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 - 39 . (canceled)  
     
     
         40 . A variant alpha-amylase, which variant has □-amylase activity, has at least 90% homology with the amino acid sequence shown in SEQ ID. NO. 3, and which comprises one or more of the following substitutions (using SEQ ID NO: 2 for numbering): 
 (a) R181A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,or V;    (b) G182A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y,or V;    (c) D183A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,or V;    (d) G184A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V;    (e) K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y,or V; and    (f) A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y,or V.    
     
     
         41 . A variant alpha-amylase, which variant has alpha-amylase activity, has at least 95% homology with the amino acid sequence shown in SEQ ID. NO. 3, and which comprises one or more of the following substitutions (using SEQ ID NO: 2 for numbering): 
 (a) R181A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V; (b) G182A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V; (c) D183A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V; (d) G184A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V; (e) K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y, or V; and (f) A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.    
     
     
         42 . The variant of  claim 40 , wherein the variant comprises a substitution of R181A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         43 . The variant of  claim 40 , wherein the variant comprises a substitution of G182A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         44 . The variant of  claim 40 , wherein the variant comprises a substitution of D183A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         45 . The variant of  claim 40 , wherein the variant comprises a substitution of G184A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         46 . The variant of  claim 40 , wherein the variant comprises a substitution of K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y, or V.  
     
     
         47 . The variant of  claim 40 , wherein the variant comprises a substitution of A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         48 . The variant of  claim 41 , wherein the variant comprises a substitution of R181A,D,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         49 . The variant of  claim 41 , wherein the variant comprises a substitution of G182A,D,R,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         50 . The variant of  claim 41 , wherein the variant comprises a substitution of D183A,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         51 . The variant of  claim 41 , wherein the variant comprises a substitution of G184A,R,D,N,C,E,Q,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         52 . The variant of  claim 41 , wherein the variant comprises a substitution of K185A,D,R,N,C,E,Q,G,H,I,L,M,F,P,S,T,W,Y, or V.  
     
     
         53 . The variant of  claim 41 , wherein the variant comprises a substitution of A186D,R,N,C,E,Q,G,H,I,L,K,M,F,P,S,T,W,Y, or V.  
     
     
         54 . A DNA construct comprising a DNA sequence encoding an alpha-amylase variant according to  claim 40 .  
     
     
         55 . A recombinant expression vector which carries a DNA construct according to  claim 54 .  
     
     
         56 . A cell which is transformed with a DNA construct according to  claim 54 .  
     
     
         57 . A DNA construct comprising a DNA sequence encoding an alpha-amylase variant according to  claim 41 .  
     
     
         58 . A recombinant expression vector which carries a DNA construct according to  claim 57 .  
     
     
         59 . A recombinant expression vector which carries a DNA construct according to  claim 57.