# US 7,604,969 B2 · Mutant glucose dehydrogenase > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,604,969 B2 - Document ID: US7604969B2 - Status: Granted - Kind: B2 - Application number: 11/665,296 - Filed: 2006-06-12 - Published: 2008-08-28 - Granted: 2009-10-20 - Earliest claimed: 2005-06-20 - Assignees: ARKRAY, Inc. - Inventors: Hideaki Yamaoka; Masashi Tsukada - Web page: https://patentgrep.com/patent/US7604969B2 ## CPC - C12Q 1/32: involving dehydrogenase - C12N 9/0006: {acting on CH-OH groups as donors (1.1)} - C12Q 1/54: involving glucose or galactose - C12Y 101/01049: Glucose-6-phosphate dehydrogenase (1.1.1.49) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract A mutant glucose dehydrogenase having an improved substrate specificity to glucose, which is a protein comprising the amino acid sequence depicted in SEQ ID NO:3 or an amino acid sequence having the substitution, deletion, insertion or addition of one or more amino acid residues excluding amino acid 365 in the amino acid sequence depicted in SEQ ID NO:3 and having a glucose dehydrogenase activity, the amino acid residue corresponding to amino acid 365 being substituted by other amino acid residue. ## Claims ### Claim 1 An isolated mutant glucose dehydrogenase of SEQ ID NO: 3, wherein said mutant glucose dehydrogenase consists of a replacement of the amino acid residue at position 365 with another amino acid residue, and/or consists of a substitution, deletion, insertion or addition of 1 to 10 amino acid residues other than the amino acid residue at position 365 with another or other amino acid residue at the 324th, 326th, 333rd, 334th, 368th, 369th, 376th, 377th, 418th, 419th, 436th, 433rd, 448th, 472nd, 475th, 525th or 529th positions in the amino acid sequence of SEQ ID NO: 3, wherein said mutant glucose dehydrogenase has an increased substrate specificity to glucose compared to the glucose dehydrogenase of SEQ ID NO:3. ### Claim 2 (depends on 1) The isolated mutant glucose dehydrogenase according to claim 1, consisting of a replacement of serine at position 365 with another amino acid. ### Claim 3 (depends on 1) The isolated mutant glucose dehydrogenase according to claim 1, which has a reduced reactivity to a disaccharide compared with the glucose dehydrogenase of SEQ ID NO: 3. ### Claim 4 (depends on 3) The isolated mutant glucose dehydrogenase according to claim 3, wherein the disaccharide is maltose. ### Claim 5 (depends on 4) The isolated mutant glucose dehydrogenase according to claim 4, which has a reactivity to maltose in a degree of 20% or less of reactivity to glucose. ### Claim 6 (depends on 1) The isolated mutant glucose dehydrogenase according to claim 1, wherein the replacement amino acid residue is an amino acid residue selected from phenylalanine, tyrosine, tryptophan and histidine residues. ### Claim 7 (depends on 1) The isolated mutant glucose dehydrogenase according to claim 1, wherein at least one position selected from the 326th, 472nd, 475th and 529th positions is replaced with another or other amino acid residues. ### Claim 8 (depends on 7) The isolated mutant glucose dehydrogenase according to claim 7, wherein the 472nd position is replaced with another or other amino acid residues. ### Claim 9 (depends on 7) The isolated mutant glucose dehydrogenase according to claim 7, wherein the 475th position is replaced with another or other amino acid residues. ### Claim 10 (depends on 7) The isolated mutant glucose dehydrogenase according to claim 7, wherein both the 472nd position and the 475th position are replaced with other amino acid residues. ### Claim 11 (depends on 7) The isolated mutant glucose dehydrogenase according to claim 7, wherein the 326th position is replaced with another or other amino acid residues. ### Claim 12 (depends on 7) The isolated mutant glucose dehydrogenase according to claim 7, wherein the 529th position is replaced with another or other amino acid residues. ### Claim 13 (depends on 8) The isolated mutant glucose dehydrogenase according to claim 8, wherein the amino acid residue at a position corresponding to the 472nd position is replaced with an amino acid residue selected from aspartic acid, glutamic acid, phenylalanine, tyrosine, isoleucine, asparagine and histidine residues. ### Claim 14 (depends on 9) The isolated mutant glucose dehydrogenase according to claim 9, wherein the amino acid residue at the 475th position is replaced with histidine or serine residue. ### Claim 15 (depends on 11) The isolated mutant glucose dehydrogenase according to claim 11, wherein the amino acid residue of the 326th position is replaced with glutamine or valine residue. ### Claim 16 (depends on 12) The isolated mutant glucose dehydrogenase according to claim 12, wherein the amino acid residue at the 529th position is replaced with tyrosine, histidine or tryptophan residue. ### Claim 17 An isolated mutant glucose dehydrogenase of the glucose dehydrogenase having the amino acid sequence of SEQ ID NO: 3, wherein said mutant glucose dehydrogenase consists of: (i) substitution of the amino acid residue at position 365 in the amino acid sequence of SEQ ID NO: 3, (ii) substitution of aspartic acid residue for another amino acid residue at position 472, and (iii) substitution of histidine residue for another amino acid residue at position 475, wherein said mutant glucose dehydrogenase has increased substrate specificity to glucose, compared to the glucose dehydrogenase comprising the amino acid sequence of SEQ ID NO: 3. ### Claim 18 (depends on 17) The isolated mutant glucose dehydrogenase according to claim 17, wherein the other amino acid residue or residues are selected from phenylalanine, tyrosine and tryptophan residues. ### Claim 19 An isolated FAD-binding type mutant glucose dehydrogenase comprising the amino acid sequence of SEQ ID NO: 7. ### Claim 20 (depends on 1) An isolated mutant glucose dehydrogenase complex comprising at least the mutant glucose dehydrogenase according to claim 1 and an electron transfer subunit. ### Claim 21 (depends on 20) The isolated glucose dehydrogenase complex according to claim 20, wherein the electron transfer subunit is cytochrome C. ### Claim 22 (depends on 1) A glucose assay kit comprising the mutant glucose dehydrogenase according to claim 1. ### Claim 23 (depends on 1) A glucose sensor comprising the mutant glucose dehydrogenase according to claim 1. ### Claim 24 (depends on 20) A glucose assay kit comprising the mutant glucose dehydrogenase complex according to claim 20. ### Claim 25 (depends on 20) A glucose sensor comprising the mutant glucose dehydrogenase complex according to claim 20.