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 Ca 2+ 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 consisting 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, and 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; D6ON; N104D; E132A,V,P; D161N; K170Q; K176R; G179N; K180T; A181 N; 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 , 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). 
     
     
         4 . 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 numbering. 
     
     
         5 . The variant of  claim 1 , wherein the parent alpha-amylase is selected from the group consisting of SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 12; SEQ ID NO: 13; or KSM-AP1378. 
     
     
         6 . The variant of  claim 1 , 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%. 
     
     
         7 . The variant of  claim 1 , wherein the parent alpha-amylase is encoded by a nucleic acid sequence, which hybridizes under high stringency conditions, with the nucleic acid sequence of SEQ ID NO: 7. 
     
     
         8 . The variant of  claim 1 , which variant has altered stability at high temperatures from 70-120° C. and/or low pH in the range from pH 4-6. 
     
     
         9 . A composition comprising an alpha-amylase variant of  claim 1 . 
     
     
         10 . A detergent composition comprising an alpha-amylase variant of  claim 1  and a surfactant. 
     
     
         11 . A detergent composition of  claim 10 , which additionally comprises one or more other enzymes selected from the group consisting of lipase, peroxidase, protease, another amylolytic enzyme, glucoamylase, maltogenic amylase, CGTase, mannanase, cutinase, laccase and/or a cellulase. 
     
     
         12 . A method for liquefying starch, comprising treating the starch with an alpha-amylase variant of  claim 1 . 
     
     
         13 . A method for producing ethanol, comprising liquefying starch with an alpha-amylase variant of  claim 1 , hydrolyzing the liquefied starch with a glucoamylase, and fermenting the hydrolyzed starch. 
     
     
         14 . A method for desizing a textile, comprising treating a sized textile with an alpha-amylase variant of  claim 1 . 
     
     
         15 . A DNA construct comprising a DNA sequence encoding an alpha-amylase variant of  claim 1 .