FLAVIN-BOUND GLUCOSE DEHYDROGENASES, A METHOD FOR PRODUCING A FLAVIN-BOUND GLUCOSE DEHYDROGENASE, AND YEAST TRANSFORMANT USED FOR THE SAME

• US 20140057331A1
• Filed: 11/30/2011
• Published: 02/27/2014
• Est. Priority Date: 12/02/2010
• Status: Active Grant

• Current Assignee: Kikkoman Corporation
• Original Assignee: Kikkoman Corporation
• Inventors: Tajima, Ryoko, Ichiyanagi, Atsushi, Hirokawa, Kozo, Yuzuki, Masanobu
• Granted Patent: US 9,469,844 B2
• Time in Patent Office: Days
• US Class Current: 435/190
• 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 following properties (i) to (iv):

(i) action;

glucose dehydrogenase activity in the presence of an electron acceptor;

(ii) molecular weight;

approximately 80-kDa molecular weight of the polypeptide chain of the protein;

(iii) substrate specificity;

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

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

C. for 15 minutes.

2. The flavin-bound glucose dehydrogenase of claim 1, consisting of the amino acid sequence of SEQ ID NO:

1 or 3, an amino acid sequence at least 85% identical to said amino acid sequence, or an amino acid sequence having one or several amino acids deleted, substituted, or added in said amino acid sequence.

3. The flavin-bound glucose dehydrogenase of claim 1, characterized by that said flavin-bound GDH derives from a microorganism classified as Mucoromycotina, preferably Mucoromycetes, more preferably Mucorales, further preferably Mucoraceae and is expressed using yeast as a host.

8. The flavin-bound glucose dehydrogenase of claim 2, characterized by that said flavin-bound GDH derives from a microorganism classified as Mucoromycotina, preferably Mucoromycetes, more preferably Mucorales, further preferably Mucoraceae and is expressed using yeast as a host.

4. A yeast transformant obtained by introducing into yeast a gene encoding a flavin-bound glucose dehydrogenase, consisting of the amino acid sequence of SEQ ID NO:

1 or 3, an amino acid sequence at least 85% identical to said amino acid sequence, or an amino acid sequence having one or several amino acids deleted, substituted, or added in said amino acid sequence.

5. The yeast transformant of claim 4, characterized by that said yeast belongs to Zygosaccharomyces, Saccharomyces, Pichia, or Candida.

6. A method for producing a flavin-bound glucose dehydrogenase, characterized by culturing the yeast transformant of claim 4 and collecting a flavin-bound GDH from said culture.

9. A method for producing a flavin-bound glucose dehydrogenase, characterized by culturing the yeast transformant of claim 5 and collecting a flavin-bound GDH from said culture.

7. A method for producing a flavin-bound glucose dehydrogenase, characterized by culturing a yeast transformant carrying DNA which encodes a protein having flavin-bound GDH enzyme activity and encoding a flavin-bound glucose dehydrogenase gene, consisting of any DNA selected from the group consisting of the following (A) to (E):

(A) DNA encoding the amino acid sequence of SEQ ID NO;

1;

(B) DNA consisting of the base sequence of SEQ ID NO;

2;

(C) DNA encoding the amino acid sequence of SEQ ID NO;

3;

(D) DNA consisting of the base sequence of SEQ ID NO;

4; and

(E) DNA having a base sequence at least 85% identical to the base sequence of SEQ ID NO;

2 or 4, and

collecting a flavin-bound glucose dehydrogenase from said culture.