Alpha-amylase variants

Abstract

The invention relates to a variant of a parent Termamyl-like alpha-amylase, which variant exhibits altered properties, in particular reduced capability of cleaving a substrate close to the branching point, and improved substrate specificity and/or improved specific activity relative to the parent alpha-amylase.

1 . A variant of a parent Termamyl-like alpha-amylase, comprising an alteration at one or more positions selected from the group of:
 W13, G48, T49, S50, Q51, A52, D53, V54, G57, G107, G108, A111, S168, M197, 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 parent Termamyl-like alpha-amylase having the amino acid sequence of SEQ ID NO: 4.   
     
     
         2 . The variant of  claim 1 , comprises a mutation in a position corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO: 4:
 V54N, A52S, A52S+V54N, T49L, T49+G107A, A52S+V54N+T49L+G107A, A52S+V54N+T49L, G107A, Q51R, Q51R+A52S, A52N; or   T49F+G107A, T49V+G107A, T49D+G107A, T49Y+G107A, T49S+G107A, T49N+G107A, T49I+G107A, T49L+A52S+G107A, T49L+A52T+G107A, T49L+A52F+G107A, T49L+A52L+G107A, T49L+A52I+G107A, T49L+A52V+G107A; or   T49V, T49I, T49D, T49N, T49S, T49Y, T49F, T49W, T49M, T49E, T49Q, T49K, T49R, A52T, A52L, A52I, A52V, A52M, A52F, A52Y, A52W, V54M, G107V, G071, G107L, G107C.   
     
     
         3 . The variant of  claim 1 , comprising a mutation in a position corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO: 4:
 W13F,L,I,V,Y,A;   G48A, V, S, T, I, L;   *48aD or *48aY (i.e., insertion of D or Y);   T49X;   *49aX (i.e., insertion of any amino acid residue)   S50X, in particular D, Y, L, T, V, I;   Q51R,K;   A52X, in particular A52S,N,T,F, L, I, V;   D53E,Q,Y,I,N,S,T,V,L;   V54X, in particular V54I,N,W,Y,F,L;   G57S,A,V,L,I,F, Y, T;   G107X, in particular G107A, V, S, T, I, L, C;   G108X, in particular G108A, V, S, T, I, L;   A111V, I, L;   S168Y;   M197X, in particular Y, F, L, I, T, A, G.   
     
     
         4 . The variant of  claim 1 , comprises the following mutations corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO: 4:
 T49X+A52X+V54N/I/L/Y/F/W+G107A.   
     
     
         5 . The variant of  claim 1 , further comprising G108A. 
     
     
         6 . The variant of  claim 1 , comprises the following mutations corresponding to at least one of the following mutations in the amino acid sequence shown in SEQ ID NO: 4:
 T49L+G107A;   T49I+G107A;   T49L+G107A+V54I;   T49I+G107A+V54I;   A52S+V54N+T49L+G107A;   A52S+V54I+T49L+G107A;   A52S+T49L+G107A;   A52T+T49L+G107A;   A52S+V54N+T49I+G107A;   A52S+V54I+T49I+G107A;   A52S+T49I+G107A;   T49L+G108A;   T49I+G108A;   T49L+G108A+V54I;   T49I+G108A+V54I.   
     
     
         7 . A variant of  claim 1 , wherein said variant has a reduced capability of cleaving an oligo-saccharide substrate close to the branching point as compared to the parent alpha-amylase. 
     
     
         8 . A variant of  claim 1 , which further exhibits improved substrate specificity and/or improved specific activity relative to the parent Termamyl-like alpha-amylase. 
     
     
         9 . A variant of  claim 1 , wherein the parent alpha-amylase is a hybrid alpha-amylase of SEQ ID NO: 4 and SEQ ID NO: 6. 
     
     
         10 . The variant of  claim 1 , wherein the parent hybrid alpha-amylase is a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the  B. licheniformis  alpha-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the alpha-amylase derived from  B. amyloliquefaciens  shown in SEQ ID NO: 6. 
     
     
         11 . The variant of  claim 1 , wherein the parent hybrid Termamyl-like alpha-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4) or LE174. 
     
     
         12 . The variant of  claim 1 , wherein the parent hybrid Termamyl-like alpha-amylase further has the following mutations: H156Y+A181T+N190F+A209V+Q264S+1201F (using the numbering of SEQ ID NO: 4) or LE429. 
     
     
         13 . A DNA construct comprising a DNA sequence encoding an alpha-amylase variant according to  claim 1 . 
     
     
         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 . 
     
     
         16 . A cell of  claim 9 , which is a microorganism, in particular a bacterium or a fungus, such as 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.    
     
     
         17 . A composition comprising:
 (i) a mixture of the alpha-amylase from  B. licheniformis  having the sequence shown in SEQ ID NO: 4 with one or more variants of  claim 1 , derived from (as the parent Termamyl-like alpha-amylase) the  B. stearothermophilus  alpha-amylase having the sequence shown in SEQ ID NO: 8; or   (ii) a mixture of the alpha-amylase from  B. stearothermophilus  having the sequence shown in SEQ ID NO: 8 with one or more variants of  claim 1  derived from one or more other parent Termamyl-like alpha-amylases; or   (iii) a mixture of one or more variants of  claim 1  derived from (as the parent Termamyl-like alpha-amylase) the  B. stearothermophilus  alpha-amylase having the sequence shown in SEQ ID NO: 8 with one or more variants according to the invention derived from one or more other parent Termamyl-like alpha-amylases.   
     
     
         18 . A composition comprising:
 a mixture of one or more variants of  claim 1  derived from (as the parent Termamyl-like alpha-amylase) the  B. stearothermophilus  alpha-amylase having the sequence shown in SEQ ID NO: 8 and a Termamyl-like alpha-amylase derived from the  B. licheniformis  alpha-amylase having the sequence shown in SEQ ID NO: 4.   
     
     
         19 . A composition comprising:
 a mixture of one or more variants of  claim 1  derived from (as the parent Termamyl-like alpha-amylase) the  B. stearothermophilus  alpha-amylase having the sequence shown in SEQ ID NO: 8 and a hybrid alpha-amylase comprising a part of the  B. amyloliquefaciens  alpha-amylase shown in SEQ ID NO: 6 and a part of the  B. licheniformis  alpha-amylase shown in SEQ ID NO: 4.   
     
     
         20 . A composition comprising:
 a mixture of one or more variants of  claim 1  derived from (as the parent Termamyl-like alpha-amylase) a hybrid alpha-amylase comprising a part of the  B. amyloliquefaciens  alpha-amylase shown in SEQ ID NO: 6 and a part of the  B. licheniformis  alpha-amylase shown in SEQ ID NO: 4.   
     
     
         21 . A composition of  claim 20 , wherein the hybrid alpha-amylase is a hybrid alpha-amylase comprising the 445 C-terminal amino acid residues of the  B. licheniformis  alpha-amylase shown in SEQ ID NO: 4 and the 37 N-terminal amino acid residues of the alpha-amylase derived from  B. amyloliquefaciens  shown in SEQ ID NO: 6. 
     
     
         22 . A composition of  claim 21 , wherein the hybrid alpha-amylase further has the following mutations:
 H156Y+A181T+N190F+A209V+Q264S (using the numbering in SEQ ID NO: 4) or LE174.   
     
     
         23 . A composition of  claim 21 , wherein the hybrid alpha-amylase further has the following mutations:
 H156Y+A181T+N190F+A209V+Q264S+1201F as shown in SEQ ID NO: 2 or LE429.   
     
     
         24 . A method for generating a variant of a parent Termamyl-like alpha-amylase, which variant exhibits a reduced capability of cleaving a substrate close to the branching point, and further exhibits improved substrate specificity and/or improved specific activity relative to the parent, the method comprising:
 (a) subjecting a DNA sequence encoding the parent Termamyl-like alpha-amylase to random mutagenesis,   (b) expressing the mutated DNA sequence obtained in step (a) in a host cell, and   (c) screening for host cells expressing a mutated alpha-amylase which has increased stability at low pH and low calcium concentration relative to the parent alpha-amylase.