Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

• US 8,691,547 B2
• Filed: 03/27/2006
• Issued: 04/08/2014
• 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 HOSSAIN
• Application Number:
  US11/886,885
• Publication Number:
  US 20120122130A1
• Time in Patent Office: 2,934 Days
• Field of Search: None
• US Class Current: 435/190
• 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 cDNA encoding a coenzyme-linked glucose dehydrogenase, comprising the sequences set forth in SEQ ID NOs. 5 to 7, the coenzyme-linked glucose dehydrogenase having the following properties (a) to (d):

(a) having 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) utilizing a FAD as a coenzyme;

(c) catalyzing a reaction in which a hydroxyl group at a 1-position of glucose is oxidized and the glucose is converted to glucono-δ

-lactone; and

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

5. A vector comprising the cDNA set forth in claim 1.

6. An isolated transformed host cell comprising the vector set forth in claim 5.

7. A method for producing a coenzyme-linked glucose dehydrogenase, comprising culturing the isolated transformed host cell set forth in claim 6, thereby expressing the coenzyme-linked glucose dehydrogenase having a glucose dehydrogenation activity, and collecting the coenzyme-linked glucose dehydrogenase having a glucose dehydrogenation activity from the cultivated product.

10. The cDNA of claim 1, wherein the cDNA is derived from an Aspergillus terreus (A. terreus).

2. A cDNA encoding a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD), the GLD comprising sequences set forth in SEQ ID NOs. 8 to 12, the coenzyme-linked glucose dehydrogenase having following properties (a) to (c):

(a) having 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 polyacrylamide gel electrophoresis (SDS-PAGE);

(b) catalyzing a reaction in which a hydroxyl group at a 1-position of glucose is oxidized and the glucose is converted to glucono-δ

-lactone; and

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

11. The cDNA of claim 2, wherein the cDNA is derived from an Aspergillus terreus (A. terreus).

3. A cDNA encoding a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) which catalyzes dehydrogenation of glucose in presence of an electron acceptor and having an activity toward maltose of 5% or less with respect to an activity toward glucose,

wherein the GLD has an amino acid sequence with a homology of at least [[60%]] 90% to the amino acid sequence set forth in SEQ ID NO:

2 or the amino acid sequence set forth in amino acid 20 to 592 of SEQ ID NO;

2, and the cDNA is derived from an Aspergillus terreus (A. terreus).

4. A cDNA encoding a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) which catalyzes dehydrogenation of glucose in presence of an electron acceptor and having an activity toward maltose of 5% or less with respect to an activity toward glucose, the cDNA comprising the nucleotide sequence set forth in SEQ ID NO. 1.

8. A cDNA encoding a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) which catalyzes dehydrogenation of glucose in presence of an electron acceptor and having an activity toward maltose of 5% or less with respect to an activity toward glucose,

wherein the GLD has an amino acid sequence with a homology of at least 90% to the amino acid sequence set forth in SEQ ID NO:

2 or the amino acid sequence set forth in amino acid 20 to 592 of SEQ ID NO;

2, the GLD is derived from a microorganism of an Aspergillus genus, Penicillium genus, or Ganoderma genus, and the GLD comprises at least one partial amino acid sequence selected from sequences of the coenzyme-linked glucose dehydrogenase set forth in SEQ ID NOs. 8 to 12.

12. A vector comprising the cDNA set forth in claim 8.

14. An isolated transformed host cell comprising the vector set forth in claim 12.

16. A method for producing a coenzyme-linked glucose dehydrogenase, comprising cultivating the isolated transformed host cell of claim 14, thereby expressing the coenzyme-linked glucose dehydrogenase having a glucose dehydrogenation activity, and collecting the coenzyme-linked glucose dehydrogenase having a glucose dehydrogenation activity from the cultivated product.

9. A cDNA encoding a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) catalyzing dehydrogenation of glucose in presence of an electron acceptor and having an activity toward maltose of 5% or less with respect to an activity toward glucose,

wherein the GLD has an amino acid sequence comprising sequences of the coenzyme-linked glucose dehydrogenase set forth in SEQ ID NOs. 8 to 12.

13. A vector comprising the cDNA set forth in claim 9.

15. An isolated transformed host cell comprising the vector set forth in claim 13.

17. A method for producing a coenzyme-linked glucose dehydrogenase, comprising cultivating the isolated transformed host cell of claim 15, thereby expressing the coenzyme-linked glucose dehydrogenase having a glucose dehydrogenation activity, and collecting the coenzyme-linked glucose dehydrogenase having a glucose dehydrogenation activity from the cultivated product.