Glucose dehydrogenase

Patent metadata

Field	Value
Publication number	US-9657325-B2
Application number	US-201615134684-A
Country	US
Kind code	B2
Filing date	Apr 21, 2016
Priority date	Oct 21, 2013
Publication date	May 23, 2017
Grant date	May 23, 2017

Abstract

Provided is a flavin adenine dinucleotide-dependent glucose dehydrogenase comprising a polypeptide having an amino acid sequence with 78% or more identity to the amino acid sequence of SEQ ID NO: 3, and having glucose dehydrogenase activity.

First claim

The invention claimed is:

1. A flavin adenine dinucleotide-dependent glucose dehydrogenase comprising a polypeptide having an amino acid sequence with 85% or more identity to the amino acid sequence of SEQ ID NO: 3, and having glucose dehydrogenase activity, wherein the polypeptide does not comprise the amino acid sequence of SEQ ID NO: 3.

2. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 , which has the following characteristics (A) to (E):
(A) Action: the flavin adenine dinucleotide-dependent glucose dehydrogenase catalyzes a reaction in which D-glucose is oxidized in the presence of an electron acceptor to produce D-glucono-δ-lactone;
(B) Molecular weight: the molecular weight of the polypeptide chain portion of the protein measured by SDS-polyacrylamide electrophoresis is 65000;
(C) Thermal stability: the residual activity after treatment at 60° C. for 15 minutes is 85% or more, the residual activity after treatment at 65° C. for 15 minutes is 50% or more, and the residual activity after treatment at 70° C. for 15 minutes is 10% or more;
(D) Optimum reaction pH: 7.0; and
(E) Substrate specificity:
the reactivity to maltose is 2% or less based on the reactivity to D-glucose taken as 100%,
the reactivity to D-galactose is 2% or less based on the reactivity to D-glucose taken as 100%, and
the reactivity to D-xylose is 10% or less based on the reactivity to D-glucose taken as 100%.

3. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 , wherein the polypeptide has an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 3.

4. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 , wherein the polypeptide has an amino acid sequence with 95% or more identity to the amino acid sequence of SEQ ID NO: 3.

5. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 , wherein the polypeptide has an amino acid sequence with 98% or more identity to the amino acid sequence of SEQ ID NO: 3.

6. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 1 , wherein the polypeptide has an amino acid sequence with 99% or more identity to the amino acid sequence of SEQ ID NO: 3.

7. An electrode comprising a flavin adenine dinucleotide-dependent glucose dehydrogenase comprising a polypeptide having an amino acid sequence with 85% or more identity to the amino acid sequence of SEQ ID NO: 3, and having glucose dehydrogenase activity, wherein the flavin-binding glucose dehydrogenase is immobilized onto the electrode.

8. The electrode according to claim 7 , which has the following characteristics (A) to (E):
(A) Action: the flavin adenine dinucleotide-dependent glucose dehydrogenase catalyzes a reaction in which D-glucose is oxidized in the presence of an electron acceptor to produce D-glucono-δ-lactone;
(B) Molecular weight: the molecular weight of the polypeptide chain portion of the protein measured by SDS-polyacrylamide electrophoresis is 65000;
(C) Thermal stability: the residual activity after treatment at 60° C. for 15 minutes is 85% or more, the residual activity after treatment at 65° C. for 15 minutes is 50% or more, and the residual activity after treatment at 70° C. for 15 minutes is 10% or more;
(D) Optimum reaction pH: 7.0; and
(E) Substrate specificity:
the reactivity to maltose is 2% or less based on the reactivity to D-glucose taken as 100%,
the reactivity to D-galactose is 2% or less based on the reactivity to D-glucose taken as 100%, and
the reactivity to D-xylose is 10% or less based on the reactivity to D-glucose taken as 100%.

9. The electrode according to claim 7 , wherein the polypeptide has an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 3.

10. The electrode according to claim 7 , wherein the polypeptide has an amino acid sequence with 95% or more identity to the amino acid sequence of SEQ ID NO: 3.

11. The electrode according to claim 7 , wherein the polypeptide has an amino acid sequence with 98% or more identity to the amino acid sequence of SEQ ID NO: 3.

12. The electrode according to claim 7 , wherein the polypeptide has an amino acid sequence with 99% or more identity to the amino acid sequence of SEQ ID NO: 3.

13. A flavin adenine dinucleotide-dependent glucose dehydrogenase comprising a polypeptide having an amino acid sequence with 85% or more identity to the amino acid sequence of SEQ ID NO: 3, and having glucose dehydrogenase activity, wherein the flavin adenine dinucleotide-dependent glucose dehydrogenase is freeze-dried.

14. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 13 , which has the following characteristics (A) to (E):
(A) Action: the flavin adenine dinucleotide-dependent glucose dehydrogenase catalyzes a reaction in which D-glucose is oxidized in the presence of an electron acceptor to produce D-glucono-δ-lactone;
(B) Molecular weight: the molecular weight of the polypeptide chain portion of the protein measured by SDS-polyacrylamide electrophoresis is 65000;
(C) Thermal stability: the residual activity after treatment at 60° C. for 15 minutes is 85% or more, the residual activity after treatment at 65° C. for 15 minutes is 50% or more, and the residual activity after treatment at 70° C. for 15 minutes is 10% or more;
(D) Optimum reaction pH: 7.0; and
(E) Substrate specificity:
the reactivity to maltose is 2% or less based on the reactivity to D-glucose taken as 100%,
the reactivity to D-galactose is 2% or less based on the reactivity to D-glucose taken as 100%, and
the reactivity to D-xylose is 10% or less based on the reactivity to D-glucose taken as 100%.

15. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 13 , wherein the polypeptide has an amino acid sequence with 90% or more identity to the amino acid sequence of SEQ ID NO: 3.

16. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 13 , wherein the polypeptide has an amino acid sequence with 95% or more identity to the amino acid sequence of SEQ ID NO: 3.

17. The flavin adenine dinucleotide-dependent glucose dehydrogenase according to claim 13 , wherein the polypeptide has an amino acid sequence with 98% or more identity to the amino acid sequence of SEQ ID NO: 3.

Assignees

• Toyo Boseki

Inventors

• Aiba Hiroshi
• Sumida Yosuke
• Yamazaki Yasuhiro
• Utashima Yuu
• Kishimoto Takahide

Classifications

• C12Y101/05

  with a quinone or similar compound as acceptor (1.1.5) · CPC title

• C12Q1/006 Primary

  for glucose · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• G01N27/3271

  Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood (amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001) · CPC title