Alpha-amylase mutants with altered properties

Abstract

The present invention relates to variants (mutants) of parent Termamyl-like alpha-amylases, which variant has alpha-amylase activity and exhibits altered stability, in particular at high temperatures and/or at low pH relative, and/or low Ca2+ to the parent alpha-amylase.

1 . A variant of an alpha-amylase having at least 60% homology to SEQ ID NO.8, comprising an alteration at one or more positions selected from the group of: 
 49, 60, 104, 132, 161, 170, 176, 179, 180, 181, 183, 200, 203, 204, 207, 212, 237, 239, 250, 280, 298, 318, 374, 385, 393, 402, 406, 427, 430, 440, 444, 447, 482,    wherein 
 (a) the alteration(s) are independently 
 (i) an insertion of an amino acid downstream of the amino acid which occupies the position,  
 (ii) a deletion of the amino acid which occupies the position, or  
 (iii) a substitution of the amino acid which occupies the position with a different amino acid,  
 
 (b) the variant has alpha-amylase activity, and  
 (c) each position corresponds to a position of the amino acid sequence of the alpha-amylase having the amino acid sequence shown in SEQ ID NO: 8:  
   
     
     
         2 . The variant of  claim 1 , which variant has one or more of the following mutations: T49I; D60N; N104D; E132A,V,P; D161N; K170Q; K176R; G179N; K180T; A181N; D183N; D200N; X203Y; D204S; D207V,E,L,G; X212I; K237P; S239W; E250G,F; N280S; X298Q; L318M; Q374R; E385V; Q393R; Y402F; H406L,W; L427I D430N; V440A; N444R,K; E447Q,K; Q482K using SEQ ID NO: 8 for the numbering.  
     
     
         3 . The variant of  claim 1  or 2, wherein the variant has the following mutations: K170Q+D207V+N280S; E132A+D207V; D207E+E250G+H406L+L427I; D207V+L318M; D60N+D207V+L318M; T49I+E132V+V440A; T49I+K176R+D207V+Y402F; Q374R+E385V+Q393R; N190F+A209V+Q264S; G48A+T49I+G107A+I201F; T49I+G107A+I201F; G48A+T49I+I201F; G48A+T49I+G107A; T49I+I201F; T49I+G107A; G48A+T49I; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K; D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444K+E447Q+Q482K; D161N+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K; N104D+D161lN+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+E447Q+Q482K; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N; H406W+D430N; N444K+E447Q+Q482K; E447Q+Q482K; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W+H406W+D430N+N444R+N444K+E447K+Q482K; N104D+D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W; D161N+G179N+K180T+A181N+D183N+D200N+D204S+K237P+S239W; H406W+D430N; N444K+E447K+Q482K; E447K+Q482K; N104D+D161N+A181N+D183N+D200N+D204S+K237P+S239W; N104D+D161N+A181N+D183N+D200N+D204S+K237P; N104D+D161N+A181N+D183N+D200N+D204S; D161N+A181N+D183N+D200N+D204S+K237P+S239W; D161N+A181N+D183N+D200N+D204S+K237P; D161N+A181N+D183N+D200N+D204S; K237P+S239W, using SEQ ID NO: 8 for the numbering.  
     
     
         4 . The variant of any of claims  1 - 3 , wherein the parent alpha-amylase is derived from a strain of  B. licheniformis  (SEQ ID NO:  8 ),  B. amyloliquefaciens  (SEQ ID NO: 10), or  B. stearothermophilus  (SEQ ID NO: 6).  
     
     
         5 . The variant of any of claims  1 - 4 , wherein the parent alpha-amylase is any of: 
 LE174; LE174+G48A+T49I+G107A+I201F; LE174+M197L; LE174+G48A+T49I+G107A+M197L+I201F.    
     
     
         6 . The variant of  claim 1 , wherein the variant is mutated in one or more of the following positions: T51I; D62N; N106D; D134A,V,P; D163N; X172Q; K179R; G184N; K185T; A186N; D188N; D205N; M208Y; D209S; X212V,E,L,G; L217I, K242P, S244W, N255G,F, N285S, S303Q, X323M; D387V, N395R; Y404F; H408L,W; X429I; D432N; V442A; X446R,K; X449Q,K; X484K, using SEQ ID NO: 4 for the numbering.  
     
     
         7 . The variant of  claim 1  or  6 , wherein the variant has the following mutations: E212V+N285S; D134A+E212V; 255G+H408L+X429I; E212V+X323M; D62N+E212V+X323M; T51I+D134V+V442A; T51I+K179R+E212V+Y404F; D387V+N395R; N195F+X212V+K269S, when using SEQ ID NO: 4 for the numbering.  
     
     
         8 . The variant of any of claims  1 - 7 , wherein the parent alpha-amylase is selected from the group comprising: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 12; SEQ ID NO: 13; or KSM-AP1378.  
     
     
         9 . The variant of any of claims  1 - 8 , wherein the parent alpha amylase is any of: SEQ ID NO. 4+D183*+G184*; SEQ ID NO. 4+D183*+G184*+N195F; SP722+D183*+G184*+M202L; SEQ ID NO. 4+D183*+G184*+N195F+M202L; SEQ ID NO.6+I181*+G182*; SEQ ID NO.6+I181*+G182*+N193F; SEQ ID NO.6+I181*+G182*+M200L; SEQ ID NO.6+I181*+G182*+N193F+M200L; SEQ ID NO.12+D183*+G184*; SEQ ID NO.12+D183*+G184*+N195F; SEQ ID NO.12+D183*+G184*+M202L; SEQ ID NO.12+D183*+G184*+N195F+M202L.  
     
     
         10 . The variant of any of claims  1 - 9 , wherein the parent alpha-amylase has an amino acid sequence which has a degree of identity to SEQ ID NO: 8 of at least 70%, more preferably at least 80%, even more preferably at least about 90%, even more preferably at least 95%, even more preferably at least 97%, and even more preferably at least 99%.  
     
     
         11 . The variant of any of claims  1 - 10 , wherein the parent alpha-amylase is encoded by a nucleic acid sequence, which hybridizes under low, preferably medium, preferred high stringency conditions, with the nucleic acid sequence of SEQ ID NO: 7.  
     
     
         12 . The variant of any of claims  1 - 11 , which variant has altered stability, in particular at high temperatures from 70-120° C. and/or low pH in the range from pH 4-6.  
     
     
         13 . A DNA construct comprising a DNA sequence encoding an alpha-amylase variant according to any one of claims  1 -12.  
     
     
         14 . A recombinant expression vector which carries a DNA construct according to  claim 13 .  
     
     
         15 . A cell which is transformed with a DNA construct according to  claim 13  or a vector according to  claim 14 .  
     
     
         16 . The cell according to  claim 15 , which is a microorganism, preferably a bacterium or a fungus.  
     
     
         17 . The cell according to  claim 16 , which cell is a gram-positive bacterium, such as  Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus  or  Bacillus thuringiensis.    
     
     
         18 . A composition comprising an alpha-amylase variant of any of claims  1 - 12 .  
     
     
         19 . The composition of  claim 18 , further comprising a  B. stearothermophilus  alpha-amylase, particular in a ratio of 1:10 to 10:1, preferably 1:2.  
     
     
         20 . The composition of  claim 18  or  19 , wherein the composition further comprises a glucoamylase, pullulanase and/or a phytase.  
     
     
         21 . A detergent composition comprising an alpha-amylase variant according to any of claims  1 - 12 .  
     
     
         22 . A detergent composition of  claim 21 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme, glucoamylase, maltogenic amylase, CGTase, mannanase, cutinase, laccase and/or a cellulase.  
     
     
         23 . Use of an alpha-amylase variant according to any of claims  1 - 12  or a composition according to any of claims  18 - 20  for starch liquefaction.  
     
     
         24 . Use of an alpha-amylase variant according to any of claims  1 - 12  or a composition according to claims  18 - 20  for ethanol production.  
     
     
         25 . Use of an alpha-amylase variant according to any one of claims  1 - 12  or a composition according to claims  18 - 20  for washing and/or dishwashing.  
     
     
         26 . Use of an alpha-amylase variant of any one of claims  1 - 12  or a composition according to claims  18 - 20  for textile desizing.