Fungal Alpha-Amylase Variants

Abstract

The inventors have developed a method of altering the amino acid sequence of a fungal alpha-amylase to obtain variants, and they have used the method to construct such variants. The variants may be useful for anti-staling in baked products. Accordingly, the invention provides a method of constructing fungal alpha-amylase variants based on a comparison of three-dimensional (3D) structures of the fungal alpha-amylase and a maltogenic alpha-amylase. One or both models includes a substrate. The invention also provides novel fungal alpha-amylase variants.

1 - 13 . (canceled)  
     
     
         14 . A method of producing a variant polypeptide, which method comprises: 
 a) providing an amino acid sequence and a three-dimensional model for a fungal alpha-amylase and for a maltogenic alpha-amylase wherein one or both models includes a substrate,    b) superimposing the two three-dimensional models,    c) selecting an amino acid residue in the fungal amylase which has a C-alpha atom located >0.8 Å from the C-alpha atom of any amino acid residue in the maltogenic alpha-amylase and <11 Å from an atom of a substrate,    d) altering the fungal amylase sequence wherein the alteration comprises substitution or deletion of the selected residue or by insertion of a residue adjacent to the selected residue, and    e) producing the polypeptide having the resulting amino acid sequence.    
     
     
         15 . The method of  claim 14  wherein the substitution or insertion is made with an amino acid residue of the same type as the corresponding residue in the maltogenic alpha-amylase sequence, wherein the type is positively charged, negatively charged, hydrophilic or hydrophobic.  
     
     
         16 . The method of  claim 14  wherein the substitution or insertion is made with a larger or smaller amino acid residue depending on whether the corresponding residue in the maltogenic alpha-amylase sequence is larger or smaller.  
     
     
         17 . The method of  claim 14  wherein, the alteration of the amino acid sequence further comprises substitution of a fungal alpha-amylase residue which has a C-alpha atom located less than 11 Å from an atom of a substrate and <0.8 Å from the C-alpha atom of a maltogenic alpha-amylase residue.  
     
     
         18 . The method of  claim 17  wherein the substitution is made with an amino acid residue of the same type as the corresponding maltogenic alpha-amylase residue, wherein the type is positive, negative, hydrophilic or hydrophobic.  
     
     
         19 . A polypeptide which: 
 a) has an amino acid sequence having at least 70% identity to SEQ ID NO: 2; and    b) compared to SEQ ID NO: 2 comprises an amino acid alteration which is a deletion, substitution or insertion at a position corresponding to 15, 32-36, 63-64, 73-77, 119-120, 125-126, 151-152, 155-156, 167-172, 211 or 233-239,    c) has the ability to hydrolyze starch.    
     
     
         20 . The polypeptide of  claim 19  wherein said polypeptide: 
 a) has an amino acid sequence having at least 80% identity to SEQ ID NO: 2; and    b) compared to SEQ ID NO: 2 comprises an amino acid alteration which is a deletion, substitution or insertion at a position corresponding to 15, 32-36, 63-64, 73-77, 119-120, 125-126, 151-152, 155-156, 167-172, 211 or 233-239,    c) has the ability to hydrolyze starch.    
     
     
         21 . The polypeptide of  claim 19  wherein said polypeptide; 
 a) has an amino acid sequence having at least 90% identity to SEQ ID NO: 2; and    b) compared to SEQ ID NO: 2 comprises an amino acid alteration which is a deletion, substitution or insertion at a position corresponding to 15, 32-36, 63-64, 73-77, 119-120, 125-126, 151-152, 155-156, 167-172, 211 or 233-239,    c) has the ability to hydrolyze starch.    
     
     
         22 . A polypeptide of  claim 19  wherein said polypeptide: 
 a) has an amino acid sequence having at least 95% identity to SEQ ID NO: 2; and    b) compared to SEQ ID NO; 2 comprises an amino acid alteration which is a deletion, substitution or insertion at a position corresponding to 15, 32-36, 63-64, 73-77, 119-120, 125-126, 151-152, 155-156, 167-172, 211 or 233-239,    c) has the ability to hydrolyze starch.    
     
     
         23 . The polypeptide of  claim 19  wherein the alteration comprises substitution or insertion with an amino acid residue of the same type as the corresponding residue in the maltogenic alpha-amylase sequence, wherein the type is positively charged, negatively charged, hydrophilic or hydrophobic.  
     
     
         24 . The polypeptide of  claim 19  wherein the alteration comprises substitution or insertion with a larger or smaller amino acid residue depending on whether the corresponding residue in the maltogenic alpha-amylase sequence is larger or smaller.  
     
     
         25 . The polypeptide of  claim 19  comprising alteration corresponding to Q35K/R, Y75A/F, Y155W, L166F, G167T, N169P, T170A, L232Y, D233G, G234D, Y252F, Y256T, 166LGDNTV171 to FTDPAGF, 168-171 (DNTV) substituted with DPAGF, 168-171 (DNTV) substituted with DPAGL, 168-171 (DNTV) substituted with DPAGC.  
     
     
         26 . The polypeptide of  claim 19  which has the amino acid sequence of SEQ ID NO: 2 comprising one or more of the following alterations: 
 Q35K/R    Y75A/F    Y155W    L166F    G167T    N169P    T170A    L232Y    D233G    G234D    Y252F    Y256T    166LGDNTV171 to FTDPAGF    168-171 (DNTV) substituted with DPAGF    168-171 (DNTV) substituted with DPAGL    168-171 (DNTV) substituted with DPAGC    D233G+G234D    Q35K+Y75F+D168Y    Q35R+Y75F    Q35R+Y75F+D168Y    168-171 (DNTV) substituted with DPAGF+Y75A    168-171 (DNTV) substituted with DPAGF+Q35K+Y75A    168-171 (DNTV) substituted with DPAGF+Q35K+Y75A+D233G+G234D    168-171 (DNTV) substituted with DPAGF+Y75A+G234D    168-171 (DNTV) substituted with DPAGF+Y75A+D233G+G234D    166-171 (LGDNTV) substituted with FTDPAGF+Y75A    166-171 (LGDNTV) substituted with FTDPAGF+Q35K+Y75A    166-171 (LGDNTV) substituted with FTDPAGF+Q35K+Y75A+D233G+G234D    
     
     
         27 . A polypeptide which: 
 a) has an amino acid sequence having at least 70% identity to SEQ ID NO: 3;    b) compared to SEQ ID NO: 3 comprises an amino acid alteration which comprises Q35K, Q35R, P70K, L151F, L151D, N233G+G234D, D75G, D75A or 166-171 (EGDTIV) substituted with FTDPAGF, and    c) has the ability to hydrolyze starch.    
     
     
         28 . The polypeptide of  claim 27  wherein said polypeptide: 
 a) has an amino acid sequence having at least 80% identity to SEQ ID NO: 3;    b) compared to SEQ ID NO: 3 comprises an amino acid alteration which comprises Q35K, Q35R, P70K, L151F, L151D, N233G+G234D, D75G, D75A or 166-171 (EGDTIV) substituted with FTDPAGF, and    c) has the ability to hydrolyze starch.    
     
     
         29 . The polypeptide of  claim 27  wherein said polypeptide: 
 a) has an amino acid sequence having at least 90% identity to SEQ ID NO: 3;    b) compared to SEQ ID NO: 3 comprises an amino acid alteration which comprises Q35K, Q35R, P70K, L151F, L151D, N233G+G234D, D75G, D75A or 166-171 (EGDTIV) substituted with FTDPAGF, and    c) has the ability to hydrolyze starch.    
     
     
         30 . The polypeptide of  claim 27  wherein said polypeptide: 
 a) has an amino acid sequence having at least 95% identity to SEQ ID NO: 3;    b) compared to SEQ ID NO: 3 comprises an amino acid alteration which comprises Q35K, Q35R, P70K, L151F, L151D, N233G+G234D, D75G, D75A or 166-171 (EGDTIV) substituted with FTDPAGF, and    c) has the ability to hydrolyze starch.    
     
     
         31 . A polypeptide which: 
 a) has an amino acid sequence having at least 70% identity to SEQ ID NO: 4;    b) compared to SEQ ID NO: 4 comprises an amino acid alteration which comprises G35K, G35R, A76del+D77del, D74del+A78del, D74A, D74G, D77A, D77G, Y157W or L168F+A169T+T171P+P172A+T173G, and    c) has the ability to hydrolyze starch.    
     
     
         32 . The polypeptide of  claim 31  wherein said polypeptide: 
 a) has an amino acid sequence having at least 80% identity to SEQ ID NO: 4;    b) compared to SEQ ID NO: 4 comprises an amino acid alteration which comprises G35K, G35R, A76del+D77del, D74del+A78del, D74A, D74G, D77A, D77G, Y157W or L168F+A169T+T171P+P172A+T173G, and    c) has the ability to hydrolyze starch.    
     
     
         33 . The polypeptide of  claim 31  wherein said polypeptide: 
 a) has an amino acid sequence having at least 90% identity to SEQ ID NO: 4;    b) compared to SEQ ID NO: 4 comprises an amino acid alteration which comprises G35K, G35R, A76del+D77del, D74del+A78del, D74A, D74G, D77A, D77G, Y157W or L168F+A169T+T171P+P172A+T173G, and    c) has the ability to hydrolyze starch.    
     
     
         34 . The polypeptide of  claim 31  wherein said polypeptide: 
 a) has an amino acid sequence having at least 95% identity to SEQ ID NO: 4;    b) compared to SEQ ID NO: 4 comprises an amino acid alteration which comprises G35K, G35R, A76del+D77del, D74del+A78del, D74A, D74G, D77A, D77G, Y157W or L168F+A169T+T171P+P172A+T173G, and    c) has the ability to hydrolyze starch.    
     
     
         35 . A process for preparing a dough or a baked from dough product which comprises adding the polypeptide of  claim 19.