# US 10,669,565 B2 · Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

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- Number: US 10,669,565 B2
- Document ID: US10669565B2
- Status: Granted
- Kind: B2
- Application number: 16/145,190
- Filed: 2018-09-28
- Published: 2019-02-07
- Granted: 2020-06-02
- Earliest claimed: 2005-03-25
- Assignees: Ikeda Food Research Co., Ltd.; PHC Corporation
- Applicants: Ikeda Food Research Co., Ltd.; PHC Corporation
- Inventors: Hironori Omura; Hirokazu Sanada; Takako Yada; Ayaka Atsumi; Tetsunari Morita; Emi Ishimaru
- Web page: https://patentgrep.com/patent/US10669565B2

## CPC

- C12Q 1/006: {for glucose}
- C12N 9/0004: {Oxidoreductases (1.)}
- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12Q 1/004: {mediator-assisted}
- C12Q 1/32: involving dehydrogenase
- C12Y 101/9901: Glucose dehydrogenase (acceptor) (1.1.99.10)
- 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)}
- C12Y 101/05: with a quinone or similar compound as acceptor (1.1.5)
- G01N 2333/904: acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 1

A method for producing a biosensor for measuring glucose in a sample liquid comprising:
(i) contacting a transformed fungal or yeast host cell with a liquid culture medium in a fermenter, wherein the transformed fungal or yeast host cell comprises a recombinant expression vector comprising a polynucleotide encoding a flavin adenine dinucleotide-linked glucose dehydrogenase (FAD-GLD) which comprises the sequences set forth in SEQ ID NOS:8 to 12, wherein SEQ ID NO:8 is GGGTSGLWANRLSE, SEQ ID NO:10 is SPLILELSGVGNP, and SEQ ID NO:12 is LLPFARGNIHI, and has the amino acid sequence of an Aspergillus FAD-GLD and wherein the FAD-GLD has an activity toward glucose of catalyzing dehydrogenation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or less with respect to the activity toward glucose;
(ii) culturing the transformed fungal or yeast host cell in the liquid culture medium in the fermenter and recombinantly expressing the FAD-GLD;
(iii) collecting the recombinantly-expressed FAD-GLD; and
(iv) 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 FAD-GLD and an electron acceptor.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

The method of claim 1, wherein the FAD-GLD has a total content of galactose, glucose, mannose, and arabinose of 10 μg or less per μg of protein.

### Claim 7 (depends on 1)

The method of claim 1, wherein the FAD-GLD has a total content of galactose, glucose, mannose, and arabinose of 2 μg or less per μg of protein.

### Claim 8 (depends on 1)

The method of claim 1, wherein collecting the FAD-GLD comprises collecting FAD-GLD secreted by the transformed fungal or yeast host cell.

### Claim 9 (depends on 1)

The method of claim 1, wherein SEQ ID NO:9 is RAGKALGGTSTINGMAYTRAEDVQID.

### Claim 10 (depends on 1)

The method of claim 1, wherein the recombinant expression vector of the transformed fungal or yeast host cell comprises cDNA sequences corresponding to one or more of a promoter, splicing region, and polyA-addition site.

### Claim 11 (depends on 1)

The method of claim 1, wherein the recombinant expression vector comprises a pKA1, pCDM8, pSVK3, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pYE82, pIND/V5-His, pFLAG-CMV-2, pEGFP-N1, or pEGFP-C1 expression vector.

### Claim 12 (depends on 1)

The method of claim 1, wherein collecting the FAD-GLD comprises isolating it from a cultivated liquid or culture medium composition containing the FAD-GLD secreted outward of a fungus body of the transformed fungal host cell.