Glucose dehydrogenase

Abstract

Modified water-soluble glucose dehydrogenases 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 affinity for glucose.

1 . An isolated modified water-soluble glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme characterized in that at least one amino acid residue in a natural water-soluble glucose dehydrogenase derived from  Acinetobacter  is replaced by another amino acid residue and it has improved affinity for glucose as compared with the natural water-soluble glucose dehydrogenase.  
     
     
         2 . The isolated modified glucose dehydrogenase of  claim 1  having high selectivity for glucose as compared with the wild-type PQQGDH of SEQ ID NO: 1.  
     
     
         3 . An isolated modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein asparagine 462 (asparagine 438 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  or an amino acid residue corresponding to said residue is replaced by another amino acid residue.  
     
     
         4 . An isolated modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein asparagine 452 (asparagine 428 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  or an amino acid residue corresponding to said residue is replaced by another amino acid residue.  
     
     
         5 . An isolated modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein lysine 455 (lysine 431 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  or an amino acid residue corresponding to said residue is replaced by another amino acid residue.  
     
     
         6 . An isolated modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein aspartate 456 (aspartate 432 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from Acinetobacter calcoaceticus or an amino acid residue corresponding to said residue is replaced by another amino acid residue.  
     
     
         7 . An isolated modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein aspartate 457 (aspartate 433 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from Acinetobacter calcoaceticus or an amino acid residue corresponding to said residue is replaced by another amino acid residue.  
     
     
         8 . An isolated modified glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein aspartate 448 (aspartate 424 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from Acinetobacter calcoaceticus or an amino acid residue corresponding to said residue is replaced by another amino acid residue.  
     
     
         9 . An isolated 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 the region corresponding to residues 268-289 (244-265 of SEQ ID NO: 1) or 448-468 (424-434 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from Acinetobacter calcoaceticus.  
     
     
         10 . An isolated modified water-soluble glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein glutamate 277 (glutamate 253 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  or an amino acid residue corresponding to said residue is replaced by another amino acid residue.  
     
     
         11 . An isolated modified water-soluble glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyrne wherein isoleucine 278 (isoleucine 254 of SEQ ID NO: 1) in the water-soluble PQQGDH derived from  Acinetobacter calcoaceticus  or an amino acid residue corresponding to said residue is replaced by another amino acid residue.  
     
     
         12 . An isolated modified water-soluble glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein at least one amino acid residue is replaced by another amino acid residue in the region defined by residues 268-289 (244-265 of SEQ ID NO: 1) or 448-468 (424-434 of SEQ ID NO: 1) in the amino acid sequence shown as SEQ ID NO: 1.  
     
     
         13 . An isolated modified PQQ glucose dehydrogenase comprising the sequence (SEQ ID NO: 14): 
 Xaa8 Thr Ala Gly Xaa1 Val Gln Xaa2 Xaa3 Xaa4 Gly Ser Val Thr Xaa5 Thr Leu Glu Asn Pro Gly    wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa5 and Xaa8 represent any natural amino acid residue, provided that when Xaa1 represents Asn, Xaa2 represents Lys, Xaa3 represents Asp, Xaa4 represents Asp and Xaa5 represents Asn, then Xaa8 does not represent Asp.    
     
     
         14 . An isolated modified PQQ glucose dehydrogenase comprising the sequence (SEQ ID NO: 3): 
 Ser Glu Gln Gly Pro Asn Ser Asp Asp Xaa6 Xaa7 Asn Leu Ile Val Lys Gly Gly Asn TyrGlyTrp    wherein Xaa6 and Xaa7 represent any natural amino acid residue, provided that when Xaa6 represents Glu, Xaa7 does not represent Ile.    
     
     
         15 . The isolated modified glucose dehydrogenase of  claim 14  wherein glutamate 277 (glutamate 253 of SEQ ID NO: 1) is replaced by another amino acid residue.  
     
     
         16 . The isolated modified glucose dehydrogenase of  claim 14  wherein isoleucine 278 (isoleucine 254 of SEQ ID NO: 1) is replaced by another amino acid residue.  
     
     
         17 . A gene encoding the modified glucose dehydrogenase of  claim 1 .  
     
     
         18 . A vector comprising the gene of  claim 17 .  
     
     
         19 . A transformant comprising the gene of  claim 17 .  
     
     
         20 . The transform ant of  claim 19  wherein the gene of  claim 17  is integrated into the main chromosome.  
     
     
         21 . A glucose assay kit comprising the modified glucose dehydrogenase of  claim 1 .  
     
     
         22 . A glucose sensor comprising the modified glucose dehydrogenase of claim