Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

• US 10,883,133 B2
• Filed: 09/28/2018
• Issued: 01/05/2021
• 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:
  US16/145,191
• Publication Number:
  US 20190048385A1
• Time in Patent Office: 830 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 biosensor for measuring glucose in a sample liquid comprising:

(i) contacting a transformed host cell with a liquid culture medium, wherein the transformed host cell comprises a recombinant expression vector comprising a polynucleotide encoding an Aspergillus species flavin adenine dinucleotide-linked glucose dehydrogenase (FAD-GLD) which comprises a sequence set forth in SEQ ID NO;

8, a sequence set forth in SEQ ID NO;

9, a sequence set forth in SEQ ID NO;

10, a sequence set forth in SEQ ID NO;

11, and a sequence set forth in SEQ ID NO;

12, so as to recombinantly express the FAD-GLD and obtaining recombinant FAD-GLD from the transformed host cell;

(ii) purifying the recombinant FAD-GLD; and

(iii) forming a biosensor comprising an electrode system and an enzyme reaction layer on an electrode of the electrode system, the enzyme reaction layer comprising the recombinant FAD-GLD and an electron acceptor, wherein the recombinant FAD-GLD has an activity toward glucose of catalyzing dehydrogenation of glucose in the presence of the electron acceptor and has an activity toward maltose of 5% or less with respect to the activity toward glucose.

2. The method of claim 1, wherein the electron acceptor is a ferricyanide compound.

3. The method of claim 1, wherein mannose is contained in the recombinant FAD-GLD.

4. The method of claim 1, wherein a N-type sugar chain or an O-type sugar chain is bound to the recombinant FAD-GLD.

5. The method of claim 1, wherein the biosensor can measure glucose concentrations of one or more of 10 mM, 20 mM, 30 mM and 40 mM.

6. The method of claim 1, wherein the recombinant FAD-GLD has a total content of galactose, glucose, mannose, and arabinose of 10 μ

g or less per μ

g of protein.

7. The method of claim 1, wherein the recombinant FAD-GLD has a total content of galactose, glucose, mannose, and arabinose of 2 μ

g or less per μ

g of protein.

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

9. The method of claim 8, wherein the recombinant expression vector comprises a promoter.

10. The method of claim 9, wherein the promoter is an amylase promoter derived from Aspergillus oryzae.

11. The method of claim 1, wherein SEQ ID NO:

9 is RAGKALGGTSTINGMAYTRAEDVQID.

12. The method of claim 8, wherein the transformed fungal host cell is a filamentous fungus.

13. The method of claim 8, wherein the transformed fungal host cell is an Aspergillus oryzae.

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

15. The method of claim 8, wherein the polynucleotide encoding the Aspergillus species FAD-GLD also encodes a signal peptide.

16. The method of claim 12, wherein obtaining the recombinant FAD-GLD comprises collecting recombinant FAD-GLD secreted outward of a fungus body.

17. The method of claim 1, wherein the recombinant FAD-GLD activity toward maltose is 2% or less with respect to its activity toward glucose.

18. The method of claim 1, wherein SEQ ID NO:

8 is GGGTSGLVVANRLSE.

19. The method of claim 1, wherein SEQ ID NO:

10 is SPLILELSGVGNP.

20. The method of claim 1, wherein SEQ ID NO:

12 is LLPFARGNIHI.

21. The method of claim 1, wherein purifying the recombinant FAD-GLD comprises salting-out, dialysis, ultrafiltration, ion-exchange chromatography and/or hydrophobic chromatography.