The activity in
the samples was measured at 37.degree. C. and 50.degree. C. using
50 mM Britton Robinson buffer pH 7.3.
[0258] The temperature dependent activity and the percentage of the
activity at 37.degree. C. relative to the activity at 50.degree. C.
is shown in the table below for the parent enzyme (SEQ ID NO: 1)
and for the variants in question.
TABLE-US-00035 NU/mg NU/mg NU (37.degree. C.)/ Variant 37.degree.
C. 50.degree. C. NU (50.degree. C.) SP690 (seq ID NO: 1) 13090
21669 60% K269Q 7804 10063 78%
B: .alpha.-Amylase Activity of Variants of the Sequence in SEQ ID
NO:2
[0259] The measurement were made using solutions of the respective
variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and
using the Phadebas assay described above. The activity in the
samples was measured at both 25.degree. C. and 37.degree. C. using
50 mM Britton Robinson buffer pH 7.3.
[0260] The temperature dependent activity and the percentage of the
activity at 25.degree. C. relative to the activity at 37.degree. C.
is shown in the table below for the parent enzyme (SEQ ID NO: 2)
and for the variants in question.
TABLE-US-00036 NU/mg NU/mg NU (25.degree. C.)/ Variant 25.degree.
C. 37.degree. C. NU (37.degree. C.) .DELTA. (D183-G184) + M323L
3049 10202 30% .DELTA. (D183-G184) + M323L + R181S 18695 36436
51%
C: .alpha.-Amylase Activity of Variants of the Sequence in SEQ ID
NO:4
[0261] The measurement were made using solutions of the respective
variants in 50 mM Britton Robinson buffer adjusted to pH 7.3 and
using the Phadebas assay described above. The activity in the
samples was measured at both 37.degree. C. using 50 mM Britton
Robinson buffer pH 7.3 and at 60.degree. C. using 50 mM CAPS buffer
pH 10.5.
[0262] The temperature dependent activity and the percentage of the
activity at 37.degree. C. relative to the activity at 60.degree. C.
is shown in the table below for the parent enzyme (SEQ ID NO: 4)
and for the variants in question.
TABLE-US-00037 NU/mg NU/mg NU (37.degree. C.)/ Variant 37.degree.
C. 60.degree. C. NU (60.degree. C.) Termamyl 7400 4350 170% Q264S
10000 4650 215%
Example 10
Construction of Variants of Parent Hybrid
BAN:1-300/Termamyl:301-483 .alpha.-Amylase
[0263] Plasmid pTVB191 contains the gene encoding hybrid
.alpha.-amylase BAN:1-300/Termamyl:301-483 as well as an origin of
replication functional in Bacillus subtilis and the cat gene
conferring chloramphenicol resistance.
[0264] Variant BM4 (F290E) was constructed using the megaprimer
approach (Sarkar and Sommer, 1990) with plasmid pTVB191 as
template.
[0265] Primer p1 (SEQ ID NO: 52) and mutagenic oligonucleotide bm4
(SEQ ID NO: 47) were used to amplify a 444 bp fragment with
polymerase chain reaction (PCR) under standard conditions.
This fragment was purified from an agarose gel and used as
`Megaprimer` in a second PCR with primer p2 (SEQ ID NO: 53)
resulting in a 531 bp fragment. This fragment was digested with
restriction endonucleases HinDIII and Tth111I. The 389 bp fragment
produced by this was ligated into plasmid pTVB191 that had been
cleaved with the same two enzymes. The resulting plasmid was
transformed into B. subtilis SHA273. Chloramphenicol resistant
clones were selected by growing the transformants on plates
containing chloramphenicol as well as insoluble starch. Clones
expressing an active .alpha.-amylase were isolated by selecting
clones that formed halos after staining the plates with iodine
vapour. The identity of the introduced mutations was confirmed by
DNA sequencing.
[0266] Variants BM5(F290K), BM6(F290A), BM8(Q360E) and BM11(N102D)
were constructed in a similar way. Details of their construction
are given below.
Variant: BM5(F290K)
[0267] mutagenic oligonucleotide: bm5 (SEQ ID NO: 48)
Primer (1st PCR): p1 (SEQ ID NO: 52)
[0268] Size of resulting fragment: 444 bp
Primer (2nd PCR): p2 (SEQ ID NO: 53)
[0269] Restriction endonucleases: HinDIII, Tth111I Size of cleaved
fragment: 389 bp
Variant: BM6(F290A)
[0270] mutagenic oligonucleotide: bm6 (SEQ ID NO: 49)
Primer (1st PCR): p1 (SEQ ID NO: 52)
[0271] Size of resulting fragment: 444 bp
Primer (2nd PCR): p2 (SEQ ID NO: 53)
[0272] Restriction endonucleases: HinDIII, Tth111I Size of cleaved
fragment: 389 bp
Variant: BM8(Q360E)
[0273] mutagenic oligonucleotide: bm8 (SEQ ID NO: 50)
Primer (1st PCR): p1 (SEQ ID NO: 52)
[0274] Size of resulting fragment: 230 bp
Primer (2nd PCR): p2 (SEQ ID NO: 53)
[0275] Restriction endonucleases: HinDIII, Tth111I Size of cleaved
fragment: 389 bp
Variant: BM11(N102D)
[0276] mutagenic oligonucleotide: BM11 (SEQ ID NO: 51)
Primer (1st PCR): p3 (SEQ ID NO: 54)
[0277] Size of resulting fragment: 577
Primer (2nd PCR): p4 (SEQ ID NO: 55)
[0278] Restriction endonucleases: HinDIII, PvuI Size of cleaved
fragment: 576
Mutagenic Oligonucleotides:
TABLE-US-00038 [0279] bm4 (SEQ ID NO: 47): F290E primer 5' GTG TTT
GAC GTC CCG CTT CAT GAG AAT TTA CAG G bm5 (SEQ ID NO: 48): F290K
primer 5' GTG TTT GAC GTC CCG CTT CAT AAG AAT TTA CAG G bm6 (SEQ ID
NO: 49): F290A primer 5' GTG TTT GAC GTC CCG CTT CAT GCC AAT TTA
CAG G bm8 (SEQ ID NO: 50): Q360E primer 5' AGG GAA TCC GGA TAC CCT
GAG GTT TTC TAC GG bm11 (SEQ ID NO: 51): N102D primer 5' GAT GTG
GTT TTG GAT CAT AAG GCC GGC GCT GAT G
Other Primers:
TABLE-US-00039 [0280] (SEQ ID NO: 52) p1: 5' CTG TTA TTA ATG CCG
CCA AAC C (SEQ ID NO: 53) p2: 5' G GAA AAG AAA TGT TTA CGG TTG CG
(SEQ ID NO: 54) p3: 5' G AAA TGA AGC GGA ACA TCA AAC ACG (SEQ ID
NO: 55) p4: 5' GTA TGA TTT AGG AGA ATT CC
Example 11
.alpha.-Amylase Activity at Alkaline pH of Variants of Parent
BAN:1-300/Termamyl:301-483 Hybrid .alpha.-Amylase
[0281] The measurements were made using solutions for the
respective enzymes and utilizing the Phadebas assay (described
above). The activity was measured after incubating for 15 minutes
at 30.degree. C. in mM Britton-Robinson buffer adjusted to the
indicated pH by NaOH.
TABLE-US-00040 NU/mg enzyme pH wt Q360E F290A F290K F290E N102D 8.0
5300 7800 8300 4200 6600 6200 9.0 1600 2700 3400 2100 1900 1900
REFERENCES CITED
[0282] Klein, C., et al., Biochemistry 1992, 31, 8740-8746, [0283]
Mizuno, H., et al., J. Mol. Biol. (1993) 234, 1282-1283, [0284]
Chang, C., et al, J. Mol. Biol. (1993) 229, 235-238, [0285] Larson,
S. B., J. Mol. Biol. (1994) 235, 1560-1584, [0286] Lawson, C. L.,
J. Mol. Biol. (1994) 236, 590-600, [0287] Qian, M., et al., J. Mol.
Biol. (1993) 231, 785-799, [0288] Brady, R. L., et al., Acta
Crystallogr. sect. B, 47, 527-535, [0289] Swift, H. J., et al.,
Acta Crystallogr. sect. B, 47, 535-544 [0290] A. Kadziola, Ph.D.
Thesis: "An alpha-amylase from Barley and its Complex with a
Substrate Analogue Inhibitor Studied by X-ray Crystallography",
Department of Chemistry University of Copenhagen 1993 [0291]
MacGregor, E. A., Food Hydrocolloids, 1987, Vol. 1, No. 5-6, p.
[0292] B. Diderichsen and L. Christiansen, Cloning of a maltogenic
.alpha.-amylase from Bacillus stearothermophilus, FEMS Microbiol.
letters: 56: pp. 53-60 (1988) [0293] Hudson et al., Practical
Immunology, Third edition (1989), Blackwell Scientific
Publications, [0294] Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989 [0295] S. L.
Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, pp.
1859-1869 [0296] Matthes et al., The EMBO J. 3, 1984, pp. 801-805.
[0297] R. K. Saiki et al., Science 239, 1988, pp. 487-491. [0298]
Morinaga et al., (1984, Biotechnology 2:646-639) [0299] Nelson and
Long, Analytical Biochemistry 180, 1989, pp. 147-151 [0300]
Hunkapiller et al., 1984, Nature 310:105-111 [0301] R. Higuchi, B.
Krummel, and R. K. Saiki (1988). A general method of in vitro
preparation and specific mutagenesis of DNA fragments: study of
protein and DNA interactions. Nucl. Acids Res. 16:7351-7367. [0302]
Dubnau et al., 1971, J. Mol. Biol. 56, pp. 209-221. [0303] Gryczan
et al., 1978, J. Bacteriol. 134, pp. 318-329. [0304] S. D. Erlich,
1977, Proc. Natl. Acad. Sci. 74, pp. 1680-1682. [0305] Boel et al.,
1990, Biochemistry 29, pp. 6244-6249.
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 58 <210> SEQ ID NO 1 <211> LENGTH: 485 <212>
TYPE: PRT <213> ORGANISM: Bacillus sp. <400> SEQUENCE:
1 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 <210> SEQ ID NO 2 <211> LENGTH: 485
<212> TYPE: PRT <213> ORGANISM: Bacillus sp.
<400> SEQUENCE: 2 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 <210> SEQ ID NO 3
<211> LENGTH: 514 <212> TYPE: PRT <213> ORGANISM:
Bacillus stearothermophilus <400> SEQUENCE: 3 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 Ala 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 Ser 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 Asp 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 Met Lys Thr Asn 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 Thr 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 Val 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 Trp Ser Ile Thr Thr 485 490 495 Arg Pro Trp Thr Asp
Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp
<210> SEQ ID NO 4 <211> LENGTH: 483 <212> TYPE:
PRT <213> ORGANISM: Bacillus licheniformis <400>
SEQUENCE: 4 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 <210> SEQ ID NO 5 <211> LENGTH: 480
<212> TYPE: PRT <213> ORGANISM: Bacillus
amyloliquefaciens <400> SEQUENCE: 5 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 <210> SEQ ID NO 6 <211>
LENGTH: 485 <212> TYPE: PRT <213> ORGANISM: Bacillus
sp. <400> SEQUENCE: 6 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 Asn Ser Asp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly
Ile Thr 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 Val Arg Thr Lys Tyr Gly 65 70 75 80
Thr Arg Ser 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 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn
Asn Arg Asn 115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala
Trp Thr Arg Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser
Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp
Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe
Arg Gly His 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 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 Gln
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 Asn Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His
Pro Ser 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 Arg Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln
Lys Tyr Ala Tyr Gly Lys 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 Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln
Val Trp Ser 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 Ile Trp Val Asn Lys 485 <210> SEQ ID NO 7
<211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM:
Bacillus sp. <400> SEQUENCE: 7 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 <210> SEQ
ID NO 8 <211> LENGTH: 485 <212> TYPE: PRT <213>
ORGANISM: Bacillus sp. <400> SEQUENCE: 8 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
<210> SEQ ID NO 9 <211> LENGTH: 1455 <212> TYPE:
DNA <213> ORGANISM: Bacillus sp. <400> SEQUENCE: 9
catcataatg gaacaaatgg tactatgatg caatatttcg aatggtattt gccaaatgac
60 gggaatcatt ggaacaggtt gagggatgac gcagctaact taaagagtaa
agggataaca 120 gctgtatgga tcccacctgc atggaagggg acttcccaga
atgatgtagg ttatggagcc 180 tatgatttat atgatcttgg agagtttaac
cagaagggga cggttcgtac aaaatatgga 240 acacgcaacc agctacaggc
tgcggtgacc tctttaaaaa ataacggcat tcaggtatat 300 ggtgatgtcg
tcatgaatca taaaggtgga gcagatggta cggaaattgt aaatgcggta 360
gaagtgaatc ggagcaaccg aaaccaggaa acctcaggag agtatgcaat agaagcgtgg
420 acaaagtttg attttcctgg aagaggaaat aaccattcca gctttaagtg
gcgctggtat 480 cattttgatg ggacagattg ggatcagtca cgccagcttc
aaaacaaaat atataaattc 540 aggggaacag gcaaggcctg ggactgggaa
gtcgatacag agaatggcaa ctatgactat 600 cttatgtatg cagacgtgga
tatggatcac ccagaagtaa tacatgaact tagaaactgg 660 ggagtgtggt
atacgaatac actgaacctt gatggattta gaatagatgc agtgaaacat 720
ataaaatata gctttacgag agattggctt acacatgtgc gtaacaccac aggtaaacca
780 atgtttgcag tggctgagtt ttggaaaaat gaccttggtg caattgaaaa
ctatttgaat 840 aaaacaagtt ggaatcactc ggtgtttgat gttcctctcc
actataattt gtacaatgca 900 tctaatagcg gtggttatta tgatatgaga
aatattttaa atggttctgt ggtgcaaaaa 960 catccaacac atgccgttac
ttttgttgat aaccatgatt ctcagcccgg ggaagcattg 1020 gaatcctttg
ttcaacaatg gtttaaacca cttgcatatg cattggttct gacaagggaa 1080
caaggttatc cttccgtatt ttatggggat tactacggta tcccaaccca tggtgttccg
1140 gctatgaaat ctaaaataga ccctcttctg caggcacgtc aaacttttgc
ctatggtacg 1200 cagcatgatt actttgatca tcatgatatt atcggttgga
caagagaggg aaatagctcc 1260 catccaaatt caggccttgc caccattatg
tcagatggtc caggtggtaa caaatggatg 1320 tatgtgggga aaaataaagc
gggacaagtt tggagagata ttaccggaaa taggacaggc 1380 accgtcacaa
ttaatgcaga cggatggggt aatttctctg ttaatggagg gtccgtttcg 1440
gtttgggtga agcaa 1455 <210> SEQ ID NO 10 <211> LENGTH:
1455 <212> TYPE: DNA <213> ORGANISM: Bacillus sp.
<400> SEQUENCE: 10 catcataatg ggacaaatgg gacgatgatg
caatactttg aatggcactt gcctaatgat 60 gggaatcact ggaatagatt
aagagatgat gctagtaatc taagaaatag aggtataacc 120 gctatttgga
ttccgcctgc ctggaaaggg acttcgcaaa atgatgtggg gtatggagcc 180
tatgatcttt atgatttagg ggaatttaat caaaagggga cggttcgtac taagtatggg
240 acacgtagtc aattggagtc tgccatccat gctttaaaga ataatggcgt
tcaagtttat 300 ggggatgtag tgatgaacca taaaggagga gctgatgcta
cagaaaacgt tcttgctgtc 360 gaggtgaatc caaataaccg gaatcaagaa
atatctgggg actacacaat tgaggcttgg 420 actaagtttg attttccagg
gaggggtaat acatactcag actttaaatg gcgttggtat 480 catttcgatg
gtgtagattg ggatcaatca cgacaattcc aaaatcgtat ctacaaattc 540
cgaggtgatg gtaaggcatg ggattgggaa gtagattcgg aaaatggaaa ttatgattat
600 ttaatgtatg cagatgtaga tatggatcat ccggaggtag taaatgagct
tagaagatgg 660 ggagaatggt atacaaatac attaaatctt gatggattta
ggatcgatgc ggtgaagcat 720 attaaatata gctttacacg tgattggttg
acccatgtaa gaaacgcaac gggaaaagaa 780 atgtttgctg ttgctgaatt
ttggaaaaat gatttaggtg ccttggagaa ctatttaaat 840 aaaacaaact
ggaatcattc tgtctttgat gtcccccttc attataatct ttataacgcg 900
tcaaatagtg gaggcaacta tgacatggca aaacttctta atggaacggt tgttcaaaag
960 catccaatgc atgccgtaac ttttgtggat aatcacgatt ctcaacctgg
ggaatcatta 1020 gaatcatttg tacaagaatg gtttaagcca cttgcttatg
cgcttatttt aacaagagaa 1080 caaggctatc cctctgtctt ctatggtgac
tactatggaa ttccaacaca tagtgtccca 1140 gcaatgaaag ccaagattga
tccaatctta gaggcgcgtc aaaattttgc atatggaaca 1200 caacatgatt
attttgacca tcataatata atcggatgga cacgtgaagg aaataccacg 1260
catcccaatt caggacttgc gactatcatg tcggatgggc cagggggaga gaaatggatg
1320 tacgtagggc aaaataaagc aggtcaagtt tggcatgaca taactggaaa
taaaccagga 1380 acagttacga tcaatgcaga tggatgggct aatttttcag
taaatggagg atctgtttcc 1440 atttgggtga aacga 1455 <210> SEQ ID
NO 11 <211> LENGTH: 1548 <212> TYPE: DNA <213>
ORGANISM: Bacillus stearothermophilus <400> SEQUENCE: 11
gccgcaccgt ttaacggcac catgatgcag tattttgaat ggtacttgcc ggatgatggc
60 acgttatgga ccaaagtggc caatgaagcc aacaacttat ccagccttgg
catcaccgct 120 ctttggctgc cgcccgctta caaaggaaca agccgcagcg
acgtagggta cggagtatac 180 gacttgtatg acctcggcga attcaatcaa
aaagggaccg tccgcacaaa atacggaaca 240 aaagctcaat atcttcaagc
cattcaagcc gcccacgccg ctggaatgca agtgtacgcc 300 gatgtcgtgt
tcgaccataa aggcggcgct gacggcacgg aatgggtgga cgccgtcgaa 360
gtcaatccgt ccgaccgcaa ccaagaaatc tcgggcacct atcaaatcca agcatggacg
420 aaatttgatt ttcccgggcg gggcaacacc tactccagct ttaagtggcg
ctggtaccat 480 tttgacggcg ttgattggga cgaaagccga aaattgagcc
gcatttacaa attccgcggc 540 atcggcaaag cgtgggattg ggaagtagac
acggaaaacg gaaactatga ctacttaatg 600 tatgccgacc ttgatatgga
tcatcccgaa gtcgtgaccg agctgaaaaa ctgggggaaa 660 tggtatgtca
acacaacgaa cattgatggg ttccggcttg atgccgtcaa gcatattaag 720
ttcagttttt ttcctgattg gttgtcgtat gtgcgttctc agactggcaa gccgctattt
780 accgtcgggg aatattggag ctatgacatc aacaagttgc acaattacat
tacgaaaaca 840 gacggaacga tgtctttgtt tgatgccccg ttacacaaca
aattttatac cgcttccaaa 900 tcagggggcg catttgatat gcgcacgtta
atgaccaata ctctcatgaa agatcaaccg 960 acattggccg tcaccttcgt
tgataatcat gacaccgaac ccggccaagc gctgcagtca 1020 tgggtcgacc
catggttcaa accgttggct tacgccttta ttctaactcg gcaggaagga 1080
tacccgtgcg tcttttatgg tgactattat ggcattccac aatataacat tccttcgctg
1140 aaaagcaaaa tcgatccgct cctcatcgcg cgcagggatt atgcttacgg
aacgcaacat 1200 gattatcttg atcactccga catcatcggg tggacaaggg
aagggggcac tgaaaaacca 1260 ggatccggac tggccgcact gatcaccgat
gggccgggag gaagcaaatg gatgtacgtt 1320 ggcaaacaac acgctggaaa
agtgttctat gaccttaccg gcaaccggag tgacaccgtc 1380 accatcaaca
gtgatggatg gggggaattc aaagtcaatg gcggttcggt ttcggtttgg 1440
gttcctagaa aaacgaccgt ttctaccatc gctcggccga tcacaacccg accgtggact
1500 ggtgaattcg tccgttggac cgaaccacgg ttggtggcat ggccttga 1548
<210> SEQ ID NO 12 <211> LENGTH: 1920 <212> TYPE:
DNA <213> ORGANISM: Bacillus licheniformis <400>
SEQUENCE: 12 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 gcaaatctta atgggacgct gatgcagtat tttgaatggt
acatgcccaa tgacggccaa 480 cattggaggc gtttgcaaaa cgactcggca
tatttggctg aacacggtat tactgccgtc 540 tggattcccc cggcatataa
gggaacgagc caagcggatg tgggctacgg tgcttacgac 600 ctttatgatt
taggggagtt tcatcaaaaa gggacggttc ggacaaagta cggcacaaaa 660
ggagagctgc aatctgcgat caaaagtctt cattcccgcg acattaacgt ttacggggat
720 gtggtcatca accacaaagg cggcgctgat gcgaccgaag atgtaaccgc
ggttgaagtc 780 gatcccgctg accgcaaccg cgtaatttca ggagaacacc
taattaaagc ctggacacat 840 tttcattttc cggggcgcgg cagcacatac
agcgatttta aatggcattg gtaccatttt 900 gacggaaccg attgggacga
gtcccgaaag ctgaaccgca tctataagtt tcaaggaaag 960 gcttgggatt
gggaagtttc caatgaaaac ggcaactatg attatttgat gtatgccgac 1020
atcgattatg accatcctga tgtcgcagca gaaattaaga gatggggcac ttggtatgcc
1080 aatgaactgc aattggacgg tttccgtctt gatgctgtca aacacattaa
attttctttt 1140 ttgcgggatt gggttaatca tgtcagggaa aaaacgggga
aggaaatgtt tacggtagct 1200 gaatattggc agaatgactt gggcgcgctg
gaaaactatt tgaacaaaac aaattttaat 1260 cattcagtgt ttgacgtgcc
gcttcattat cagttccatg ctgcatcgac acagggaggc 1320 ggctatgata
tgaggaaatt gctgaacggt acggtcgttt ccaagcatcc gttgaaatcg 1380
gttacatttg tcgataacca tgatacacag ccggggcaat cgcttgagtc gactgtccaa
1440 acatggttta agccgcttgc ttacgctttt attctcacaa gggaatctgg
ataccctcag 1500 gttttctacg gggatatgta cgggacgaaa ggagactccc
agcgcgaaat tcctgccttg 1560 aaacacaaaa ttgaaccgat cttaaaagcg
agaaaacagt atgcgtacgg agcacagcat 1620 gattatttcg accaccatga
cattgtcggc tggacaaggg aaggcgacag ctcggttgca 1680 aattcaggtt
tggcggcatt aataacagac ggacccggtg gggcaaagcg aatgtatgtc 1740
ggccggcaaa acgccggtga gacatggcat gacattaccg gaaaccgttc ggagccggtt
1800 gtcatcaatt cggaaggctg gggagagttt cacgtaaacg gcgggtcggt
ttcaatttat 1860 gttcaaagat agaagagcag agaggacgga tttcctgaag
gaaatccgtt tttttatttt 1920 <210> SEQ ID NO 13 <400>
SEQUENCE: 13 000 <210> SEQ ID NO 14 <211> LENGTH: 1455
<212> TYPE: DNA <213> ORGANISM: Bacillus sp.
<400> SEQUENCE: 14 catcataatg ggacaaatgg gacgatgatg
caatactttg aatggcactt gcctaatgat 60 gggaatcact ggaatagatt
aagagatgat gctagtaatc taagaaatag aggtataacc 120 gctatttgga
ttccgcctgc ctggaaaggg acttcgcaaa atgatgtggg gtatggagcc 180
tatgatcttt atgatttagg ggaatttaat caaaagggga cggttcgtac taagtatggg
240 acacgtagtc aattggagtc tgccatccat gctttaaaga ataatggcgt
tcaagtttat 300 ggggatgtag tgatgaacca taaaggagga gctgatgcta
cagaaaacgt tcttgctgtc 360 gaggtgaatc caaataaccg gaatcaagaa
atatctgggg actacacaat tgaggcttgg 420 actaagtttg attttccagg
gaggggtaat acatactcag actttaaatg gcgttggtat 480 catttcgatg
gtgtagattg ggatcaatca cgacaattcc aaaatcgtat ctacaaattc 540
cgaggtgatg gtaaggcatg ggattgggaa gtagattcgg aaaatggaaa ttatgattat
600 ttaatgtatg cagatgtaga tatggatcat ccggaggtag taaatgagct
tagaagatgg 660 ggagaatggt atacaaatac attaaatctt gatggattta
ggatcgatgc ggtgaagcat 720 attaaatata gctttacacg tgattggttg
acccatgtaa gaaacgcaac gggaaaagaa 780 atgtttgctg ttgctgaatt
ttggaaaaat gatttaggtg ccttggagaa ctatttaaat 840 aaaacaaact
ggaatcattc tgtctttgat gtcccccttc attataatct ttataacgcg 900
tcaaatagtg gaggcaacta tgacatggca aaacttctta atggaacggt tgttcaaaag
960 catccaatgc atgccgtaac ttttgtggat aatcacgatt ctcaacctgg
ggaatcatta 1020 gaatcatttg tacaagaatg gtttaagcca cttgcttatg
cgcttatttt aacaagagaa 1080 caaggctatc cctctgtctt ctatggtgac
tactatggaa ttccaacaca tagtgtccca 1140 gcaatgaaag ccaagattga
tccaatctta gaggcgcgtc aaaattttgc atatggaaca 1200 caacatgatt
attttgacca tcataatata atcggatgga cacgtgaagg aaataccacg 1260
catcccaatt caggacttgc gactatcatg tcggatgggc cagggggaga gaaatggatg
1320 tacgtagggc aaaataaagc aggtcaagtt tggcatgaca taactggaaa
taaaccagga 1380 acagttacga tcaatgcaga tggatgggct aatttttcag
taaatggagg atctgtttcc 1440 atttgggtga aacga 1455 <210> SEQ ID
NO 15 <211> LENGTH: 60 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Forward Primer FSA <400> SEQUENCE: 15 caaaatcgta
tctacaaatt cmrkrsyarg dvktgggatt sggaagtaga ttcggaaaat 60
<210> SEQ ID NO 16 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Reverse Primer RSA <400>
SEQUENCE: 16 gaatttgtag atacgatttt g 21 <210> SEQ ID NO 17
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer sequence <400> SEQUENCE: 17 cgattgctga cgctgttatt tgcg
24 <210> SEQ ID NO 18 <211> LENGTH: 24 <212>
TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 18 cttgttccct tgtcagaacc aatg 24 <210> SEQ ID NO 19
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer 101458 <400> SEQUENCE: 19 gtcatagttg ccgaaatctg
tatcgacttc 30 <210> SEQ ID NO 20 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer 101638 <400>
SEQUENCE: 20 cccagtccca cgtacgtccc ctgaatttat atattttg 38
<210> SEQ ID NO 21 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Oligo 1 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (12)..(12)
<223> OTHER INFORMATION: n is a, c, g, or t <400>
SEQUENCE: 21 cccagtccca gntctttccc ctgaatttat atattttg 38
<210> SEQ ID NO 22 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer X2 <400> SEQUENCE: 22
gcgtggacaa agtttgattt tcctg 25 <210> SEQ ID NO 23 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA01
<400> SEQUENCE: 23 cctaatgatg ggaatcactg g 21 <210> SEQ
ID NO 24 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer DA03 <400> SEQUENCE: 24 gcattggatg
cttttgaaca accg 24 <210> SEQ ID NO 25 <211> LENGTH: 26
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer DA07 <400>
SEQUENCE: 25 cgcaaaatga tatcgggtat ggagcc 26 <210> SEQ ID NO
26 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer DA20 <400> SEQUENCE: 26 gtgatgaacc
acswaggtgg agctgatgc 29 <210> SEQ ID NO 27 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA14
<400> SEQUENCE: 27 gatggtgtat ggrycaatca cgacaattcc 30
<210> SEQ ID NO 28 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA15 <400> SEQUENCE: 28
ggtgtatggg ataactcacg acaattcc 28 <210> SEQ ID NO 29
<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA16 <400> SEQUENCE: 29 ggtgtatggg atctctcacg acaattcc
28 <210> SEQ ID NO 30 <211> LENGTH: 32 <212>
TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA17 <400> SEQUENCE: 30
gggatcaatc acgaaatttc caaaatcgta tc 32 <210> SEQ ID NO 31
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA18 <400> SEQUENCE: 31 gggatcaatc acgactcttc
caaaatcgta tc 32 <210> SEQ ID NO 32 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer DA06 <400>
SEQUENCE: 32 ggaaattatg attatatcat gtatgcagat gtag 34 <210>
SEQ ID NO 33 <211> LENGTH: 30 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Primer DA09 <400> SEQUENCE: 33 gctgaatttt
ggtcgaatga tttaggtgcc 30 <210> SEQ ID NO 34 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA11
<400> SEQUENCE: 34 gctgaatttt ggtcgaatga tttaggtgcc 30
<210> SEQ ID NO 35 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA21 <400> SEQUENCE: 35
gaattttgga agtacgattt aggtcgg 27 <210> SEQ ID NO 36
<211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA12 <400> SEQUENCE: 36 ggaaaaacga trycggtgcc
ttggagaac 29 <210> SEQ ID NO 37 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer DA13 <400>
SEQUENCE: 37 gatttaggtg cctrycagaa ctattta 27 <210> SEQ ID NO
38 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer DA08 <400> SEQUENCE: 38 cccccttcat
gagaatcttt ataacg 26 <210> SEQ ID NO 39 <211> LENGTH:
25 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA04
<400> SEQUENCE: 39 gaatccgaac ctcattacac attcg 25 <210>
SEQ ID NO 40 <211> LENGTH: 28 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Primer DA05 <400> SEQUENCE: 40 cggatggact
cgagaaggaa ataccacg 28 <210> SEQ ID NO 41 <211> LENGTH:
31 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA10
<400> SEQUENCE: 41 cgtagggcaa aatcaggccg gtcaagtttg g 31
<210> SEQ ID NO 42 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA22 <400> SEQUENCE: 42
cataactgga aatcgcccgg gaacagttac g 31 <210> SEQ ID NO 43
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA19 <400> SEQUENCE: 43 ctggaaataa awccggaaca gttacg
26 <210> SEQ ID NO 44 <211> LENGTH: 32 <212>
TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA23 <400> SEQUENCE: 44
ggaaataaac caggacccgt tacgatcaat gc 32 <210> SEQ ID NO 45
<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA32 <400> SEQUENCE: 45 gaggcttgga ctaggtttga ttttccag
28 <210> SEQ ID NO 46 <211> LENGTH: 30 <212>
TYPE: DNA <213> ORGANISM: Primer DA31 <400> SEQUENCE:
46 gctgaatttt ggcgcaatga tttaggtgcc 30 <210> SEQ ID NO 47
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer bm4 <400> SEQUENCE: 47 gtgtttgacg tcccgcttca
tgagaattta cagg 34 <210> SEQ ID NO 48 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer bm5 <400>
SEQUENCE: 48 gtgtttgacg tcccgcttca taagaattta cagg 34 <210>
SEQ ID NO 49 <211> LENGTH: 34 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Primer bm6 <400> SEQUENCE: 49 gtgtttgacg
tcccgcttca tgccaattta cagg 34 <210> SEQ ID NO 50 <211>
LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer bm8
<400> SEQUENCE: 50 agggaatccg gataccctga ggttttctac gg 32
<210> SEQ ID NO 51 <211> LENGTH: 34 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer bm11 <400> SEQUENCE: 51
gatgtggttt tggatcataa ggccggcgct gatg 34 <210> SEQ ID NO 52
<211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer p1 <400> SEQUENCE: 52 ctgttattaa tgccgccaaa cc 22
<210> SEQ ID NO 53 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer p2 <400> SEQUENCE: 53
ggaaaagaaa tgtttacggt tgcg 24 <210> SEQ ID NO 54 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer p3
<400> SEQUENCE: 54 gaaatgaagc ggaacatcaa acacg 25 <210>
SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Primer p4 <400> SEQUENCE: 55 gtatgattta
ggagaattcc 20 <210> SEQ ID NO 56 <211> LENGTH: 33
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer bl1 <400>
SEQUENCE: 56 ccagcgcgcc taggtcacgc tgccaatatt cag 33 <210>
SEQ ID NO 57 <211> LENGTH: 33 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Primer bl2 <400> SEQUENCE: 57 ccagcgcgcc
taggtcatcc tgccaatatt cag 33 <210> SEQ ID NO 58 <211>
LENGTH: 2084 <212> TYPE: DNA <213> ORGANISM: Bacillus
amyloliquefaciens <400> SEQUENCE: 58 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 ccgtaaatgg cacgctgatg 360 cagtattttg aatggtatac
gccgaacgac ggccagcatt ggaaacgatt gcagaatgat 420 gcggaacatt
tatcggatat cggaatcact gccgtctgga ttcctcccgc atacaaagga 480
ttgagccaat ccgataacgg atacggacct tatgatttgt atgatttagg agaattccag
540 caaaaaggga cggtcagaac gaaatacggc acaaaatcag agcttcaaga
tgcgatcggc 600 tcactgcatt cccggaacgt ccaagtatac ggagatgtgg
ttttgaatca taaggctggt 660 gctgatgcaa cagaagatgt aactgccgtc
gaagtcaatc cggccaatag aaatcaggaa 720 acttcggagg aatatcaaat
caaagcgtgg acggattttc gttttccggg ccgtggaaac 780 acgtacagtg
attttaaatg gcattggtat catttcgacg gagcggactg ggatgaatcc 840
cggaagatca gccgcatctt taagtttcgt ggggaaggaa aagcgtggga ttgggaagta
900 tcaagtgaaa acggcaacta tgactattta atgtatgctg atgttgacta
cgaccaccct 960 gatgtcgtgg cagagacaaa aaaatggggt atctggtatg
cgaatgaact gtcattagac 1020 ggcttccgta ttgatgccgc caaacatatt
aaattttcat ttctgcgtga ttgggttcag 1080 gcggtcagac aggcgacggg
aaaagaaatg tttacggttg cggagtattg gcagaataat 1140 gccgggaaac
tcgaaaacta cttgaataaa acaagcttta atcaatccgt gtttgatgtt 1200
ccgcttcatt tcaatttaca ggcggcttcc tcacaaggag gcggatatga tatgaggcgt
1260 ttgctggacg gtaccgttgt gtccaggcat ccggaaaagg cggttacatt
tgttgaaaat 1320 catgacacac agccgggaca gtcattggaa tcgacagtcc
aaacttggtt taaaccgctt 1380 gcatacgcct ttattttgac aagagaatcc
ggttatcctc aggtgttcta tggggatatg 1440 tacgggacaa aagggacatc
gccaaaggaa attccctcac tgaaagataa tatagagccg 1500 attttaaaag
cgcgtaagga gtacgcatac gggccccagc acgattatat tgaccacccg 1560
gatgtgatcg gatggacgag ggaaggtgac agctccgccg ccaaatcagg tttggccgct
1620 ttaatcacgg acggacccgg cggatcaaag cggatgtatg ccggcctgaa
aaatgccggc 1680 gagacatggt atgacataac gggcaaccgt tcagatactg
taaaaatcgg atctgacggc 1740 tggggagagt ttcatgtaaa cgatgggtcc
gtctccattt atgttcagaa ataaggtaat 1800 aaaaaaacac ctccaagctg
agtgcgggta tcagcttgga ggtgcgttta ttttttcagc 1860 cgtatgacaa
ggtcggcatc aggtgtgaca aatacggtat gctggctgtc ataggtgaca 1920
aatccgggtt ttgcgccgtt tggctttttc acatgtctga tttttgtata atcaacaggc
1980 acggagccgg aatctttcgc cttggaaaaa taagcggcga tcgtagctgc
ttccaatatg 2040 gattgttcat cgggatcgct gcttttaatc acaacgtggg atcc
2084
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 58 <210>
SEQ ID NO 1 <211> LENGTH: 485 <212> TYPE: PRT
<213> ORGANISM: Bacillus sp. <400> SEQUENCE: 1 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 <210> SEQ ID NO 2 <211> LENGTH: 485 <212>
TYPE: PRT <213> ORGANISM: Bacillus sp. <400> SEQUENCE:
2 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 <210> SEQ ID NO 3 <211> LENGTH: 514
<212> TYPE: PRT <213> ORGANISM: Bacillus
stearothermophilus <400> SEQUENCE: 3 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 Ala 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 Ser 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 Asp
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 Met
Lys Thr Asn 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 Thr 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 Val 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
Trp Ser Ile Thr Thr 485 490 495 Arg Pro Trp Thr Asp Glu Phe Val Arg
Trp Thr Glu Pro Arg Leu Val 500 505 510 Ala Trp <210> SEQ ID
NO 4 <211> LENGTH: 483 <212> TYPE: PRT <213>
ORGANISM: Bacillus licheniformis <400> SEQUENCE: 4 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
<210> SEQ ID NO 5 <211> LENGTH: 480 <212> TYPE:
PRT <213> ORGANISM: Bacillus amyloliquefaciens <400>
SEQUENCE: 5 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 <210> SEQ ID NO 6 <211>
LENGTH: 485 <212> TYPE: PRT <213> ORGANISM: Bacillus
sp. <400> SEQUENCE: 6 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 Asn Ser Asp Ala Ser 20 25 30 Asn Leu Lys Ser Lys Gly
Ile Thr 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 Val Arg Thr Lys Tyr Gly 65 70 75 80
Thr Arg Ser 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 Ala Thr Glu Met Val Arg Ala Val Glu Val Asn Pro Asn
Asn Arg Asn 115 120 125 Gln Glu Val Thr Gly Glu Tyr Thr Ile Glu Ala
Trp Thr Arg Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr His Ser
Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160 His Phe Asp Gly Val Asp
Trp Asp Gln Ser Arg Arg Leu Asn Asn Arg 165 170 175 Ile Tyr Lys Phe
Arg Gly His 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 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 Gln
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 Asn Ile Phe Asn Gly Thr Val Val Gln Arg 305 310 315 320 His
Pro Ser 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 Arg Ser 370 375 380 Lys Ile Asp Pro Ile Leu Glu Ala Arg Gln
Lys Tyr Ala Tyr Gly Lys 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 Ser Lys Trp Met Phe Val Gly Arg Asn Lys Ala Gly 435 440 445 Gln
Val Trp Ser 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 Ile Trp Val Asn Lys 485 <210> SEQ ID NO 7
<211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM:
Bacillus sp. <400> SEQUENCE: 7 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 <210> SEQ
ID NO 8 <211> LENGTH: 485 <212> TYPE: PRT <213>
ORGANISM: Bacillus sp. <400> SEQUENCE: 8 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
<210> SEQ ID NO 9 <211> LENGTH: 1455 <212> TYPE:
DNA <213> ORGANISM: Bacillus sp. <400> SEQUENCE: 9
catcataatg gaacaaatgg tactatgatg caatatttcg aatggtattt gccaaatgac
60 gggaatcatt ggaacaggtt gagggatgac gcagctaact taaagagtaa
agggataaca 120 gctgtatgga tcccacctgc atggaagggg acttcccaga
atgatgtagg ttatggagcc 180 tatgatttat atgatcttgg agagtttaac
cagaagggga cggttcgtac aaaatatgga 240 acacgcaacc agctacaggc
tgcggtgacc tctttaaaaa ataacggcat tcaggtatat 300 ggtgatgtcg
tcatgaatca taaaggtgga gcagatggta cggaaattgt aaatgcggta 360
gaagtgaatc ggagcaaccg aaaccaggaa acctcaggag agtatgcaat agaagcgtgg
420 acaaagtttg attttcctgg aagaggaaat aaccattcca gctttaagtg
gcgctggtat 480 cattttgatg ggacagattg ggatcagtca cgccagcttc
aaaacaaaat atataaattc 540 aggggaacag gcaaggcctg ggactgggaa
gtcgatacag agaatggcaa ctatgactat 600 cttatgtatg cagacgtgga
tatggatcac ccagaagtaa tacatgaact tagaaactgg 660 ggagtgtggt
atacgaatac actgaacctt gatggattta gaatagatgc agtgaaacat 720
ataaaatata gctttacgag agattggctt acacatgtgc gtaacaccac aggtaaacca
780 atgtttgcag tggctgagtt ttggaaaaat gaccttggtg caattgaaaa
ctatttgaat 840 aaaacaagtt ggaatcactc ggtgtttgat gttcctctcc
actataattt gtacaatgca 900 tctaatagcg gtggttatta tgatatgaga
aatattttaa atggttctgt ggtgcaaaaa 960 catccaacac atgccgttac
ttttgttgat aaccatgatt ctcagcccgg ggaagcattg 1020 gaatcctttg
ttcaacaatg gtttaaacca cttgcatatg cattggttct gacaagggaa 1080
caaggttatc cttccgtatt ttatggggat tactacggta tcccaaccca tggtgttccg
1140 gctatgaaat ctaaaataga ccctcttctg caggcacgtc aaacttttgc
ctatggtacg 1200 cagcatgatt actttgatca tcatgatatt atcggttgga
caagagaggg aaatagctcc 1260 catccaaatt caggccttgc caccattatg
tcagatggtc caggtggtaa caaatggatg 1320 tatgtgggga aaaataaagc
gggacaagtt tggagagata ttaccggaaa taggacaggc 1380 accgtcacaa
ttaatgcaga cggatggggt aatttctctg ttaatggagg gtccgtttcg 1440
gtttgggtga agcaa 1455 <210> SEQ ID NO 10 <211> LENGTH:
1455 <212> TYPE: DNA <213> ORGANISM: Bacillus sp.
<400> SEQUENCE: 10 catcataatg ggacaaatgg gacgatgatg
caatactttg aatggcactt gcctaatgat 60 gggaatcact ggaatagatt
aagagatgat gctagtaatc taagaaatag aggtataacc 120 gctatttgga
ttccgcctgc ctggaaaggg acttcgcaaa atgatgtggg gtatggagcc 180
tatgatcttt atgatttagg ggaatttaat caaaagggga cggttcgtac taagtatggg
240 acacgtagtc aattggagtc tgccatccat gctttaaaga ataatggcgt
tcaagtttat 300 ggggatgtag tgatgaacca taaaggagga gctgatgcta
cagaaaacgt tcttgctgtc 360 gaggtgaatc caaataaccg gaatcaagaa
atatctgggg actacacaat tgaggcttgg 420 actaagtttg attttccagg
gaggggtaat acatactcag actttaaatg gcgttggtat 480 catttcgatg
gtgtagattg ggatcaatca cgacaattcc aaaatcgtat ctacaaattc 540
cgaggtgatg gtaaggcatg ggattgggaa gtagattcgg aaaatggaaa ttatgattat
600 ttaatgtatg cagatgtaga tatggatcat ccggaggtag taaatgagct
tagaagatgg 660 ggagaatggt atacaaatac attaaatctt gatggattta
ggatcgatgc ggtgaagcat 720 attaaatata gctttacacg tgattggttg
acccatgtaa gaaacgcaac gggaaaagaa 780 atgtttgctg ttgctgaatt
ttggaaaaat gatttaggtg ccttggagaa ctatttaaat 840 aaaacaaact
ggaatcattc tgtctttgat gtcccccttc attataatct ttataacgcg 900
tcaaatagtg gaggcaacta tgacatggca aaacttctta atggaacggt tgttcaaaag
960 catccaatgc atgccgtaac ttttgtggat aatcacgatt ctcaacctgg
ggaatcatta 1020 gaatcatttg tacaagaatg gtttaagcca cttgcttatg
cgcttatttt aacaagagaa 1080 caaggctatc cctctgtctt ctatggtgac
tactatggaa ttccaacaca tagtgtccca 1140 gcaatgaaag ccaagattga
tccaatctta gaggcgcgtc aaaattttgc atatggaaca 1200 caacatgatt
attttgacca tcataatata atcggatgga cacgtgaagg aaataccacg 1260
catcccaatt caggacttgc gactatcatg tcggatgggc cagggggaga gaaatggatg
1320 tacgtagggc aaaataaagc aggtcaagtt tggcatgaca taactggaaa
taaaccagga 1380 acagttacga tcaatgcaga tggatgggct aatttttcag
taaatggagg atctgtttcc 1440 atttgggtga aacga 1455 <210> SEQ ID
NO 11 <211> LENGTH: 1548 <212> TYPE: DNA <213>
ORGANISM: Bacillus stearothermophilus <400> SEQUENCE: 11
gccgcaccgt ttaacggcac catgatgcag tattttgaat ggtacttgcc ggatgatggc
60 acgttatgga ccaaagtggc caatgaagcc aacaacttat ccagccttgg
catcaccgct 120 ctttggctgc cgcccgctta caaaggaaca agccgcagcg
acgtagggta cggagtatac 180 gacttgtatg acctcggcga attcaatcaa
aaagggaccg tccgcacaaa atacggaaca 240 aaagctcaat atcttcaagc
cattcaagcc gcccacgccg ctggaatgca agtgtacgcc 300 gatgtcgtgt
tcgaccataa aggcggcgct gacggcacgg aatgggtgga cgccgtcgaa 360
gtcaatccgt ccgaccgcaa ccaagaaatc tcgggcacct atcaaatcca agcatggacg
420 aaatttgatt ttcccgggcg gggcaacacc tactccagct ttaagtggcg
ctggtaccat 480 tttgacggcg ttgattggga cgaaagccga aaattgagcc
gcatttacaa attccgcggc 540 atcggcaaag cgtgggattg ggaagtagac
acggaaaacg gaaactatga ctacttaatg 600 tatgccgacc ttgatatgga
tcatcccgaa gtcgtgaccg agctgaaaaa ctgggggaaa 660 tggtatgtca
acacaacgaa cattgatggg ttccggcttg atgccgtcaa gcatattaag 720
ttcagttttt ttcctgattg gttgtcgtat gtgcgttctc agactggcaa gccgctattt
780 accgtcgggg aatattggag ctatgacatc aacaagttgc acaattacat
tacgaaaaca 840 gacggaacga tgtctttgtt tgatgccccg ttacacaaca
aattttatac cgcttccaaa 900 tcagggggcg catttgatat gcgcacgtta
atgaccaata ctctcatgaa agatcaaccg 960 acattggccg tcaccttcgt
tgataatcat gacaccgaac ccggccaagc gctgcagtca 1020 tgggtcgacc
catggttcaa accgttggct tacgccttta ttctaactcg gcaggaagga 1080
tacccgtgcg tcttttatgg tgactattat ggcattccac aatataacat tccttcgctg
1140 aaaagcaaaa tcgatccgct cctcatcgcg cgcagggatt atgcttacgg
aacgcaacat 1200 gattatcttg atcactccga catcatcggg tggacaaggg
aagggggcac tgaaaaacca 1260 ggatccggac tggccgcact gatcaccgat
gggccgggag gaagcaaatg gatgtacgtt 1320 ggcaaacaac acgctggaaa
agtgttctat gaccttaccg gcaaccggag tgacaccgtc 1380 accatcaaca
gtgatggatg gggggaattc aaagtcaatg gcggttcggt ttcggtttgg 1440
gttcctagaa aaacgaccgt ttctaccatc gctcggccga tcacaacccg accgtggact
1500 ggtgaattcg tccgttggac cgaaccacgg ttggtggcat ggccttga 1548
<210> SEQ ID NO 12 <211> LENGTH: 1920 <212> TYPE:
DNA <213> ORGANISM: Bacillus licheniformis <400>
SEQUENCE: 12 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 gcaaatctta atgggacgct
gatgcagtat tttgaatggt acatgcccaa tgacggccaa 480 cattggaggc
gtttgcaaaa cgactcggca tatttggctg aacacggtat tactgccgtc 540
tggattcccc cggcatataa gggaacgagc caagcggatg tgggctacgg tgcttacgac
600 ctttatgatt taggggagtt tcatcaaaaa gggacggttc ggacaaagta
cggcacaaaa 660 ggagagctgc aatctgcgat caaaagtctt cattcccgcg
acattaacgt ttacggggat 720 gtggtcatca accacaaagg cggcgctgat
gcgaccgaag atgtaaccgc ggttgaagtc 780 gatcccgctg accgcaaccg
cgtaatttca ggagaacacc taattaaagc ctggacacat 840 tttcattttc
cggggcgcgg cagcacatac agcgatttta aatggcattg gtaccatttt 900
gacggaaccg attgggacga gtcccgaaag ctgaaccgca tctataagtt tcaaggaaag
960 gcttgggatt gggaagtttc caatgaaaac ggcaactatg attatttgat
gtatgccgac 1020 atcgattatg accatcctga tgtcgcagca gaaattaaga
gatggggcac ttggtatgcc 1080 aatgaactgc aattggacgg tttccgtctt
gatgctgtca aacacattaa attttctttt 1140 ttgcgggatt gggttaatca
tgtcagggaa aaaacgggga aggaaatgtt tacggtagct 1200 gaatattggc
agaatgactt gggcgcgctg gaaaactatt tgaacaaaac aaattttaat 1260
cattcagtgt ttgacgtgcc gcttcattat cagttccatg ctgcatcgac acagggaggc
1320 ggctatgata tgaggaaatt gctgaacggt acggtcgttt ccaagcatcc
gttgaaatcg 1380 gttacatttg tcgataacca tgatacacag ccggggcaat
cgcttgagtc gactgtccaa 1440 acatggttta agccgcttgc ttacgctttt
attctcacaa gggaatctgg ataccctcag 1500 gttttctacg gggatatgta
cgggacgaaa ggagactccc agcgcgaaat tcctgccttg 1560 aaacacaaaa
ttgaaccgat cttaaaagcg agaaaacagt atgcgtacgg agcacagcat 1620
gattatttcg accaccatga cattgtcggc tggacaaggg aaggcgacag ctcggttgca
1680 aattcaggtt tggcggcatt aataacagac ggacccggtg gggcaaagcg
aatgtatgtc 1740 ggccggcaaa acgccggtga gacatggcat gacattaccg
gaaaccgttc ggagccggtt 1800 gtcatcaatt cggaaggctg gggagagttt
cacgtaaacg gcgggtcggt ttcaatttat 1860 gttcaaagat agaagagcag
agaggacgga tttcctgaag gaaatccgtt tttttatttt 1920 <210> SEQ ID
NO 13 <400> SEQUENCE: 13 000 <210> SEQ ID NO 14
<211> LENGTH: 1455 <212> TYPE: DNA <213>
ORGANISM: Bacillus sp. <400> SEQUENCE: 14 catcataatg
ggacaaatgg gacgatgatg caatactttg aatggcactt gcctaatgat 60
gggaatcact ggaatagatt aagagatgat gctagtaatc taagaaatag aggtataacc
120 gctatttgga ttccgcctgc ctggaaaggg acttcgcaaa atgatgtggg
gtatggagcc 180 tatgatcttt atgatttagg ggaatttaat caaaagggga
cggttcgtac taagtatggg 240 acacgtagtc aattggagtc tgccatccat
gctttaaaga ataatggcgt tcaagtttat 300 ggggatgtag tgatgaacca
taaaggagga gctgatgcta cagaaaacgt tcttgctgtc 360 gaggtgaatc
caaataaccg gaatcaagaa atatctgggg actacacaat tgaggcttgg 420
actaagtttg attttccagg gaggggtaat acatactcag actttaaatg gcgttggtat
480 catttcgatg gtgtagattg ggatcaatca cgacaattcc aaaatcgtat
ctacaaattc 540 cgaggtgatg gtaaggcatg ggattgggaa gtagattcgg
aaaatggaaa ttatgattat 600 ttaatgtatg cagatgtaga tatggatcat
ccggaggtag taaatgagct tagaagatgg 660 ggagaatggt atacaaatac
attaaatctt gatggattta ggatcgatgc ggtgaagcat 720 attaaatata
gctttacacg tgattggttg acccatgtaa gaaacgcaac gggaaaagaa 780
atgtttgctg ttgctgaatt ttggaaaaat gatttaggtg ccttggagaa ctatttaaat
840 aaaacaaact ggaatcattc tgtctttgat gtcccccttc attataatct
ttataacgcg 900 tcaaatagtg gaggcaacta tgacatggca aaacttctta
atggaacggt tgttcaaaag 960 catccaatgc atgccgtaac ttttgtggat
aatcacgatt ctcaacctgg ggaatcatta 1020 gaatcatttg tacaagaatg
gtttaagcca cttgcttatg cgcttatttt aacaagagaa 1080 caaggctatc
cctctgtctt ctatggtgac tactatggaa ttccaacaca tagtgtccca 1140
gcaatgaaag ccaagattga tccaatctta gaggcgcgtc aaaattttgc atatggaaca
1200 caacatgatt attttgacca tcataatata atcggatgga cacgtgaagg
aaataccacg 1260 catcccaatt caggacttgc gactatcatg tcggatgggc
cagggggaga gaaatggatg 1320 tacgtagggc aaaataaagc aggtcaagtt
tggcatgaca taactggaaa taaaccagga 1380 acagttacga tcaatgcaga
tggatgggct aatttttcag taaatggagg atctgtttcc 1440 atttgggtga aacga
1455 <210> SEQ ID NO 15 <211> LENGTH: 60 <212>
TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Forward Primer FSA <400>
SEQUENCE: 15 caaaatcgta tctacaaatt cmrkrsyarg dvktgggatt sggaagtaga
ttcggaaaat 60 <210> SEQ ID NO 16 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Reverse Primer RSA
<400> SEQUENCE: 16 gaatttgtag atacgatttt g 21 <210> SEQ
ID NO 17 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer sequence <400> SEQUENCE: 17 cgattgctga
cgctgttatt tgcg 24 <210> SEQ ID NO 18 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer sequence <400>
SEQUENCE: 18 cttgttccct tgtcagaacc aatg 24 <210> SEQ ID NO 19
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer 101458 <400> SEQUENCE: 19 gtcatagttg ccgaaatctg
tatcgacttc 30 <210> SEQ ID NO 20 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer 101638 <400>
SEQUENCE: 20 cccagtccca cgtacgtccc ctgaatttat atattttg 38
<210> SEQ ID NO 21 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Oligo 1 <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION: (12)..(12)
<223> OTHER INFORMATION: n is a, c, g, or t <400>
SEQUENCE: 21 cccagtccca gntctttccc ctgaatttat atattttg 38
<210> SEQ ID NO 22 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer X2 <400> SEQUENCE: 22
gcgtggacaa agtttgattt tcctg 25 <210> SEQ ID NO 23 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA01
<400> SEQUENCE: 23 cctaatgatg ggaatcactg g 21 <210> SEQ
ID NO 24 <211> LENGTH: 24 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer DA03 <400> SEQUENCE: 24 gcattggatg
cttttgaaca accg 24 <210> SEQ ID NO 25 <211> LENGTH:
26
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer DA07 <400>
SEQUENCE: 25 cgcaaaatga tatcgggtat ggagcc 26 <210> SEQ ID NO
26 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer DA20 <400> SEQUENCE: 26 gtgatgaacc
acswaggtgg agctgatgc 29 <210> SEQ ID NO 27 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA14
<400> SEQUENCE: 27 gatggtgtat ggrycaatca cgacaattcc 30
<210> SEQ ID NO 28 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA15 <400> SEQUENCE: 28
ggtgtatggg ataactcacg acaattcc 28 <210> SEQ ID NO 29
<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA16 <400> SEQUENCE: 29 ggtgtatggg atctctcacg acaattcc
28 <210> SEQ ID NO 30 <211> LENGTH: 32 <212>
TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA17 <400> SEQUENCE: 30
gggatcaatc acgaaatttc caaaatcgta tc 32 <210> SEQ ID NO 31
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA18 <400> SEQUENCE: 31 gggatcaatc acgactcttc
caaaatcgta tc 32 <210> SEQ ID NO 32 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer DA06 <400>
SEQUENCE: 32 ggaaattatg attatatcat gtatgcagat gtag 34 <210>
SEQ ID NO 33 <211> LENGTH: 30 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Primer DA09 <400> SEQUENCE: 33 gctgaatttt
ggtcgaatga tttaggtgcc 30 <210> SEQ ID NO 34 <211>
LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA11
<400> SEQUENCE: 34 gctgaatttt ggtcgaatga tttaggtgcc 30
<210> SEQ ID NO 35 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA21 <400> SEQUENCE: 35
gaattttgga agtacgattt aggtcgg 27 <210> SEQ ID NO 36
<211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA12 <400> SEQUENCE: 36 ggaaaaacga trycggtgcc
ttggagaac 29 <210> SEQ ID NO 37 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer DA13 <400>
SEQUENCE: 37 gatttaggtg cctrycagaa ctattta 27 <210> SEQ ID NO
38 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer DA08 <400> SEQUENCE: 38 cccccttcat
gagaatcttt ataacg 26 <210> SEQ ID NO 39 <211> LENGTH:
25 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA04
<400> SEQUENCE: 39 gaatccgaac ctcattacac attcg 25 <210>
SEQ ID NO 40 <211> LENGTH: 28 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Primer DA05 <400> SEQUENCE: 40 cggatggact
cgagaaggaa ataccacg 28 <210> SEQ ID NO 41 <211> LENGTH:
31 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer DA10
<400> SEQUENCE: 41 cgtagggcaa aatcaggccg gtcaagtttg g 31
<210> SEQ ID NO 42 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA22 <400> SEQUENCE: 42
cataactgga aatcgcccgg gaacagttac g 31 <210> SEQ ID NO 43
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA19 <400> SEQUENCE: 43 ctggaaataa awccggaaca gttacg
26 <210> SEQ ID NO 44 <211> LENGTH: 32 <212>
TYPE: DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer DA23 <400> SEQUENCE: 44
ggaaataaac caggacccgt tacgatcaat gc 32 <210> SEQ ID NO 45
<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer DA32 <400> SEQUENCE: 45 gaggcttgga ctaggtttga ttttccag
28 <210> SEQ ID NO 46
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Primer DA31 <400> SEQUENCE: 46 gctgaatttt ggcgcaatga
tttaggtgcc 30 <210> SEQ ID NO 47 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer bm4 <400>
SEQUENCE: 47 gtgtttgacg tcccgcttca tgagaattta cagg 34 <210>
SEQ ID NO 48 <211> LENGTH: 34 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Primer bm5 <400> SEQUENCE: 48 gtgtttgacg
tcccgcttca taagaattta cagg 34 <210> SEQ ID NO 49 <211>
LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer bm6
<400> SEQUENCE: 49 gtgtttgacg tcccgcttca tgccaattta cagg 34
<210> SEQ ID NO 50 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer bm8 <400> SEQUENCE: 50
agggaatccg gataccctga ggttttctac gg 32 <210> SEQ ID NO 51
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer bm11 <400> SEQUENCE: 51 gatgtggttt tggatcataa
ggccggcgct gatg 34 <210> SEQ ID NO 52 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Primer p1 <400>
SEQUENCE: 52 ctgttattaa tgccgccaaa cc 22 <210> SEQ ID NO 53
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Primer p2 <400> SEQUENCE: 53 ggaaaagaaa tgtttacggt tgcg 24
<210> SEQ ID NO 54 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Primer p3 <400> SEQUENCE: 54
gaaatgaagc ggaacatcaa acacg 25 <210> SEQ ID NO 55 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer p4
<400> SEQUENCE: 55 gtatgattta ggagaattcc 20 <210> SEQ
ID NO 56 <211> LENGTH: 33 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Primer bl1 <400> SEQUENCE: 56 ccagcgcgcc
taggtcacgc tgccaatatt cag 33 <210> SEQ ID NO 57 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Primer bl2
<400> SEQUENCE: 57 ccagcgcgcc taggtcatcc tgccaatatt cag 33
<210> SEQ ID NO 58 <211> LENGTH: 2084 <212> TYPE:
DNA <213> ORGANISM: Bacillus amyloliquefaciens <400>
SEQUENCE: 58 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 ccgtaaatgg cacgctgatg
360 cagtattttg aatggtatac gccgaacgac ggccagcatt ggaaacgatt
gcagaatgat 420 gcggaacatt tatcggatat cggaatcact gccgtctgga
ttcctcccgc atacaaagga 480 ttgagccaat ccgataacgg atacggacct
tatgatttgt atgatttagg agaattccag 540 caaaaaggga cggtcagaac
gaaatacggc acaaaatcag agcttcaaga tgcgatcggc 600 tcactgcatt
cccggaacgt ccaagtatac ggagatgtgg ttttgaatca taaggctggt 660
gctgatgcaa cagaagatgt aactgccgtc gaagtcaatc cggccaatag aaatcaggaa
720 acttcggagg aatatcaaat caaagcgtgg acggattttc gttttccggg
ccgtggaaac 780 acgtacagtg attttaaatg gcattggtat catttcgacg
gagcggactg ggatgaatcc 840 cggaagatca gccgcatctt taagtttcgt
ggggaaggaa aagcgtggga ttgggaagta 900 tcaagtgaaa acggcaacta
tgactattta atgtatgctg atgttgacta cgaccaccct 960 gatgtcgtgg
cagagacaaa aaaatggggt atctggtatg cgaatgaact gtcattagac 1020
ggcttccgta ttgatgccgc caaacatatt aaattttcat ttctgcgtga ttgggttcag
1080 gcggtcagac aggcgacggg aaaagaaatg tttacggttg cggagtattg
gcagaataat 1140 gccgggaaac tcgaaaacta cttgaataaa acaagcttta
atcaatccgt gtttgatgtt 1200 ccgcttcatt tcaatttaca ggcggcttcc
tcacaaggag gcggatatga tatgaggcgt 1260 ttgctggacg gtaccgttgt
gtccaggcat ccggaaaagg cggttacatt tgttgaaaat 1320 catgacacac
agccgggaca gtcattggaa tcgacagtcc aaacttggtt taaaccgctt 1380
gcatacgcct ttattttgac aagagaatcc ggttatcctc aggtgttcta tggggatatg
1440 tacgggacaa aagggacatc gccaaaggaa attccctcac tgaaagataa
tatagagccg 1500 attttaaaag cgcgtaagga gtacgcatac gggccccagc
acgattatat tgaccacccg 1560 gatgtgatcg gatggacgag ggaaggtgac
agctccgccg ccaaatcagg tttggccgct 1620 ttaatcacgg acggacccgg
cggatcaaag cggatgtatg ccggcctgaa aaatgccggc 1680 gagacatggt
atgacataac gggcaaccgt tcagatactg taaaaatcgg atctgacggc 1740
tggggagagt ttcatgtaaa cgatgggtcc gtctccattt atgttcagaa ataaggtaat
1800 aaaaaaacac ctccaagctg agtgcgggta tcagcttgga ggtgcgttta
ttttttcagc 1860 cgtatgacaa ggtcggcatc aggtgtgaca aatacggtat
gctggctgtc ataggtgaca 1920 aatccgggtt ttgcgccgtt tggctttttc
acatgtctga tttttgtata atcaacaggc 1980 acggagccgg aatctttcgc
cttggaaaaa taagcggcga tcgtagctgc ttccaatatg 2040 gattgttcat
cgggatcgct gcttttaatc acaacgtggg atcc 2084
* * * * *