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 method for preparing a dough or baked product prepared from the dough comprising adding to the dough a cyclodextrin glucanotransferase variant which forms linear oligosaccharides when acting on starch in an amount effective to retard the staling of the baked product prepared from the dough. 
     
     
         2 . The method of  claim 1 , wherein the cyclodextrin glucanotransferase variant is derived from a strain of  Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter  or  Thermoanaerobacterium.    
     
     
         3 . A method for producing a variant of a parent cyclodextrin glucanotransferase, comprising modifying the amino acid sequence of a parent cyclodextrin glucanotransferase by substituting, inserting or deleting one or more amino acids of said amino acid sequence, wherein said substitution is a substitution an amino acid residue which is present in a corresponding position in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO:2 but which is not present in the amino acid sequence of the parent cyclodextrin glucanotransferase; wherein said insertion is an insertion of an amino acid residue which is present in a corresponding position in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO:1 but which is not present in the amino acid sequence of the parent cyclodextrin glucanotransferase; and wherein said deletion is a deletion of an amino acid residue which is present in the parent cyclodextrin glucanotransferase but which is not present in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2, and
 wherein the cyclodextrin glucanotransferase variant forms linear oligosaccharides when acting on starch.   
     
     
         4 . The method of  claim 3 , wherein the parent cyclodextrin glucanotransferase is from a strain of  Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter  or  Thermoanaerobacterium.    
     
     
         5 . A cyclodextrin glucanotransferase variant prepared by the method of  claim 3 . 
     
     
         6 . A method for producing a variant of a parent cyclodextrin glucanotransferase, comprising
 (a) modifying the amino acid sequence of a parent cyclodextrin glucanotransferase by substituting, inserting or deleting one or more amino acids of said amino acid sequence, wherein said substitution is a substitution an amino acid residue which is present in a corresponding position in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2 but which is not present in the amino acid sequence of the parent cyclodextrin glucanotransferase; wherein said insertion is an insertion of an amino acid residue which is present in a corresponding position in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2 but which is not present in the amino acid sequence of the parent cyclodextrin glucanotransferase; and wherein said deletion is a deletion of an amino acid residue which is present in the parent cyclodextrin glucanotransferase but which is not present in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2,   (b) testing the variant cyclodextrin glucanotransferase for the ability to form linear oligosaccharides when acting on starch;   (c) producing the variant cyclodextrin glucanotransferase by cultivating a host cell comprising a nucleic acid sequence encoding the variant cyclodextrin glucanotransferase; and   (d) recovering the cyclodextrin glucanotransferase variant.   
     
     
         7 . The method of  claim 6 , wherein the cyclodextrin glucanotransferase is derived from a strain of  Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter  or  Thermoanaerobacterium.    
     
     
         8 . A cyclodextrin glucanotransferase variant prepared by the method of  claim 6 . 
     
     
         9 . A method for producing a variant of a parent cyclodextrin glucanotransferase, comprising:
 (a) cultivating a host cell comprising a nucleic acid sequence encoding a variant of a cyclodextrin glucanotransferase, wherein said cyclodextrin glucanotransferase variant comprises and insertion, substitution or deletion of one or more amino acids, wherein said substitution is a substitution an amino acid residue which is present in a corresponding position in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2 but which is not present in the amino acid sequence of the parent cyclodextrin glucanotransferase; wherein said insertion is an insertion of an amino acid residue which is present in a corresponding position in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2 but which is not present in the amino acid sequence of the parent cyclodextrin glucanotransferase; and wherein said deletion is a deletion of an amino acid residue which is present in the parent cyclodextrin glucanotransferase but which is not present in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2, and wherein the cyclodextrin glucanotransferase variant forms linear oligosaccharides when acting on starch.   (b) recovering the cyclodextrin glucanotransferase variant.   
     
     
         10 . The method of  claim 9 , wherein the cyclodextrin glucanotransferase is derived from a strain of  Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter  or  Thermoanaerobacterium.    
     
     
         11 . A cyclodextrin glucanotransferase variant prepared by the method of  claim 9 . 
     
     
         12 . A method for producing a variant of a parent cyclodextrin glucanotransferase, comprising:
 (a) cultivating a host cell comprising a nucleic acid sequence encoding a variant of a cyclodextrin glucanotransferase, wherein said cyclodextrin glucanotransferase variant comprises and insertion, substitution or deletion of one or more amino acids, wherein said substitution is a substitution an amino acid residue which is present in a corresponding position in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2 but which is not present in the amino acid sequence of the parent cyclodextrin glucanotransferase; wherein said insertion is an insertion of an amino acid residue which is present in a corresponding position in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2 but which is not present in the amino acid sequence of the parent cyclodextrin glucanotransferase; and wherein said deletion is a deletion of an amino acid residue which is present in the parent cyclodextrin glucanotransferase but which is not present in the amino acid sequence of amino acids 1 to 686 of SEQ ID NO: 2;   (b) transforming a host cell with the nucleic acid sequence encoding the variant;   (c) cultivating the transformed host cell to express the variant;   (d) testing the variant cyclodextrin glucanotransferase for the ability to form linear oligosaccharides when acting on starch;   (e) producing the variant cyclodextrin glucanotransferase by cultivating a host cell comprising a nucleic acid sequence encoding the variant cyclodextrin glucanotransferase;   (f) recovering the cyclodextrin glucanotransferase variant.   
     
     
         13 . The method of  claim 12 , wherein the cyclodextrin glucanotransferase is derived from a strain of  Bacillus, Brevibacterium, Clostridium, Corynebacterium, Klebsiella, Micrococcus, Thermoanaerobacter  or  Thermoanaerobacterium.    
     
     
         14 . A cyclodextrin glucanotransferase variant prepared by the method of  claim 12 . 
     
     
         15 . An isolated polypeptide which:
 a) has an amino acid sequence having at least 70% identity to a parent  Bacillus  or  Thermoanerobacter  cyclodextrin glucanotransferase (CGTase);   b) comprises an amino acid modification compared to the parent CGTase in a region corresponding to amino acids 190-194 of the amino acid sequence shown in SEQ ID NO: 2, wherein the modification is selected from the group consisting of: an insertion of DAGF (SEQ ID NO: 28), an insertion of DPGF (SEQ ID NO: 29), an insertion of DPF; an insertion of DPAAGF (SEQ ID NO: 30), an insertion of DPAAGGF (SEQ ID NO: 31) and a substitution at a position corresponding to T189 of SEQ ID NO: 2; and   c) has the ability to form linear oligosaccharides as an initial product when acting on starch.   
     
     
         16 . The polypeptide of  claim 15 , wherein the amino acid modification comprises an insertion of DAGF (SEQ ID NO: 28) in a region corresponding to amino acids 190-194 of the amino acid sequence shown in SEQ ID NO: 2. 
     
     
         17 . The polypeptide of  claim 15 , wherein the amino acid modification comprises an insertion of DPGF (SEQ ID NO: 29) in a region corresponding to amino acids 190-194 of the amino acid sequence shown in SEQ ID NO: 2. 
     
     
         18 . The polypeptide of  claim 15 , wherein the amino acid modification comprises an insertion of DPF in a region corresponding to amino acids 190-194 of the amino acid sequence shown in SEQ ID NO: 2. 
     
     
         19 . The polypeptide of  claim 15 , wherein the amino acid modification comprises an insertion of DPAAGF (SEQ ID NO: 30) in a region corresponding to amino acids 190-194 of the amino acid sequence shown in SEQ ID NO: 2. 
     
     
         20 . The polypeptide of  claim 15 , wherein the amino acid modification comprises an insertion of DPAAGGF (SEQ ID NO: 31) in a region corresponding to amino acids 190-194 of the amino acid sequence shown in SEQ ID NO: 2. 
     
     
         21 . The polypeptide of  claim 15 , wherein the amino acid modification comprises a substitution at a position corresponding to T189 of SEQ ID NO: 2.