Starch debranching enzymes

Abstract

The invention relates to a genetically engineered variant of a parent starch debranching enzyme, i.e. a pullulanase or an isamylase, the enzyme variant having an improved thermostability at a pH in the range of 4-6 compared to the parent enzyme and/or an increased activity towards amylopectin and/or glycogen compared to the parent enzyme, to methods for producing such starch debranching enzyme variants with improved thermostability and/or altered substrate specificity, and to a method for converting starch to one or more sugars using at least one such enzyme variant.

1 . A genetically engineered variant of a parent starch debranching enzyme, the enzyme variant having an improved thermostability at a pH in the range of 4-6 compared to the parent enzyme.  
     
     
         2 . The enzyme variant of  claim 1 , wherein the enzyme is a pullulanase.  
     
     
         3 . The enzyme variant of  claim 1 , wherein the enzyme is an isoamylase.  
     
     
         4 . The enzyme variant of any of claims  1 - 3 , wherein the enzyme variant has an improved thermostability as defined by differential scanning calorimetry (DSC) using the method described herein.  
     
     
         5 . The enzyme variant of any of claims  1 - 3 , wherein the enzyme variant has an improved thermostability as defined by an increased half-time (T ½ ) of at least about 5%, preferably at least about 10%, more preferably at least about 15%, more preferably at least about 25%, most preferably at least about 50%, such as at least 100%, in the “T ½  assay for liquefaction” described herein, using a pH of 5.0 and a temperature of 95° C.  
     
     
         6 . The enzyme variant of any of claims  1 - 3 , wherein the enzyme variant has an improved thermostability as defined by an increased residual enzyme activity of at least about 5%, preferably at least about 10%, more preferably at least about 15%, more preferably at least about 25%, most preferably at least about 50%, in the “assay for residual activity after liquefaction” described herein, using a pH of 5.0 and a temperature of 95° C.  
     
     
         7 . The enzyme variant of any of claims  1 - 3 , wherein the enzyme variant has an improved thermostability as defined by an increased half-time (T ½ ) of at least about 5%, preferably at least about 10%, more preferably at least about 15%, more preferably at least about 25%, most preferably at least about 50%, such as at least 100%, in the “T ½  assay for saccharification” described herein, using a pH of 4.5 and a temperature of 70° C.  
     
     
         8 . The enzyme variant of any of claims  1 - 3 , wherein the enzyme variant has an improved thermostability as defined by an increased residual enzyme activity of at least about 5%, preferably at least about 10%, more preferably at least about 15%, more preferably at least about 25%, most preferably at least about 50%, in the “assay for residual activity after saccharification” described herein, using a pH of 4.5 and a temperature of 63° C.  
     
     
         9 . The enzyme variant of  claim 8 , having increased residual enzyme activity when assayed at a temperature of 70° C.  
     
     
         10 . A genetically engineered variant of a parent starch debranching enzyme, the enzyme variant having an increased activity towards amylopectin and/or glycogen compared to the parent enzyme.  
     
     
         11 . The enzyme variant of  claim 10 , wherein the parent enzyme is a pullulanase.  
     
     
         12 . The enzyme variant of  claim 11 , wherein the pullulanase is produced by a bacterium of the genus Bacillus, e.g.  Bacillus acidopullulyticus  or  Bacillus deramificans,  or Pyrococcus, or Klebsiella, e.g.  Klebsiella pneumoniae  or  Klebsiella aerogenes    
     
     
         13 . The enzyme variant of  claim 10 , wherein the parent enzyme is an isoamylase, e.g. an isoamylase produced by a bacterium of the genus Pseudomonas, e.g.  Pseudomonas amyloderamosa,  or from Sulfolobus, e.g.  Sulfolobus acidocaldarius  or  Sulfolobus solfataricus,  or Rhodothermus, e.g.  Rhodothermus marinus,    
     
     
         14 . The enzyme variant of any of claims  10 - 13 , wherein the enzyme variant further has improved thermostability as defined in any of claims  1 - 9 .  
     
     
         15 . A method for producing a starch debranching enzyme variant with increased thermostability, the method comprising the steps of: 
 identifying one or more amino acid residues and/or amino acid regions associated with thermostability in a first parent starch debranching enzyme,    identifying one or more homologous amino acid residues and/or amino acid regions in a second parent starch debranching enzyme by means of alignment of the amino acid sequences of the first and second parent starch debranching enzymes, and    mutating one or more of the homologous amino acid residues and/or amino acid regions in the second parent starch debranching enzyme to produce an enzyme variant with increased thermostability.    
     
     
         16 . The method of  claim 15 , wherein increased thermostability is obtained by mutating homologous amino acid residues and/or amino acid regions located in a loop, preferably loop 2 or 3.  
     
     
         17 . The method of  claim 15 , wherein mutation is performed at at least one amino acid residue corresponding to positions 369-397, 419-458, 484-525, 553-568, 582-608, 613-616, 661-670 and 708-739 of SEQ ID NO: 1, positions 465-493, 515-554, 580-621, 649-664, 680-711, 714-717, 757-765 and 804-834 of SEQ ID NO: 2, positions 176-195, 218-257, 283-334, 359-381, 395-413, 416-419, 461-470 and 537-568 of SEQ ID NO: 3, and positions 210-230, 250-292, 319-376, 401-437, 461-479, 482-485, 533-580 and 605-636 of SEQ ID NO: 4.  
     
     
         18 . The method of  claim 15 , wherein increased thermostability is obtained by replacing at least one non-proline residue with a proline residue.  
     
     
         19 . The method of  claim 15 , wherein increased thermostability is obtained by at least one Gly to Ala substitution.  
     
     
         20 . The method of  claim 15 , wherein increased thermostability is obtained by substituting at least one Asn or Gln residue with another amino acid residue, preferably an amino acid residue selected from the group consisting of Leu, Ile, Phe, Ala, Thr, Ser and Tyr.  
     
     
         21 . The method of any of claims  18 - 20 , wherein substitution is performed in loop 3, 5 and/or 6.  
     
     
         22 . A method for producing a starch debranching enzyme variant with altered substrate specificity, the method comprising the steps of: 
 identifying one or more amino acid residues in at least one amino acid loop associated with specificity towards a desired substrate in a first parent starch debranching enzyme,    identifying one or more homologous amino acid residues in at least one corresponding loop in a second parent starch debranching enzyme by means of alignment of the amino acid sequences of the first and second parent starch debranching enzymes, and    mutating one or more of the homologous amino acid residues in at least one loop in the second parent starch debranching enzyme to produce an enzyme variant with altered substrate specificity.    
     
     
         23 . The method of  claim 22 , wherein mutation is performed at at least one amino acid residue corresponding to positions 369-397, 419-458, 484-525, 553-568, 582-608, 613-616, 661-670 and 708-739 of SEQ ID NO: 1, positions 465-493, 515-554, 580-621, 649-664, 680-711, 714-717, 757-765 and 804-834 of SEQ ID NO: 2, positions 176-195, 218-257, 283-334, 359-381, 395-413, 416-419, 461-470 and 537-568 of SEQ ID NO: 3, and positions 210-230, 250-292, 319-376, 401-437, 461-479, 482-485, 533-580 and 605-636 of SEQ ID NO: 4.  
     
     
         24 . The method of  claim 22 , wherein mutation is performed in loop 1, 2, 4, 5 and/or 7.  
     
     
         25 . The method of any of claims  22 - 24 , wherein the mutation is a loop transfer.  
     
     
         26 . The method of any of claims  22 - 25 , wherein the first parent starch debranching enzyme is an isoamylase and the second parent starch debranching enzyme is a pullulanase.  
     
     
         27 . The method of any of claims  22 - 26 , wherein the altered substrate specificity is an increased activity towards amylopectin and/or glycogen.  
     
     
         28 . The method of any of claims  15 - 27 , wherein homologous amino acid residues and/or amino acid regions suitable for mutation are identified in the second parent starch debranching enzyme by means of alignment of the amino acid sequence of the second parent starch branching enzyme with the most homologous sequence in a key alignment, wherein said key alignment comprises an alignment of the amino acid sequences of at least two relevant starch debranching enzymes.  
     
     
         29 . The method of  claim 28 , wherein the key alignment comprises an alignment of at least two amino acid sequences selected from the group consisting of SEQ ID NO 1, 2, 3 and 4.  
     
     
         30 . The method of any of claims  15 - 29 , wherein mutation of the second parent starch debranching enzyme is performed in at least one loop of the second parent starch debranching enzyme which is homologous with a corresponding loop identified in Table 1 herein.  
     
     
         31 . A method for converting starch to one or more sugars, the method comprising debranching the starch using at least one enzyme variant as defined in any of claims  1 - 30 .  
     
     
         32 . The method of  claim 31 , wherein the starch conversion comprises liquefaction at a temperature of at least about 95° C. using at least one enzyme variant as defined in any of claims  1 - 6  and at least one α-amylase.  
     
     
         33 . The method of  claim 32 , wherein the enzyme variant is a pullulanase or an isoamylase.  
     
     
         34 . The method of any of claims  31 - 33 , wherein the starch conversion comprises saccharification at a temperature of at least about 63° C., preferably at least about 70° C., using at least one enzyme variant as defined in any of claims  1 - 4  or  7 - 9  and at least one glucoamylase.  
     
     
         35 . The method of  claim 34 , wherein the enzyme variant is a pullulanase.