Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

• US 10,738,341 B2
• Filed: 03/21/2018
• Issued: 08/11/2020
• Est. Priority Date: 03/25/2005
• Status: Active Grant

• Current Assignee: PHC Corporation (PHC Holdings Corporation)
• 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:
  US15/928,027
• Publication Number:
  US 20190177763A1
• Time in Patent Office: 874 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 glucose dehydrogenase (GLD) comprising:

(i) contacting a transformed host cell, which has been transformed with a recombinant expression vector comprising a polynucleotide encoding an Aspergillus species flavin adenine dinucleotide-linked glucose dehydrogenase comprising the sequences set forth in SEQ ID NOs. 8 to 12, with a liquid culture medium;

then

(ii) cultivating the host cell and recombinantly expressing the GLD;

and

(iii) obtaining recombinant GLD secreted from the host cell.

2. The method of claim 1, wherein the recombinant GLD has an activity toward maltose of 5% or less with respect to its activity toward glucose.

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

g or less per μ

g of protein.

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

g or less per μ

g of protein.

5. The method of claim 1, wherein the host cell is a eukaryotic host cell.

6. The method of claim 5, wherein the eukaryotic host cell transformed is a fungal cell.

7. The method of claim 5, wherein the eukaryotic host cell transformed is a yeast cell.

8. The method of claim 6, wherein the obtaining of the recombinant GLD comprises isolating the recombinant GLD from culture medium containing the recombinant GLD secreted outward of a fungus body.

9. The method of claim 1, wherein the Aspergillus species flavin adenine dinucleotide-linked glucose dehydrogenase is an Aspergillus oryzae species flavin adenine dinucleotide-linked glucose dehydrogenase.

10. The method of claim 6, wherein the fungal cell is an Aspergillus species cell.

11. The method of claim 10, wherein SEQ ID NO:

9 is RAGKALGGTSTINGMAYTRAEDVQID.

12. The method of claim 1, further comprising making a glucose biosensor by forming an enzyme reaction layer comprising the obtained recombinant GLD, which biosensor comprises an electrode system and the enzyme reaction layer on an electrode of the electrode system.

13. The method of claim 12, wherein the recombinant GLD has an activity toward maltose of 3% or less with respect to its activity toward glucose.

14. The method of claim 13, wherein the enzyme reaction layer comprises an electron acceptor.

15. The method of claim 13, wherein the enzyme reaction layer comprises a hydrophilic polymer.

16. The method of claim 12, wherein the host cell is a eukaryotic host cell.

17. The method of claim 16, wherein the eukaryotic host cell is a transformed fungal cell.

18. The method of claim 16, wherein the eukaryotic host cell is a transformed yeast cell.

19. The method of claim 17, wherein the obtaining of the recombinant GLD comprises isolating the recombinant GLD from culture medium containing the recombinant GLD secreted outward of a fungus body.

20. The method of claim 12, wherein the Aspergillus species flavin adenine dinucleotide-linked glucose dehydrogenase is an Aspergillus oryzae species flavin adenine dinucleotide-linked glucose dehydrogenase.

21. The method of claim 17, wherein the fungal cell is an Aspergillus species cell.

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

9 is RAGKALGGTSTINGMAYTRAEDVQID.