Ikeda Food Research Co., Ltd.

Panasonic Healthcare Holdings Co., Ltd.

Michinari Honda

Ryo Takenaka

Takako Yada

Koji Miyamoto

Hironori Omura

Hirokazu Sanada

Takafumi Takumi

Fuminao Kobayashi

Tsuyoshi Kameda

Sayaka Taki

Takahiro Fujii

Emi Watanabe

The present invention addresses the problem of providing: a glucose dehydrogenase; a polynucleotide encoding the enzyme; a method for producing the enzyme; a method for measuring glucose using the enzyme; a measurement reagent composition; and a biosensor. The present invention pertains to a protein that has any of amino acid sequences (a), (b) and (c) and has a glucose dehydrogenase activity, etc.: (a) an amino acid sequence represented by SEQ ID NO: 3, 6, 15 or 16; (b) an amino acid sequence derived from the amino acid sequence represented by SEQ ID NO: 3, 6, 15 or 16 by deleting, substituting or adding 1-3 amino acids; and (c) an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 3 or 15 or 85% or more identity to the amino acid sequence represented by SEQ ID NO: 6 or 16.

Flavin-conjugated glucose dehydrogenase

[Problem] [Solution to Problem to problem]

Modified glucose dehydrogenase

An object of the present invention is to provide: a novel gene (polynucleotide) encoding an FAD-conjugated glucose dehydrogenase having excellent properties that it has excellent reactivity to glucose, excellent thermal stability, and excellent substrate-recognition performance and also has a low activity for maltose; a process for the production of the enzyme using a transformant cell transfected with the gene; and a method for the determination of glucose, a reagent composition for use in the determination of glucose, a biosensor for use in the determination of glucose and others, each characterized by using the enzyme obtained. The invention relates to a polynucleotide encoding an FAD-conjugated glucose dehydrogenase, comprising a polypeptide containing an amino acid sequence: X1-X2-X3-X4-X5-X6 (wherein X1 and X2 independently represent an aliphatic amino acid residue; X3 and X6 independently represent a branched amino acid residue; and X4 and X5 independently represent a heterocyclic amino acid residue or an aromatic amino acid residue); and others.

FAD-conjugated glucose dehydrogenase gene

Biosensor comprising glucose dehydrogenase

Disclosed herein are a gene (polynucleotide) encoding an FAD-conjugated glucose dehydrogenase which can be characterized by reactivity to glucose, thermal stability, substrate-recognition performance, and low activity for maltose; a process for the production of the enzyme using a transformant cell transfected with the gene; a method for the determination of glucose; a reagent composition for use in the determination of glucose; and a biosensor for use in the determination of glucose. An embodiment is a polynucleotide encoding an FAD-conjugated glucose dehydrogenase, comprising a polypeptide whose amino acid sequence comprises X1-X2-X3-X4-X5-X6, wherein X1 and X2 independently represent an aliphatic amino acid residue; X3 and X6 independently represent a branched amino acid residue; and X4 and X5 independently represent a heterocyclic amino acid residue or an aromatic amino acid residue.

The purpose of the present invention is to provide an FAD-conjugated glucose dehydrogenase that is hard to be inhibited by the inhibitors such as 1,10-phenanthroline. The present invention relates to a modified glucose dehydrogenase (GLD), comprising an amino acid sequence of a wild-type FAD-conjugated glucose dehydrogenase (GLD) represented by SEQ ID NO: 1 having a substitution of at least one amino acid residue selected from the group consisting of amino acid residues at positions 298, 338, 340, 341, 343, 352, 354, 424, 426, 431 and 432, wherein the modified GLD has a reduced susceptibility to an inhibitor, as compared with the wild-type GLD, especially to said modified GLD, which has 40% or more of a relative activity when determined in a system wherein the inhibitor coexists at a final concentration of 1 mM based on an enzymatic activity when determined in a system wherein the inhibitor does not coexist.

The problem to be resolved is to provide an electron mediator and a fusion body with high affinity with an enzyme, a measuring method using extracellular secretion type cytochrome and an enzyme, an electrode, and a sensor. The present invention relates to an electron mediator for glucose oxidoreductase comprising extracellular secretion type cytochrome, a fusion body in which the electron mediator is fused with glucose oxidoreductase, a composition for glucose measurement including the electron mediator or fusion body, a gene encoding a new extracellular secretion type cytochrome, and a measurement method using extracellular secretion type cytochrome and an enzyme, an electrode, and a sensor.

Protein electron mediator

The invention provides a flavin-binding glucose dehydrogenase exhibiting reduced fluctuation of activity depending on temperature environment, and a method for measuring glucose concentration using the flavin-binding glucose dehydrogenase. The flavin-binding glucose dehydrogenase has the following properties (1) to (3): (1) activity: which exhibits glucose dehydrogenase activity in the presence of an electron acceptor; (2) substrate specificity: which exhibits an activity of 10% or less against maltose, D-galactose, D-fructose, sorbitol, lactose and sucrose when the activity against D-glucose is defined as 100%; and (3) temperature characteristics: which exhibits lower fluctuation of activity in a wide temperature range of 10 to 50&#xb0; C.

Flavin-binding glucose dehydrogenase

[Object] To provide an FAD-conjugated glucose dehydrogenase having a higher specificity for glucose. [Means for Resolution] A modified glucose dehydrogenase (GLD) which includes a substitution of at least one amino acid residue selected from the group consisting of amino acid residues at positions 37, 69, 72, 73, 76, 78, 102, 217, 228, 240, 356, 407, 424, 437, 527, and 530 in an amino acid sequence of a wild-type FAD-conjugated glucose dehydrogenase (GLD) represented by SEQ ID NO: 1, and has a decreased ratio of activity for xylose/activity for glucose as compared with the wild-type GLD; a polynucleotide encoding the modified GLD; a recombinant vector containing the polynucleotide; a transformed cell produced by using the recombinant vector; etc.

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Hiroshima, Japan

Michinari Honda