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, S193, N195 H107, K108, G109,D166, W167, D168, Q169, S170, 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 claims  1 - 3 , 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 any of claims  1 - 4 , 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 any of claims  1 - 5 , 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, S193, 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 claims  1 - 5 , 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 claims  1 - 11 , 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 claims  1 - 5 , 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, S170, 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 claims  13 - 15 , 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 any one of claims  1 - 16 .  
     
     
         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 lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus  or  Bacillus thuringiensis.    
     
     
         23 . Use of an α-amylase variant according to any one of claims  1 - 16  for washing and/or dishwashing.  
     
     
         24 . A detergent additive comprising an α-amylase variant according to any one of claims  1 - 16 , optionally in the form of a non-dusting granulate, stabilized liquid or protected enzyme.  
     
     
         25 . A detergent additive according to  claim 24  which contains 0.02-200 mg of enzyme protein/g of the additive.  
     
     
         26 . A detergent additive according to claims  24  or  25 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.  
     
     
         27 . A detergent composition comprising an α-amylase variant according to any of claims  1 - 16 .  
     
     
         28 . A detergent composition according to  claim 27  which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.  
     
     
         29 . A manual or automatic dishwashing detergent composition comprising an α-amylase variant according to any one of claims  1 - 16 .  
     
     
         30 . A dishwashing detergent composition according to  claim 29  which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase.  
     
     
         31 . A manual or automatic laundry washing composition comprising an α-amylase variant according to any one of claims  1 - 16 .  
     
     
         32 . A laundry washing composition according to  claim 31 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, an amylolytic enzyme and/or a cellulase.  
     
     
         33 . 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).  
 
 
     
     
         34 . The method according to  claim 33 , wherein a medium temperature α-amylase is compared with a high temperature α-amylase.  
     
     
         35 . The method according to  claim 33 , wherein a low temperature α-amylase is compared with a medium or high temperature α-amylase.  
     
     
         36 . The method according to claims  33 - 35 , wherein the α-amylases are at least 70%, preferably 80%, up to 90%, such as up to 95%, especially 95% homologous.  
     
     
         37 . The method according to  claim 36 , wherein the α-amylases compared are Termamyl-like α-amylases.  
     
     
         38 . The method according to  claim 28 , wherein the α-amylases compared are any of the α-amylases shown in SEQ ID NO: 1 to SEQ ID NO: 8.  
     
     
         39 . The method according to any of  claims 33  to  38 , wherein the stability profile of the α-amylases compared are the Ca 2+  dependency profile.