FAD-conjugated glucose dehydrogenase gene

• US 8,882,978 B2
• Filed: 06/18/2013
• Issued: 11/11/2014
• Est. Priority Date: 06/29/2006
• Status: Expired due to Fees

• Current Assignee: PHC Corporation (PHC Holdings Corporation)
• Original Assignee: Ikeda Food Research Co., Ltd.
• Inventors: Yada, Takako, Miyamoto, Koji, Honda, Michinari
• Primary Examiner(s): FRONDA, CHRISTIAN L
• Application Number:
  US13/920,445
• Publication Number:
  US 20140027280A1
• Time in Patent Office: 511 Days
• Field of Search: None
• US Class Current: 204/403.14
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: C12Y 101/9901 Glucose dehydrogenase (acceptor) (1.1.99.10)
• CPC Class Codes: G01N 2333/902 Oxidoreductases (1.)
• CPC Class Codes: G01N 2333/904 acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: G01N 27/301 Reference electrodes
• CPC Class Codes: G01N 27/302 pH sensitive, e.g. quinhydron, antimony or hydrogen electrodes ion selective electrodes G01N27/333, glass electrodes G01N27/36
• CPC Class Codes: G01N 27/3271 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 Class Codes: G01N 33/66 involving blood sugars, e.g. galactose
• CPC Class Codes: Y10T 29/49117 Conductor or circuit manufacturing

Abstract

Disclosed herein are a gene (polynucleotide) encoding an FAD-conjugated glucose dehydrogenase which can be characterized by reactivity to glucose, thermal stability, substrate-recognition performance, and low activity for maltose; a process for the production of the enzyme using a transformant cell transfected with the gene; a method for the determination of glucose; a reagent composition for use in the determination of glucose; and a biosensor for use in the determination of glucose. An embodiment is a polynucleotide encoding an FAD-conjugated glucose dehydrogenase, comprising a polypeptide whose amino acid sequence comprises X1-X2-X3-X4-X5-X6, wherein X1 and X2 independently represent an aliphatic amino acid residue; X3 and X6 independently represent a branched amino acid residue; and X4 and X5 independently represent a heterocyclic amino acid residue or an aromatic amino acid residue.

Claims

1. A biosensor comprising a recombinant FAD-conjugated glucose dehydrogenase, wherein the recombinant FAD-conjugated glucose dehydrogenase is selected from the group consisting of:

(a) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO;

1; and

(b) a polypeptide which comprises an amino acid sequence having a homology of 90% or more to the amino acid sequence (a) and has an FAD-conjugated glucose dehydrogenase activity,

and wherein the biosensor is capable of detecting glucose.

2. The biosensor of claim 1, wherein the biosensor comprises a reaction layer comprising the recombinant FAD-conjugated glucose dehydrogenase.

3. The biosensor of claim 2, wherein the reaction layer further comprises a hydrophilic polymer and an electron acceptor.

4. The biosensor of claim 3, wherein the electron receptor is an osmium compound, a quinone compound or a ferricyan compound.

5. The biosensor of claim 3, wherein the reaction layer is in contact with an electrode system comprising a working electrode, a counter electrode, and a reference electrode, each on an insulating base plate.

6. The biosensor of claim 1, which is capable of detecting glucose by detection of an oxidation current.

7. The biosensor of claim 1, which is capable of detecting glucose by a change in color intensity.

8. The biosensor of claim 1, which is capable of detecting glucose by a pH change.

9. A method of making a biosensor, comprising:

forming an electrode system comprising a working electrode, a counter electrode, and a reference electrode, each on an insulating base plate, and

forming a reaction layer on the electrode system comprising a hydrophilic polymer, the recombinant FAD-conjugated glucose dehydrogenase of claim 1, and an electron receptor.

10. The process of making a biosensor of claim 9, wherein the electron receptor is an osmium compound, a quinone compound or a ferricyan compound.