Mutant alpha-amylases

Abstract

The invention relates to a mutant α-amylase obtained by making replacement or deletion of at least one of amino acid residues such as the 167th Gln, 169th Tyr and 178th Ala in the amino acid sequence set forth in SEQ ID NO:1 in an α-amylase having said amino acid sequence, or an α-amylase having a homology of at least 70% to said amino acid sequence, a gene encoding the mutant α-amylase, a vector, transformed cells, a process for producing a mutant α-amylase, comprising culturing the transformed cells, and a detergent composition comprising the mutant α-amylase. The mutant α-amylase of the invention has excellent properties of high resistance to chelating agents, high specific activity in an alkaline region and excellent stability to heat, and is hence useful for detergents for automatic dish washer, laundry detergents and the like.

1 . A mutant α-amylase obtained by making a substitution or deletion of at least one amino acid residue of specific positions in SEQ ID NO:1, or by making a substitution or deletion of at least one amino acid residue corresponding to the above-mentioned amino acid residue in a sequence having at least 70% homology to SEQ ID NO:1, 
 wherein said at least one amino acid residue is selected from the group consisting of:    the 11 th  Tyr, 16 th  Glu, 49 th  Asn, 84 th  Glu, 144 th  Ser, 167 th  Gln, 169 th  Tyr, 178 th  Ala, 188 th  Glu, 190 th  Asn, 205 th  His and 209 th  Gln, and    said mutant α-amylase possesses increased heat resistance and maintains resistance to chelating agents when compared to SEQ ID NO:1, and    said mutant (α-amylase comprises an amino acid sequence which is at least 95% homologous to SEQ ID NO:1.    
     
     
         2 . A mutant α-amylase obtained by making a substitution of an amino acid sequence corresponding to 11 to 100 amino acid residues from the amino terminal Asp residue of the amino acid sequence set forth in SEQ ID NO:1 or an amino acid sequence corresponding to 11 to 100 amino acid residues from the amino terminal Asp residue of SEQ ID NO:1 of a sequence having at least 70% homology to SEQ ID NO:1, with an amino acid sequence corresponding to 11 to 100 amino acid residues from the amino terminal Asp residue of SEQ ID NO:2, 
 wherein said mutant α-amylase possesses increased heat resistance and maintains resistance to chelating agents when compared to SEQ ID NO:1.    
     
     
         3 . A mutant α-amylase obtained by making a substitution of an amino terminal sequence from 1 st  Asp through 19 th  Gly of SEQ ID NO:1 or an amino terminal sequence corresponding to 1 st  Asp through 19 th  Gly of SEQ ID NO:1 of a sequence having at least 70% homology to SEQ ID NO:1, with an amino acid sequence from 1 st  His to 21 st  Gly of SEQ ID NO:2, wherein said mutant α-amylase possess increased heat resistance and maintains resistance to chelating agents when compared to SEQ ID NO:1.  
     
     
         4 . A mutant (α-amylase obtained by introducing a first mutation and a second mutation into SEQ ID NO:1 or an amino acid sequence having at least 70% homology to SEQ ID NO:1, 
 wherein said first mutation consists of a substitution or a deletion of at least one amino acid residue selected from the group consisting of the 11 th  Tyr, 16 th  Glu, 49 th  Asn, 84 th  Glu, 144 th  Ser, 167 th  Gln, 169 th  Tyr, 178 th  Ala, 188 th  Glu, 190 th  Asn, 205 th  His and 209 th  Gln, and    wherein said second mutation consists of a substitution of an amino acid sequence corresponding to 11 to 100 amino acid residues from the amino terminal Asp residue of the amino acid sequence set forth in SEQ ID NO:1, and    wherein said mutant α-amylase possesses increased heat resistance and maintains resistance to chelating agents when compared to SEQ ID NO:1.    
     
     
         5 . A mutant (α-amylase obtained by introducing a first mutation and a second mutation into SEQ ID NO:1 or by making a substitution or deletion of at least one amino acid residue corresponding to the above-mentioned amino acid residue in an amino acid sequence having at least 70% homology to SEQ ID NO:1, wherein said first mutation consists of: 
 the substitution of an amino acid residue selected from the group consisting of: the 11 th  Tyr of SEQ ID NO:1 with Phe, the 16 th  Glu of SEQ ID NO:1 with Pro, the 49 th  Asn of SEQ ID NO:1 with Ser, the 167 Gln of SEQ ID NO:1 with Glu, the 169 th  Tyr of SEQ ID NO:1 with Lys, the 190 th  Asn of SEQ ID NO:1 with Phe, the 205 th  His of SEQ ID NO:1 with Arg, and the 209 th  Gln of SEQ ID NO:1 with Val,    and wherein said second mutation consists of:    substituting an amino terminal sequence from 1 st  Asp through 19 th  Gly of SEQ ID NO:1 with an amino acid sequence from 1 st  His to 21 st  Gly of SEQ ID NO:2.    
     
     
         6 . A nucleotide sequence encoding the mutant α-amylase according to  claim 1  or a vector containing said gene.  
     
     
         7 . A cell transformed by the vector according to claim 6.  
     
     
         8 . A process for producing a mutant α-amylase, comprising culturing the transformed cells according to  claim 7 .  
     
     
         9 . A detergent composition comprising the mutant α-amylase according to  claim 1 .  
     
     
         10 . A mutant α-amylase obtained by making a substitution or deletion of at least one amino acid residue of specific positions in SEQ ID NO:1, or by making a substitution or deletion of at least one amino acid residue corresponding to the above-mentioned amino acid residue in a sequence having at least 70% homology to SEQ ID NO:1, 
 wherein said at least one amino acid residue is selected from the group consisting of:    the 11 th  Tyr, 16 th  Glu, 49 th  Asn, 84 th  Glu, 144 th  Ser, 167 th  Gin, 169 th  Tyr, 178 th  Ala, 188 th  Glu, 190 th  Asn, 205 th  His and 209 th  Gln, and    wherein said mutant (α-amylase possesses increased heat resistance, which is improved by combining mutations when compared to SEQ ID NO:1, and maintains resistance to chelating agents and oxidizing agents when compared to SEQ ID NO:1, and    said mutant (α-amylase comprises an amino acid sequence which is at least 95%. homologous to SEQ ID NO:1.    
     
     
         11 . A mutant (α-amylase obtained by making a substitution or deletion of at least one amino acid residue of specific positions in SEQ ID NO:1, or by making a substitution or deletion of at least one amino acid residue corresponding to the above-mentioned amino acid residue in a sequence having at least 70% homology to SEQ ID NO:1, 
 wherein said at least one amino acid residue is selected from the group consisting of:    the 11 th  Tyr, 16 th  Glu, 49 th  Asn, 84 th  Glu, 144 th  Ser, 167 th  Gin, 169 th  Tyr, 178 th  Ala, 188 th  Glu, 190 th  Asn, 205 th  His and 209 th  Gln, and    wherein said mutant α-amylase: 
 (i) possesses increased heat resistance when compared to SEQ ID NO:1;  
 (ii) maintains resistance to chelating agents when compared to SEQ ID NO:1;  
 (iii) maintains high specific activity under alkaline pH region when compared to SEQ ID NO:1; and  
 (iv) comprises an amino acid sequence which is at least 95% homologous to SEQ ID NO:1.  
   
     
     
         12 . The mutant α-amylase of  claim 13 , wherein said mutant α-amylase acts in an optimum temperature range of 50° C. to 60° C.  
     
     
         13 . The mutant α-amylase according to  claim 10 , wherein the 11 th  Tyr of SEQ ID NO:1 is substituted with Phe, the 16 th  Glu of SEQ ID NO:1 is substituted with Pro, the 49 th  Asn of SEQ ID NO:1 is substituted with Ser, the 167 Gln of SEQ ID NO:1 is substituted with Glu, the 169 th  Tyr of SEQ ID NO:1 is substituted with Lys, the 190 th  Asn of SEQ ID NO:1 is substituted with Phe, the 205 th  His of SEQ ID NO:1 is substituted with Arg, and the 209 th  Gln of SEQ ID NO:1 is substituted with Val.  
     
     
         14 . The mutant α-amylase according to  claim 11 , wherein the 11 th  Tyr of SEQ ID NO:1 is replaced with Phe.  
     
     
         15 . The mutant α-amylase according to  claim 11 , wherein the 16 th  Glu of SEQ ID NO:1 is replaced with Pro.  
     
     
         16 . The mutant α-amylase according to  claim 11 , wherein the 49 th  Asn of SEQ ID NO:1 is replaced with Ser.  
     
     
         17 . The mutant α-amylase according to  claim 11 , wherein the 167 Gln of SEQ ID NO:1 is replaced with Glu.  
     
     
         18 . The mutant α-amylase according to  claim 11 , wherein the 169 th  Tyr of SEQ ID NO:1 is replaced with Lys.  
     
     
         19 . The mutant α-amylase according to  claim 11 , wherein the 190 th  Asn of SEQ ID NO:1 is replaced with Phe.  
     
     
         20 . The mutant α-amylase according to  claim 13 , wherein the 205 th  His of SEQ ID NO:1 is replaced with Arg.  
     
     
         21 . The mutant α-amylase according to  claim 13 , wherein the 209 th  Gln of SEQ ID NO:1 is replaced with Val.  
     
     
         22 . A mutant α-amylase obtained by making a substitution or deletion of at least one amino acid residue of specific positions in SEQ ID NO:1, 
 wherein said at least one amino acid residue is selected from the group consisting of:    the 11 th  Tyr, 16 th  Glu, 49 th  Asn, 84 th  Glu, 144 th  Ser, 167 th  Gln, 169 th  Tyr, 178 th  Ala, 188 th  Glu, 190 th  Asn, 205 th  His and 209 th  Gln, and    said mutant α-amylase possesses increased heat resistance and maintains resistance to chelating agents when compared to SEQ ID NO:1, and    said mutant α-amylase comprises an amino acid sequence which is at least 95% homologous to SEQ ID NO:1.    
     
     
         23 . A mutant (α-amylase obtained by making a substitution or deletion of at least one amino acid residue of specific positions in SEQ ID NO:4, 
 wherein said at least one amino acid residue is selected from the group consisting of:    the 11 th  Tyr, 16 th  Glu, 49 th  Asn, 84 th  Glu, 167 th  Gln, 169 th  Tyr, 178 th  Ala, 188 th  Glu, 190 th  Asn, 205 th  His and 209 th  Gln, and    said mutant α-amylase possesses increased heat resistance and maintains resistance to chelating agents when compared to SEQ ID NO:4, and    said mutant α-amylase comprises an amino acid sequence which is at least 95% homologous to SEQ ID NO:4.