Amylolytic enzyme variants

Abstract

The inventors have discovered some striking, and not previously predicted structural similarities and differences between the structure of Novamyl and the reported structures of CGTases, and based on this they have constructed variants of maltogenic alpha-amylase having CGTase activity and variants of CGTase having maltogenic alpha-amylase activity. Further, on the basis of sequence homology between Novamyl® and CGTases, the inventors have constructed hybrid enzymes with one or more improvements to specific properties of the parent enzymes, using recombinant DNA methodology.

1 . A polypeptide which: 
 a) has at least 70% identity to amino acids 1-686 of SEQ ID NO: 1;    b) comprises an amino acid modification which is an insertion, substitution or deletion compared to SEQ ID NO: 1 in a region corresponding to amino acids 40-43, 78-85, 136-139, 173-180, 189-195 or 259-268; and    c) has the ability to form cyclodextrin when acting on starch.    
     
     
         2 . The polypeptide of  claim 1  wherein the modification comprises a substitution with or insertion of the amino acid residue present at the corresponding position of a cyclodextrin glucanotransferase (CGTase) and/or deletion of an amino acid residue which is not present at that position in a cyclodextrin glucanotransferase (CGTase).  
     
     
         3 . The polypeptide of  claim 1  or  2  wherein the CGTase is derived from a strain of Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter or Thermoanaerobacterium.  
     
     
         4 . The polypeptide of any of claims  1 - 3  wherein the modification comprises a deletion in the region 190-195, preferably the deletion Δ (191-195).  
     
     
         5 . The polypeptide of any of claims  1 - 4  which further comprises a substitution of amino acid 188 and/or 189, preferably F188L and/or Y189Y.  
     
     
         6 . A method of constructing a variant of a parent maltogenic alpha-amylase, which method comprises: 
 a) modifying the amino acid sequence by an insertion, substitution or deletion in a region corresponding to amino acids 40-43, 78-85, 136-139, 173-180, 189-195 or 259-268 of SEQ ID NO: 1;    b) optionally modifying the amino acid sequence by an insertion, substitution or deletion at a position other than a);    c) optionally repeating a)-b) recursively;    d) preparing the variant resulting from a)-c);    e) testing the variant by hydrolyzing starch and analyzing the initial products;    f) optionally repeating a)-e) recursively; and    g) selecting a variant having the ability to form cyclodextrin from starch.    
     
     
         7 . A polypeptide which: 
 a) has an amino acid sequence having at least 70% identity to a parent cyclodextrin glucanotransferase (CGTase);    b) comprises an amino acid modification which is an insertion, substitution or deletion compared to the parent CGTase in a region corresponding to amino acids 40-43, 78-85, 136-139, 173-180, 189-195 or 259-268 of SEQ ID NO: 1; and    c) has the ability to form linear oligosaccharides as an initial product when acting on starch.    
     
     
         8 . The polypeptide of  claim 7  wherein the modification comprises a substitution with or insertion of the amino acid residue present at the corresponding position of SEQ ID NO: 1 and/or deletion of an amino acid residue which is not present at that position in SEQ ID NO: 1.  
     
     
         9 . The polypeptide of  claim 7  or  8 , wherein the modification is an insertion of the amino acid sequence DPAGF (Asp-Pro-Ala-Gly-Phe) at a position corresponding to D190-F194 of SEQ ID NO: 1.  
     
     
         10 . The polypeptide of any of claims  7 - 9 , wherein the CGTase is derived from a strain of Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micro-coccus, Thermoanaerobacter or Thermoanaerobacterium.  
     
     
         11 . The polypeptide of any of claims  7 - 10 , which further comprises a modification in a region corresponding to amino acids 37-39, 44-45, 135, 140-145, 181-186, 269-273, or 377-383 of SEQ ID NO: 1.  
     
     
         12 . A method of constructing a variant of a parent cyclodextrin glucanotransferase (CGTase), which method comprises: 
 a) modifying the amino acid sequence by an insertion, substitution or deletion in a region corresponding to amino acids 40-43, 78-85, 136-139, 173-180, 189-195 or 259-268 of SEQ ID NO: 1;    b) optionally modifying the amino acid sequence by an insertion, substitution or deletion at a position other than a);    c) optionally repeating a)-b) recursively;    d) preparing the variant resulting from a)-c);    e) testing the variant by hydrolyzing starch and analyzing the initial products;    f) optionally repeating a)-e) recursively; and    g) selecting a variant having the ability to form linear oligosaccharides as an initial product from starch.    
     
     
         13 . A nucleic acid sequence encoding the polypeptide of any of claims  1 - 5  or  7 - 11 , preferably operably linked to one or more control sequences which direct the expression of the variant in a suitable expression host.  
     
     
         14 . A recombinant expression vector comprising the nucleic acid sequence of  claim 13 , a promoter, and transcriptional and translational stop signals, and preferably further comprising a selectable marker.  
     
     
         15 . A transformed host cell comprising the nucleic acid sequence of  claim 13  or the vector of  claim 14 .  
     
     
         16 . A method for producing the polypeptide of any of claims  1 - 5  or  7 - 11 , comprising: 
 a) cultivating a host cell comprising a nucleic acid construct comprising a nucleic acid sequence encoding said variant under conditions conducive to expression of the variant; and  
 b) recovering the variant.  
 
     
     
         17 . A method for constructing a maltogenic alpha-amylase, comprising: 
 a) recombining DNA encoding a cyclodextrin glucanotransferase (CGTase) and DNA encoding a maltogenic alpha-amylase;    b) using the recombinant DNA to express a polypeptide; and    c) testing the polypeptide to select a polypeptide having the ability to form linear oligosaccharides when acting on starch.    
     
     
         18 . The method of  claim 17 , further comprising amplifying the recombinant DNA with primers encoding a partial amino acid sequence of amino acids 1-686 of SEQ ID NO: 1 in combination with primers encoding a partial sequence of the CGTase, said primers preferably comprising at least 5 amino acid residues, more preferably comprising one or more of positions 188-196, most preferably comprising positions 190-194.  
     
     
         19 . The method of  claim 17  or  18 , wherein the primers encoding a partial sequence of SEQ ID NO: 1 comprise a partial sequence of the DNA sequence shown in SEQ ID NO: 1.  
     
     
         20 . The method of any of claims  17 - 19 , wherein the mature forms of the CGTase and the maltogenic alpha-amylase have at least 40% identity, preferably at least 50%, more preferably at least 60%.  
     
     
         21 . The method of any of claims  17 - 20 , wherein the CGTase is derived from a strain of Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micro-coccus, Thermoanaerobacter or Thermoanaerobacterium.  
     
     
         22 . A method of selecting DNA encoding maltogenic alpha-amylase in a DNA pool, comprising: 
 a) amplifying DNA encoding maltogenic alpha-amylase by a polymerase chain reaction (PCR) using primers encoding a partial amino acid sequence of amino acids 1-686 of SEQ ID NO: 1, preferably comprising at least 5 amino acid residues, preferably comprising one or more of positions 188-196, more preferably comprising positions 190-194,    b) cloning and expressing the amplified DNA, and    c) screening for maltogenic alpha-amylase activity.    
     
     
         23 . A process for preparing a dough or a baked product prepared from the dough which comprises adding the polypeptide of any of claims  7 - 11  or a variant constructed by the method of any of claims  12  or  17 - 20  to the dough in an amount which is effective to retard the staling of the bread.  
     
     
         24 . A process for preparing cyclodextrin, comprising treating liquefied starch with the polypeptide of any of claims  1 - 5  or the variant constructed by the method of  claim 6 .  
     
     
         25 . A method for producing a variant, comprising: 
 a) constructing the variant by the method of  claim 6  or  12 ;    b) transforming a microorganism with a DNA sequence encoding the variant;    c) cultivating the transformed microorganism under conditions which are conducive for producing the variant, and    d) recovering the variant from the resulting culture broth.