Flavin-bound glucose dehydrogenases, a method for producing a flavin-bound glucose dehydrogenase, and yeast transformant used for the same

• US 9,469,844 B2
• Filed: 11/30/2011
• Issued: 10/18/2016
• Est. Priority Date: 12/02/2010
• Status: Active Grant

• Current Assignee: Kikkoman Corporation
• Original Assignee: Kikkoman Corporation
• Inventors: Yuzuki, Masanobu, Tajima, Ryoko, Ichiyanagi, Atsushi, Hirokawa, Kozo
• Primary Examiner(s): PAK, YONG D
• Application Number:
  US13/991,087
• Publication Number:
  US 20140057331A1
• Time in Patent Office: 1,784 Days
• US Class Current: 1/1
• CPC Class Codes: C12N 1/16 Yeasts; Culture media therefor
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12Y 101/9901 Glucose dehydrogenase (acceptor) (1.1.99.10)

Abstract

A heat-resistant flavin-bound glucose dehydrogenase having a high substrate specificity for D-glucose, an method for producing the same, and a transformant used for the same. A flavin-bound glucose dehydrogenase gene encoding a flavin-bound glucose dehydrogenase derived from Mucor is introduced into yeast, Zygosaccharomyces, to obtain a transformant. Subsequently, the yeast transformant is cultured to obtain a flavin-bound glucose dehydrogenase from the culture. The heat-resistant flavin-bound glucose dehydrogenase is less susceptible to the effects of dissolved oxygen and allows accurate measurement of glucose even in the presence of sugar compounds other than glucose in a sample.

Claims

1. A flavin-bound glucose dehydrogenase having the amino acid sequence of SEQ ID NO:

3, and having the following properties (i) to (iv);

(i) action;

glucose dehydrogenase activity in the presence of an electron acceptor;

(ii) molecular weight;

the molecular weight of the polypeptide chain portion of the protein is approximately 80 kDa;

(iii) substrate specificity;

lower reactivity to maltose, D-galactose, and D-xylose than that to D-glucose; and

(iv) 50% or above residual activity after heat treatment at 50°

C. for 15 minutes, wherein the flavin-bound glucose dehydrogenase is produced by a yeast transformant.

3. The flavin-bound glucose dehydrogenase of claim 1, wherein said flavin-bound glucose dehydrogenase is obtained from a microorganism classified as Mucoromycotina.

5. The flavin-bound glucose dehydrogenase of claim 1, wherein said glucose dehydrogenase is obtained from a microorganism classified as Mucoromycetes.

6. The flavin-bound glucose dehydrogenase of claim 1, wherein said glucose dehydrogenase is obtained from a microorganism classified as Mucorales.

7. The flavin-bound glucose dehydrogenase of claim 1, wherein said glucose dehydrogenase is obtained from a microorganism classified as Mucoraceae.

2. A flavin-bound glucose dehydrogenase having an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

3, and having the following properties (i) to (iv);

(i) action;

glucose dehydrogenase activity in the presence of an electron acceptor;

(ii) molecular weight;

the molecular weight of the polypeptide chain portion of the protein is approximately 80 kDa;

(iii) substrate specificity;

lower reactivity to maltose, D-galactose, and D-xylose than that to D-glucose; and

(iv) 50% or above residual activity after heat treatment at 50°

C. for 15 minutes, wherein the flavin-bound glucose dehydrogenase is produced by a yeast transformant.

4. The flavin-bound glucose dehydrogenase of claim 2, wherein said flavin-bound glucose dehydrogenase is obtained from a microorganism classified as Mucoromycotina.

8. The flavin-bound glucose dehydrogenase of claim 2, wherein said glucose dehydrogenase is obtained from a microorganism classified as Mucoromycetes.

9. The flavin-bound glucose dehydrogenase of claim 2, wherein said glucose dehydrogenase is obtained from a microorganism classified as Mucorales.

10. The flavin-bound glucose dehydrogenase of claim 2, wherein said glucose dehydrogenase is obtained from a microorganism classified as Mucoraceae.