Glucose Dehydrogenase/Cytochrome Fusion Protein

• US 20070267301A1
• Filed: 09/28/2004
• Published: 11/22/2007
• Est. Priority Date: 09/30/2003
• Status: Active Grant

• Current Assignee: ARKRAY, Inc
• Original Assignee: ARKRAY, Inc, Ultizyme International Ltd.
• Inventors: Sode, Koji
• Granted Patent: US 8,354,112 B2
• Time in Patent Office: Days
• US Class Current: 205/777.500
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• 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 1/006 for glucose
• CPC Class Codes: C12Y 101/05002 Quinoprotein glucose dehydrogenase (1.1.5.2)

Abstract

A fusion protein of pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) and a cytochrome is disclosed. PQQGDH is, for example, a water-soluble PQQGDH derived from Acinetobacter calcoaceticus. The cytochrome is, for example, an electron transfer domain of quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni. The fusion protein of the present invention shows intramolecular electron transfer from PQQ, a redox center, to the cytochrome, which allow construction of a direct electron transfer-type glucose sensor which requires no electron mediators.

Claims

1. A fusion protein of pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) and a cytochrome.

2. The fusion protein according to claim 1, wherein the PQQGDH is a water-soluble PQQGDH derived from Acinetobacter calcoaceticus.

3. The fusion protein according to claim 1 or 2, wherein the cytochrome has been fused to the C-terminal side of PQQGDH.

4. The fusion protein according to claim 1, wherein the cytochrome is cytochrome c or cytochrome B562.

5. The fusion protein according to claim 1, wherein the cytochrome is derived from a quinohemoprotein which is a protein having both PQQ and a heme in one molecule.

6. The fusion protein according to claim 1, wherein the cytochrome is derived from a quinohemoprotein alcohol dehydrogenase.

7. The fusion protein according to claim 1, wherein the cytochrome is derived from quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni.

8. The fusion protein according to claim 1, which is either (a) or (b):

(a) a protein comprising an amino acid sequence represented by SEQ ID NO;

2;

(b) a protein comprising an amino acid sequence in which one or more amino acid residues have been deleted, substituted or added in the amino acid sequence (a) and having a glucose dehydrogenase activity and an electron transfer ability.

9. A gene encoding the fusion protein according to claim 1.

10. A vector containing the gene according to claim 9.

11. A transformant containing the gene according to claim 9.

12. A transformant in which the gene according to claim 9 has been integrated into its main chromosome.

13. An enzyme electrode comprising the fusion protein according to claim 1 attached thereto.

14. A method of measuring the glucose concentration in a sample comprising the steps of:

contacting the sample with the enzyme electrode according to claim 13;

and

measuring electrons generated from the oxidation of glucose.

15. A glucose sensor comprising an enzyme electrode according to claim 13 as a working electrode.