Glucose dehydrogenase
Disclosed is a modified glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme, wherein one or more amino acid residues in a region of 186-206 amino acid of water-soluble PQQGDH derived from Acinetobacter calcoaceticus or in an equivalent region from other species are replaced with other amino acid residues. Also disclosed is a gene coding for the modified glucose dehydrogenase of the invention, a vector comprising the gene of the invention and a transformant comprising the vector, as well as a glucose assay kit and a glucose sensor comprising the modified glucose dehydrogenase of the invention.
1. An isolated mutant water-soluble glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme, wherein said mutant is a mutant of a glucose dehydrogenase comprising the amino acid sequence of SEQ ID NO: 1, and wherein said mutant consists of an amino acid substitution selected from the group consisting of:
(1) glutamine at position 168 of SEQ ID NO:1 is substituted with glycine, glutamic acid, leucine, phenylalanine, serine or aspartic acid, optionally combined with (a) a substitution wherein aspartate at position 143 of SEQ ID NO: 1 is substituted with glutamic acid or (b) a substitution wherein asparagine at position 428 of SEQ ID NO:1 is substituted with threonine;
(2) leucine at position 169 of SEQ ID NO:1 substituted with alanine, glycine, methionine, tryptophan or lysine, optionally combined with (a) a substitution wherein aspartate at position 143 of SEQ ID NO:1 is substituted with glutamic acid or (b) a substitution wherein asparagine at position 428 of SEQ ID NO:1 is substituted with threonine ; and
(3) aspartate at position 143 of SEQ ID NO:1 is substituted with glutamic acid and asparagine at position 428 of SEQ ID NO: 1 is substituted with threonine.
2. A glucose assay kit comprising the modified glucose dehydrogenase as claimed in claim 1 .
3. A glucose sensor comprising the modified glucose dehydrogenase as claimed in claim 1 .
4. The mutant glucose dehydrogenase as claimed in claim 1 , wherein glutamine at position 168 of SEQ ID NO:1 is substituted with glycine, glutamic acid, leucine, phenylalanine, serine or aspartic acid.
5. The mutant glucose dehydrogenase as claimed in claim 1 , wherein leucine at position 169 of SEQ ID NO:1 is substituted wit alanine, glycine, methionine, tryptophan or lysine.
6. The mutant glucose dehydrogenase as claimed in claim 1 , wherein aspartate at position 143 of SEQ ID NO:1 is substituted with glutamic acid, and asparagine at position 428 of SEQ ID NO:1 is substituted with threonine.
7. The mutant glucose dehydrogenase as claimed in claim 1 , wherein glutamine at position 168 of SEQ ID NO:1 is substituted with glycine, glutamic acid, leucine, phenylalanine, serine or aspartic acid in SEQ ID NO:1, and aspartate at position 143 of SEQ ID NO:1 is substituted wit glutamic acid.
8. The mutant glucose dehydrogenase as claimed in claim 1 , wherein glutamine at position 168 of SEQ ID NO:1 is substituted with glycine, glutamic acid, leucine, phenylalanine, serine or aspartic acid in SEQ ID NO:1, and asparagine at 428 of SEQ ID NO:1 is substituted with threonine.
9. The mutant glucose dehydrogenase as claimed in claim 1 , wherein leucine at position 169 of SEQ ID NO:1 is substituted with alanine, glycine, methionine, tryptophan or lysine and aspartate at position 143 of SEQ ID NO:1 is substituted wit glutamic acid.
10. The mutant glucose dehydrogenase as claimed in claim 1 , wherein leucine at 169 of SEQ ID NO:1 is substituted with alanine, glycine, methionine, tryptophan or lysine and asparagine at position 428 of SEQ ID NO:1 is substituted with threonine.
11. An isolated mutant water-soluble glucose dehydrogenase having pyrroloquinoline quinone as a coenzyme, wherein said mutant is a mutant of a glucose dehydrogenase comprising the amino acid sequence of SEQ ID NO:1, and wherein said mutant consist of an amino acid substitution wherein glutamine at position 168 of SEQ ID NO:1 is substituted with glycine, glutamic acid, leucine, phenylalanine, serine or aspartic acid.