Alpha-amylase Mutants

Abstract

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.