Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

• US 10,815,515 B2
• Filed: 09/28/2018
• Issued: 10/27/2020
• Est. Priority Date: 03/25/2005
• Status: Active Grant

• Current Assignee: Ikeda Food Research Co., Ltd.
• Original Assignee: Ikeda Food Research Co., Ltd.
• Inventors: Omura, Hironori, Sanada, Hirokazu, Yada, Takako, Atsumi, Ayaka, Morita, Tetsunari, Ishimaru, Emi
• Primary Examiner(s): Chowdhury, Iqbal H
• Application Number:
  US16/145,168
• Publication Number:
  US 20190040440A1
• Time in Patent Office: 760 Days
• Field of Search: None
• CPC Class Codes: C12N 9/0004 Oxidoreductases (1.)
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12Y 101/05 with a quinone or similar compound as acceptor (1.1.5)
• CPC Class Codes: C12Y 101/9901 Glucose dehydrogenase (acceptor) (1.1.99.10)
• CPC Class Codes: G01N 2333/904 acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: G01N 27/3271 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

Abstract

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.

Claims

1. A method for producing a recombinant flavin adenine dinucleotide-linked glucose dehydrogenase (GLD) comprising:

(i) contacting a transformed fungal or yeast host cell with a liquid culture medium in a fermenter, wherein the transformed host cell comprises a recombinant expression vector comprising a polynucleotide encoding a GLD comprising the sequences set forth in SEQ ID NOs. 8 to 12, wherein in SEQ ID NO;

8, Xaa at residue #2 is A, and/or Xaa at residue #8 is V, and/or Xaa at residue #9 is I;

wherein in SEQ ID NO;

10, Xaa at residue #3 is L, and/or Xaa at residue #4 is L, and/or Xaa at residue #10 is V; and

wherein in SEQ ID NO;

12, Xaa at residue #5 is S, and/or Xaa at residue #8 is N, and/or Xaa at residue #9 is I;

(ii) culturing the transformed fungal or yeast host cell and recombinantly expressing the GLD; and

(iii) collecting recombinant GLD secreted from the transformant transformed fungal or yeast host cell.

2. The method of claim 1, wherein the transformed host cell is a transformant fungal host cell.

3. The method of claim 1, wherein the transformed host cell is a transformant yeast host cell.

4. The method of claim 2, wherein the collecting of the recombinant GLD comprises isolating the recombinant GLD from culture medium containing the recombinant GLD secreted by a fungus body of the transformed fungal host cell.

5. The method of claim 1, wherein the recombinant GLD has the amino acid sequence of an Aspergillus species GLD.

6. The method of claim 5, wherein the Aspergillus species is an Aspergillus oryzae.

7. The method of claim 6, wherein SEQ ID NO:

9 is RAGKALGGTSTINGMAYTRAEDVQID.

8. The method of claim 4, wherein the recombinant expression vector comprises a polynucleotide encoding the GLD and a signal peptide of the GLD.

9. The method of claim 2, wherein the fungal host cell is an Aspergillus cell.