Glucose dehydrogenases

Abstract

Modified water-soluble glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme are provided wherein at least one amino acid residue is replaced by another amino acid residue in a specific region. Modified water-soluble PQQGDHs of the present invention have improved thermal stability.

1 . A modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein an amino acid residue corresponding to serine 231 in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  is replaced by another amino acid residue.  
     
     
         2 . A modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein an amino acid residue corresponding to glutamine 209 in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  is replaced by another amino acid residue.  
     
     
         3 . A modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein an amino acid residue corresponding to glutamate 210 in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  is replaced by another amino acid residue.  
     
     
         4 . A modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein an amino acid residue corresponding to aspartate 420 in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  is replaced by another amino acid residue.  
     
     
         5 . A modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein an amino acid residue corresponding to alanine 421 in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  is replaced by another amino acid residue.  
     
     
         6 . A modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein at least one amino acid residue is replaced by another amino acid residue in one or more regions selected from the group consisting of the regions defined by residues 48-53, 60-62, 69-71, 79-82, 91-101, 110-115, 127-135, 147-150, 161-169, 177-179, 186-221, 227-244, 250-255, 261-263, 271-275, 282-343, 349-377, 382-393, 400-403, 412-421, 427-432, 438-441 and 449-468 in the amino acid sequence shown as SEQ ID NO: 1, characterized in that it has higher thermal stability than that of the water-soluble glucose dehydrogenase derived from  Acinetobacter calcoaceticus.    
     
     
         7 . The modified glucose dehydrogenase of  claim 3  wherein at least one amino acid residue is replaced by another amino acid residue in the region defined by residues 227-244 in the amino acid sequence shown as SEQ ID NO: 1.  
     
     
         8 . The modified glucose dehydrogenase of  claim 7  wherein serine 231 in the amino acid sequence shown as SEQ ID NO: 1 is replaced by another amino acid residue.  
     
     
         9 . The modified glucose dehydrogenase of  claim 3  wherein at least one amino acid residue is replaced by another amino acid residue in the region defined by residues 186-221 in the amino acid sequence shown as SEQ ID NO: 1.  
     
     
         10 . The modified glucose dehydrogenase of  claim 9  wherein an amino acid residue corresponding to glutamine 209 in the amino acid sequence shown as SEQ ID NO: 1 is replaced by another amino acid residue.  
     
     
         11 . The modified glucose dehydrogenase of  claim 9  wherein an amino acid residue corresponding to glutamate 210 in the amino acid sequence shown as SEQ ID NO: 1 is replaced by another amino acid residue.  
     
     
         12 . The modified glucose dehydrogenase of  claim 3  wherein at least one amino acid residue is replaced by another amino acid residue in the region defined by residues 412-421 in the amino acid sequence shown as SEQ ID NO: 1.  
     
     
         13 . The modified glucose dehydrogenase of  claim 12  wherein an amino acid residue corresponding to aspartate 420 in the amino acid sequence shown as SEQ ID NO: 1 is replaced by another amino acid residue.  
     
     
         14 . The modified glucose dehydrogenase of  claim 12  wherein an amino acid residue corresponding to alanine 421 in the amino acid sequence shown as SEQ ID NO: 1 is replaced by another amino acid residue.  
     
     
         15 . A glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme comprising the sequence:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   Asn Leu Asp Gly Xaa23l Ile Pro Lys Asp Asn Pro 
                     
                 
                     
                     
                 
                     
                   Ser Phe Asn Gly Val Val Ser 
                 
                     
                     
                 
             
                
                
                
                
                
               
            
           
         
       
       wherein Xaa231 represents a natural amino acid residue other than Ser.  
     
     
         16 . A glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme comprising the sequence:  
       
         
           
                 
                 
               
                     
                 
                   Gly Asp Gln Gly Arg Asn Gln Leu Ala Tyr Leu Phe 
                     
                 
                     
                 
                   Leu Pro Asn Gln Ala Gln His Thr Pro Thr Gln Xaa209 
                 
                     
                 
                   Xaa210 Leu Asn Gly Lys Asp Tyr His Thr Tyr Met Gly 
                 
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
       
       wherein Xaa209 and Xaa210 represent any natural amino acid residue, provided that when Xaa209 represents Gln, Xaa210 does not represent Glu.  
     
     
         17 . A glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme comprising the sequence:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   Pro Thr Tyr Ser Thr Thr Tyr Asp Xaa420 Xaa421 
                     
                 
                     
                     
                 
             
                
                
                
               
            
           
         
       
       wherein Xaa420 and Xaa421 represent any natural amino acid residue, provided that when Xaa420 represents Asp, Xaa421 does not represent Ala.  
     
     
         18 . A gene encoding the modified glucose dehydrogenase of any one of  claim 1 .  
     
     
         19 . A vector comprising the gene of  claim 18 .  
     
     
         20 . A transformant comprising the gene of  claim 18 .  
     
     
         21 . The transformant of  claim 20  wherein the gene is integrated into the main chromosome.  
     
     
         22 . A glucose assay kit comprising the modified glucose dehydrogenase of any one of  claim 1 .  
     
     
         23 . A glucose sensor comprising the modified glucose dehydrogenase of any one of  claim 1.