Fungamyl-like Alpha-Amylase Variants

Abstract

The invention relates to a variant of a parent Fungamyl-like fungal alpha-amylase, which exhibits improved thermal stability at acidic pH suitable for, e.g., starch processes.

1 . A variant of a parent Fungamyl-like alpha-amylase, comprising an alteration at one or more regions selected from the group of:
 Region 98-110,   Region 150-160,   Region 161-167,   Region 280-288,   Region 448-455,   Region 468-475.   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 region or position corresponds to a region position of the amino acid sequence of the parent Fungamyl-like alpha-amylase having the amino acid sequence of SEQ ID NO: 2. 
   
     
     
         2 . The variant of  claim 1 , wherein the variant is one or more of the following substitution: Q153S. 
     
     
         3 . The variant of  claim 1 , which variant has improved thermostability and/or increased stability at acidic pH. 
     
     
         4 . A DNA construct comprising a DNA sequence encoding an alpha-amylase variant of any of  claims 1 - 3 . 
     
     
         5 . A recombinant expression vector which carries a DNA construct according to  claim 4 . 
     
     
         6 . A cell which is transformed with a DNA construct according to  claim 4  or a vector according to  claim 5 . 
     
     
         7 . A cell according to  claim 6 , wherein the cell is a microorganism, such as a bacterium or a fungus. 
     
     
         8 . The cell according to  claim 7 , which is a protease deficient strain of  Aspergillus , in particular  A. oryzae.    
     
     
         9 . A composition for producing high maltose syrup comprising an Fungamyl-like alpha-amylase variant of  claims 1 - 3 . 
     
     
         10 . The composition of  claim 9 , further comprising beta-amylase activity. 
     
     
         11 . A dough improving composition, comprising an alpha-amylase variant of any of  claims 1 - 3 . 
     
     
         12 . A brewing composition comprising an alpha-amylase variant of any of  claims 1 - 3 . 
     
     
         13 . The brewing composition of  claim 12 , further comprising one or ore enzymes selected from the group of beta-amylase and isoamylase. 
     
     
         14 . A composition for producing alcohol, comprising an alpha-amylase variant of any of  claims 1 - 3 . 
     
     
         15 . A process of liquefying starch, wherein an alpha-amylase variant of  claims 1 - 3  is used for treating starch. 
     
     
         16 . A process of producing high maltose syrups, wherein an alpha-amylase variant of  claims 1 - 3  is used for liquefying starch. 
     
     
         17 . A brewing process, wherein an alpha-amylase variant of  claims 1 - 3  is added during fermentation of wort. 
     
     
         18 . An alcohol production process, wherein an alpha-amylase variant of  claim 1 - 3  is used for liquefaction starch in a distillery mash. 
     
     
         19 . A process, wherein a dough product comprising an alpha-amylase variant of  claims 1 - 3  is baked. 
     
     
         20 . Use of an alpha-amylase variant of any of  claims 1 - 3  or a composition of  claim 9  for starch liquefaction. 
     
     
         21 . Use of an alpha-amylase variant of any of  claims 1 - 3  or a composition of  claim 9  for producing alcohol. 
     
     
         22 . Use of an alpha-amylase variant of any of  claims 1 - 3  or a composition of  claim 9  for brewing. 
     
     
         23 . Use of an alpha-amylase variant of any of  claims 1 - 3  or a composition of  claim 9  for baking. 
     
     
         24 . A method for generating an alpha-amylase variant of a parent Fungamyl-like alpha-amylase, which variant has increased thermostability, in particular at acidic pH relative to the parent, the method comprising:
 (a) subjecting a DNA sequence encoding the parent Fungamyl-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 improved thermostability at acidic pH relative to the parent Fungamyl-like alpha-amylase.   
     
     
         25 . Process for producing a maltose syrup comprising the steps of:
 1) liquefying starch in the presence of an alpha-amylase, followed by   2) dextrinization the presence of a fungal alpha-amylase variant of  claim 1 - 3 ;   3) recovery of the syrup; and optional purification.   
     
     
         26 . Process for producing syrup, in particular maltose syrup, comprising the steps of:
 1) liquefying starch at a temperature of 140-160° C. at a pH of 4-6, followed by   2) dextrinization at a temperature in the range from 60-95° C. at a pH 4-6 in the presence of a fungal alpha-amylase variant of  claims 1 - 3 ; and   3) recovery of the syrup; and optional purification.   
     
     
         27 . The process of  claim 26 , wherein the liquefying starch is treated at a temperature of 65-85° C., in particular 70-80° C. 
     
     
         28 . The process of  claim 27 , wherein an effective amount of glucoamylase is added in step 2). 
     
     
         29 . Process for producing maltose syrup, comprising the steps of:
 1) liquefying starch at a temperature of 95-110° C. at a pH of 4-6 in the presence of a  Bacillus  alpha-amylase, followed by   2) dextrinization at a temperature in the range from 60-95° C. at a pH 4-6 in the presence of a fungal alpha-amylase variant of  claims 1 - 3 ; and   3) recovery of the syrup; and optional purification.   
     
     
         30 . An immobilized variant of  claims 1 - 3 .