Alpha-Amylase Variants

Abstract

The invention relates to a variant of a parent Termamyl-like alpha-amylase, comprising mutations in two, three, four, five or six regions/positions. The variants have increased stability at high temperatures (relative to the parent). The invention also relates to a DNA construct comprising a DNA sequence encoding an alpha-amylase variant of the invention, a recombinant expression vector which carries a DNA construct of the invention, a cell which is transformed with a DNA construct of the invention, the use of an alpha-amylase variant of the invention for washing and/or dishwashing, textile desizing, starch liquefaction, a detergent additive comprising an alpha-amylase variant of the invention, a manual or automatic dishwashing detergent composition comprising an alpha-amylase variant of the invention, a method for generating a variant of a parent Termamyl-like alpha-amylase, which variant exhibits increased.

1 . A variant of a parent Termamyl-like α-amylase, which variant α-amylase has been altered in comparison to the parent α-amylase in one or more solvent exposed amino acid residues on the surface of the α-amylase to increase the overall hydrophobicity of the α-amylase and/or to increase the overall numbers of methyl groups in the sidechains of said solvent exposed amino acid residues on the surface. 
     
     
         2 . The variant according to  claim 1 , wherein one or more solvent exposed amino acid residues on a concave surface with inwards bend are altered to more hydrophobic amino acid residues. 
     
     
         3 . The variant according to  claim 1 , wherein one or more solvent exposed amino acid residues on a convex surface are altered to increase the number of methyl groups in the sidechain. 
     
     
         4 . A variant of a parent Termamyl-like α-amylase, comprising an alteration at one or more positions selected from the group of: 
       E376, S417, A420, S356, Y358; 
       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 α-amylase activity and (c) each position corresponds to a position of the amino acid sequence of the parent Termamyl-like α-amylase having the amino acid sequence of SEQ ID NO: 4. 
     
     
         5 . The variant according to  claim 1 , wherein the parent Termamyl-like α-amylase is any of the α-amylases selected from the group depicted in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, and 8. 
     
     
         6 . The variant according to  claim 1 , wherein the parent Termamyl-like α-amylase has an amino acid sequence which has a degree of identity to SEQ ID NO: 4 of at least 65%, preferably 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 97%. 
     
     
         7 . The variant according to  claim 1 , wherein the parent α-amylase further has a mutation in one or more of the following positions: K176, I201 and H205 (using the numbering in SEQ ID NO: 4). 
     
     
         8 . The variant according to  claim 1 , wherein the variant has increased stability at pHs below 7.0 (acidic pH) and/or at low calcium concentration and/or at temperatures in the range from 95 to 160° C. (high temperatures) relative to the parent α-amylase. 
     
     
         9 . The variant according to  claim 1 , which variant has one or more of the following substitutions: E376K, S417T, A420Q, R, S356A, Y358F. 
     
     
         10 . A DNA construct comprising a DNA sequence encoding an α-amylase variant according to  claim 1 . 
     
     
         11 . A recombinant expression vector which carries a DNA construct according to  claim 10 . 
     
     
         12 . A cell which is transformed with a DNA construct according to  claim 10 . 
     
     
         13 . A cell according to  claim 12 , which is a microorganism. 
     
     
         14 . A cell according to  claim 13 , which is a bacterium or a fungus. 
     
     
         15 . The cell according to  claim 14 , which is a grampositive 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.    
     
     
         16 . A detergent additive comprising an α-amylase variant according to  claim 1 , optionally in the form of a non-dusting granulate, stabilised liquid or protected enzyme. 
     
     
         17 . A detergent additive according to  claim 16  which contains 0.02-200 mg of enzyme protein/g of the additive. 
     
     
         18 . A detergent additive according to  claim 16 , which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase. 
     
     
         19 . A detergent composition comprising an α-amylase variant according to  claim 1 . 
     
     
         20 . A detergent composition according to  claim 19  which additionally comprises another enzyme such as a protease, a lipase, a peroxidase, another amylolytic enzyme and/or a cellulase. 
     
     
         21 . A manual or automatic dishwashing detergent composition comprising an α-amylase variant according to  claim 1 . 
     
     
         22 . A manual or automatic laundry washing composition comprising an α-amylase variant according to  claim 1 . 
     
     
         23 . A composition comprising:
 (i) a mixture of the α-amylase from  B. licheniformis  having the sequence shown in SEQ ID NO: 4 with one or more variants according to  claim 1  derived from (as the parent Termamyl-like α-amylase) the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID NO: 3; or   (ii) a mixture of the α-amylase from  B. stearothermophilus  having the sequence shown in SEQ ID NO: 3 with one or more variants according to  claim 1  derived from one or more other parent Termamyl-like α-amylases; or   (iii) a mixture of one or more variants according of  claim 1  derived from (as the parent Termamyl-like α-amylase) the  B. stearothermophilus  α-amylase having the sequence shown in SEQ ID NO: 3 with one or more variants according to the invention derived from one or more other parent Termamyl-like α-amylases.   
     
     
         24 . A method of using an α-amylase variant according to  claim 1  or a composition according to  claim 23  for washing and/or dishwashing. 
     
     
         25 . A method for generating a variant of a parent Termamyl-like α-amylase, which variant exhibits increased stability at high temperatures relative to the parent, the method comprising:
 (a) subjecting a DNA sequence encoding the parent Termamyl-like α-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 α-amylase which has increased stability at high temperatures relative to the parent Termamyl-like α-amylase.