Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

• US 10,626,434 B2
• Filed: 09/28/2018
• Issued: 04/21/2020
• 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): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US16/145,174
• Publication Number:
  US 20190040441A1
• Time in Patent Office: 571 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) transforming a cell with a vector comprising a polynucleotide encoding a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) so as to recombinantly express the GLD,

(ii) obtaining GLD secreted by the transformed cell, 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 GLD and an electron acceptor,

wherein;

(a) the 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, and

(b) the GLD has an amino acid sequence 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.

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 GLD.

4. The method of claim 1, wherein a N-type sugar chain or an O-type sugar chain is bound to the 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 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 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 SEQ ID NO:

9 is RAGKALGGTSTINGMAYTRAEDVQID.

9. The method of claim 1, further comprising cultivating the transformed cell.