Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

• US 9,957,543 B2
• Filed: 03/23/2016
• Issued: 05/01/2018
• Est. Priority Date: 03/25/2005
• Status: Expired due to Fees

• 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 HOSSAIN
• Application Number:
  US15/079,002
• Publication Number:
  US 20160273018A1
• Time in Patent Office: 769 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 biosensor for measuring glucose in a sample liquid, comprising

an electrode system and an enzyme reaction layer on an electrode of the electrode system,

the enzyme reaction layer comprising an electron acceptor and a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD), 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;

(b) the GLD has an amino acid sequence comprising the sequences set forth in SEQ ID NOs. 8 to 12; and

(c) the total content of galactose, glucose, mannose, and arabinose contained in the GLD is 80 μ

g or less per μ

g of protein.

2. The biosensor of claim 1, wherein the total content of galactose, glucose, mannose, and arabinose contained in the GLD is 10 μ

g or less per μ

g of protein.

3. The biosensor of claim 1, wherein the electron acceptor comprises ferricyanide in an amount so that the ferricyanide concentration is within a range of 2 mM to 500 mM after addition of the sample liquid to the biosensor.

4. The biosensor of claim 1, wherein:

(i) the enzyme reaction layer further comprises a hydrophilic polymer, and

(ii) the enzyme reaction layer is configured so that dissolution of the enzyme reaction layer by the sample liquid results in reduction of the electron acceptor by a reaction of the GLD and glucose and electrochemical oxidation of the electron acceptor, and produces a measurable oxidation current by which the concentration of glucose in the sample liquid may be determined.

5. A biosensor for measuring glucose in a sample liquid, comprising

an electrode system and an enzyme reaction layer on an electrode of the electrode system,

the enzyme reaction layer comprising a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD),

wherein the GLD comprises the sequences set forth in SEQ ID NOs. 8 to 12, and has the following properties (a) to (d):

(a) a subunit molecular weight of approximately 63 kDa, wherein the term “

subunit molecular weight”

refers to a subunit molecular weight determined by subjecting a coenzyme-linked glucose dehydrogenase without sugar chains to polyacrylamide gel electrophoresis (SDS-PAGE);

(b) an activity toward glucose of catalytically oxidizing a hydroxyl group at the 1-position of the glucose so that the glucose is converted to glucono-δ

-lactone;

(c) an activity toward maltose of 5% or less with respect to the activity toward glucose; and

(d) the total content of galactose, glucose, mannose, and arabinose contained in the GLD is 80 μ

g or less per μ

g of protein.

6. The biosensor of claim 5, wherein the total content of galactose, glucose, mannose, and arabinose contained in the GLD is 10 μ

g or less per μ

g of protein.

7. The biosensor of claim 5, wherein the enzyme reaction layer comprises ferricyanide in an amount so that the ferricyanide concentration is within a range of 2 mM to 500 mM after addition of the sample liquid to the biosensor.

8. The biosensor of claim 5, wherein the enzyme reaction layer:

(i) further comprises a hydrophilic polymer and an electron acceptor, and

(ii) the enzyme reaction layer is configured so that dissolution of the enzyme reaction layer by the sample liquid results in reduction of the electron acceptor by a reaction of the GLD and glucose and electrochemical oxidation of the electron acceptor, and produces a measurable oxidation current by which the concentration of glucose in the sample liquid may be determined.

9. A biosensor for measuring glucose in a sample liquid, comprising:

an electrode system and an enzyme reaction layer on an electrode of the electrode system,

the enzyme reaction layer comprising a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD), wherein

the nucleic acid encoding the GLD comprises the sequences set forth in SEQ ID NOs. 5 to 7, and

the total content of galactose, glucose, mannose, and arabinose contained in the GLD is 80 μ

g or less per μ

g of protein.

10. The biosensor of claim 9, wherein the GLD has the following properties (a) to (d):

(a) a subunit molecular weight of approximately 63 kDa, wherein the subunit molecular weight is determined by subjecting a coenzyme-linked glucose dehydrogenase without sugar chains to polyacrylamide gel electrophoresis (SDS-PAGE);

(b) an ability to use a FAD as a coenzyme;

(c) an activity toward glucose of catalytically oxidizing a hydroxyl group at the 1-position of the glucose so that the glucose is converted to glucono-δ

-lactone; and

(d) an activity toward maltose of 5% or less with respect to the activity toward glucose.

11. The biosensor of claim 9, wherein the total content of galactose, glucose, mannose, and arabinose contained in the GLD is 10 μ

g or less per μ

g of protein.

12. The biosensor of claim 9, wherein the enzyme reaction layer comprises ferricyanide in an amount so that the ferricyanide concentration is within a range of 2 mM to 500 mM after addition of the sample liquid to the biosensor.

13. The biosensor of claim 9, wherein:

(i) the enzyme reaction layer further comprises a hydrophilic polymer and an electron acceptor, and

(ii) the enzyme reaction layer is configured so that dissolution of the enzyme reaction layer by the sample liquid results in reduction of the electron acceptor by a reaction of the GLD and glucose and electrochemical oxidation of the electron acceptor, and produces a measurable oxidation current by which the concentration of glucose in the sample liquid may be determined.