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Flavin adenine dinucleotide-binding glucose dehydrogenase

A glucose dehydrogenase and flavin adenine technology, applied in the field of glucose dehydrogenase, can solve the problems of difficult extraction and separation operations, low glucose selectivity, and easy dissociation of PQQ by enzymes

Inactive Publication Date: 2009-06-10
AMANO ENZYME INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PQQ-GDH has the following problems: (1) PQQ is easily dissociated by enzymes, (2) has low selectivity to glucose, and (3) is difficult to extract and separate because it is generally present in membrane fractions Wait

Method used

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  • Flavin adenine dinucleotide-binding glucose dehydrogenase
  • Flavin adenine dinucleotide-binding glucose dehydrogenase
  • Flavin adenine dinucleotide-binding glucose dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0122] 1. Screening of FAD-GDH-producing bacteria

[0123] (1-1) Culture of Aspergillus oryzae

[0124] Seven strains of Aspergillus oryzae (BB-56, IAM2603, IAM2628, IAM2683, IAM2736, IAM2706, NBRC30113) were cultured by the following method.

[0125] (1-1-1) Pre-culture

[0126] 0.2% (w / v) yeast extract (Becton, Dichinson), 1.0% (w / v) soy peptone (DMV), 2.0% (w / v) glucose were dispensed into a 300 mL conical flask (Wako Pure Chemical Industries, Ltd.), 0.1% (w / v) KH 2 PO 4 (Wako Pure Chemical Industries, Ltd.) and 0.05% (w / v) MgSO 4 ·7H 2 50 mL of a medium consisting of O (pH 5.7) (SIGMA-ALDRICH Japan) was sterilized for 20 minutes at 121° C. and 0.12 MPa. After cooling, each strain of Aspergillus oryzae was inoculated with a 5×5 mm square slant, and pre-cultured at 30° C. and 200 rpm for 3 days.

[0127] (1-1-2) Formal training

[0128] 15.0% (w / v) glucose, 3.0% (w / v) Meast P1G (ASAHI FOOD & HEALTHCARE company), 6.0% (w / v) soy peptone, 0.3% ( w / v)KH 2 PO 4 , 0.2%(...

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Abstract

The object is to provide a novel enzyme which enables to determine the glucose level more accurately, a bacterium capable of producing the enzyme, and use of the enzyme. Disclosed is a flavin adenine dinucleotide-binding glucose dehydrogenase having the following properties (1) to (3):(1) the enzyme has an activity of catalyzing a reaction for oxidizing a hydroxyl group in glucose in the presence of an electron acceptor to produce glucono-delta-lactone; (2) the enzyme has a molecular weight of about 100 kDa as measured by SDS-polyacrylamide gel electrophoresis or about 400 kDa as measured by gel filtration chromatography; and (3) the enzyme is less reactive to maltose, D-fructose, D-mannose and D-galactose. Also disclosed is a microorganism Aspergillus oryzae which can produce the enzyme. Further disclosed are a glucose determination method, a reagent for the determination of glucose, and a kit for the determination of glucose, each utilizing the enzyme.

Description

technical field [0001] The present invention relates to a coenzyme-binding glucose dehydrogenase and its use. The present invention specifically relates to a glucose dehydrogenase using flavin adenine dinucleotide as a coenzyme, a bacterium for producing the enzyme, a method for producing the enzyme, a method for measuring glucose using the enzyme, and the like. Background technique [0002] In recent years, a simple self-glucose meter using an electrochemical biosensor has been widely used. A biosensor is formed by forming electrodes and an enzyme reaction layer on an insulating substrate. Examples of enzymes that can be used there include glucose dehydrogenase (GDH), glucose oxidase (GO), and the like. It has been pointed out that the method using GO is susceptible to the influence of dissolved oxygen in the measurement sample, and that the dissolved oxygen affects the measurement result. [0003] On the other hand, GDH (PQQ-GDH) using pyrroloquinoline quinone (PQQ) as ...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N1/14C12Q1/32C12R1/69G01N21/75
CPCC12Q1/54C12R1/69C12N9/0006C12Q1/32C12Y101/9901C12R2001/69C12N1/145
Inventor 田中宪彰结城健介
Owner AMANO ENZYME INC
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