E. COLI TRANSFORMANT, METHOD FOR PRODUCING FLAVIN-BOUND GLUCOSE DEHYDROGENASE USING THE SAME, AND MUTANT FLAVIN-BOUND GLUCOSE DEHYDROGENASES

• US 20130309750A1
• Filed: 11/30/2011
• Published: 11/21/2013
• Est. Priority Date: 12/01/2010
• Status: Active Grant

• Current Assignee: Kikkoman Corporation
• Original Assignee: Kikkoman Corporation
• Inventors: Yoshihara, Eriko, Tajima, Ryoko, Ichiyanagi, Atsushi, Hirokawa, Kozo
• Granted Patent: US 9,238,802 B2
• Time in Patent Office: Days
• US Class Current: 435/190
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)

Abstract

A flavin-bound glucose dehydrogenase (FAD-GDH) with high substrate specificity for D-glucose. A gene encoding a mutant FAD-GDH with its N-terminal region, containing an amino acid sequence corresponding to MKITAAIITVATAFASFASA that exists in the N-terminal region, deleted from the amino acid sequence of a wild-type FAD-GDH derived from Mucor is introduced into E. coli to obtain an E. coli transformant. Subsequently, this E. coli transformant is cultured to obtain an FAD-GDH with a specific N-terminal region deleted. The transformant allows the production of a large amount of GDH in a short time as compared with the original microorganism. An FAD-GDH that 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. An E. coli transformant obtained by introducing into Escherichia coli a gene encoding a mutant flavin-bound glucose dehydrogenase lacking the N-terminal region, comprising an amino acid sequence corresponding to MKITAAIITVATAFASFASA that exists in the N-terminal region, from the amino acid sequence of wild-type flavin-bound glucose dehydrogenase derived from a microorganism classified as Mucoromycotina, preferably Mucoromycetes, more preferably Mucorales, further preferably Mucoraceae.

3. A method for producing a flavin-bound glucose dehydrogenase, characterized by culturing the E. coli transformant of claim 1 and collecting a flavin-bound glucose dehydrogenase from said culture.

2. An E. coli transformant obtained by introducing into Escherichia coli a gene encoding a mutant flavin-bound glucose dehydrogenase lacking the N-terminal region, comprising an amino acid sequence corresponding to MKITAAIITVATAFASFASA that exists in the N-terminal region, from the amino acid sequence of 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. A method for producing flavin-bound glucose dehydrogenase, characterized by culturing E. coli transformant of claim 2 and collecting a flavin-bound glucose dehydrogenase from said culture.

4. A flavin-bound glucose dehydrogenase lacking the N-terminal region, comprising an amino acid sequence corresponding to MKITAAIITVATAFASFASA that exists in the N-terminal region, from the amino acid sequence of 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.