Alpha-amylase Mutants
The invention relates to a variant of a parent Termamyl-like α-amylase, which exhibits an alteration in at least one of the following properties relative to said parent α-amylase: i) improved pH stability at a pH from 8 to 10.5; and/or ii) improved Ca 2+ stability at pH 8 to 10.5, and/or iii) increased specific activity at temperatures from 10 to 60° C.
1 . A variant of a parent Termamyl-like α-amylase, which variant has α-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 α-amylase:
i) improved pH stability at a pH from 8 to 10.5; and/or
ii) improved Ca 2+ stability at pH 8 to 10.5, and/or
iii) increased specific activity at temperatures from 10 to 60° C., preferably 20-50° C., especially 30-40° 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 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 α-amylase is selected from:
the Bacillus strain NCIB 12512 α-amylase having the sequence shown in SEQ ID NO: 1;
the B. amyloliquefaciens α-amylase having the sequence shown in SEQ ID NO: 5;
the B. licheniformis α-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° C., preferably 20-50° C., especially 30-40° 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 α-amylase is the B. licheniformis α-amylase having the sequence shown in SEQ ID NO: 4.
17 . A DNA construct comprising a DNA sequence encoding an α-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 α-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 α-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 α-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 α-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 α-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 α-amylase by comparing the molecular dynamics of two or more α-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 α-amylase is compared with a high temperature α-amylase.
34 . The method according to claim 32 , wherein a low temperature α-amylase is compared with a medium or high temperature α-amylase.
35 . The method according to claim 32 , wherein the α-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 α-amylases compared are Termamyl-like α-amylases.
37 . The method according to claim 27 , wherein the α-amylases compared are any of the α-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 α-amylases compared are the Ca 2+ dependency profile.