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Mutant glucose dehydrogenase

A glucose dehydrogenase and glucose technology, applied in the direction of oxidoreductase, biochemical equipment and methods, applications, etc., can solve problems such as inability to measure blood sugar levels

Active Publication Date: 2012-05-16
ARKRAY INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wild-type CyGDH and PQQGDH have the following disadvantages: Since they react not only with glucose but also with maltose, it is impossible to measure accurate blood sugar levels when the concentration of maltose in the patient's blood is high
However, it cannot be said that it is a substance that has a complete effect of avoiding the influence of maltose.

Method used

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  • Mutant glucose dehydrogenase
  • Mutant glucose dehydrogenase
  • Mutant glucose dehydrogenase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Plasmid expressing GDH or CyGDH of Burkholderia cepacia

[0083] Plasmids expressing the α subunit and γ subunit of GDH were prepared as plasmids expressing GDH of Burkholderia cepacia, and plasmids expressing α subunit, β subunit, and γ subunit were prepared as plasmids expressing CyGDH.

[0084] Plasmids expressing the α subunit and γ subunit of GDH

[0085] The plasmid pTrc99A / γ+α described in WO02 / 036779 (corresponding to EP1331272A1, US2004023330A1, CN1484703A) was used as a plasmid expressing the α subunit and the γ subunit. This plasmid is a DNA fragment isolated from the chromosomal DNA of Burkholderia cepacia KS1 strain (FERM BP-7306), which continuously contains the GDH γ subunit structural gene and α subunit structural gene, and is inserted as the cloning site of the vector pTrc99A Point the NcoI / HindIII in the resulting plasmid. The GDHγα gene in this plasmid is controlled by the trc promoter. pTrc99A / γ+α retains the ampicillin resistance gene...

Embodiment 2

[0103] Example 2 Substrate interaction by introducing mutations into the CyGDHα subunit gene site exploration

[0104] (1) Introduce mutations to 326, 365 and 472

[0105] On the GDHα subunit gene contained in pTrc99Aγαβ obtained in Example 1, the 326-position serine residue, the 365-position serine residue, and the 472-position alanine residue of the α subunit encoding the gene Variations are introduced by substitution of other amino acid residues.

[0106] Specifically, using a commercially available site-specific mutation introduction kit (Stratagenc, QuikChangell Site-Directed Mutagenesis Kit), the GDHα subunit gene contained in the plasmids pTrc99A / γ+α and pTrc99Aγαβ described in Example 1 The codon for serine at position 326 (TCG), the codon for serine at position 365 (TCG), and the codon for alanine at position 472 (GCG) were substituted with codons for other amino acids.

[0107] The sequences of the forward primer and the reverse primer used for the above amino ...

Embodiment 3

[0121] Example 3 Analysis of the substrate specificity of variant GDH

[0122] Using the mutant GDH expression plasmid obtained in Example 2, a mutant GDH was produced and substrate specificity was studied.

[0123] (1) training

[0124] For the Escherichia coli DH5α strain into which the mutation was introduced, 2 ml each of LB medium (containing 50 μg / ml ampicillin and 30 μg / ml kanamycin) was used and cultured overnight at 37° C. in an L-shaped tube with shaking. These culture solutions were inoculated into a 500 ml slanted flask containing 150 ml of LB medium (containing 50 μg / ml ampicillin and 30 μg / ml kanamycin), and cultured with shaking at 37°C. After 3 hours from the start of the culture, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.1 mM, and cultured for another 2 hours.

[0125] (2) Preparation of crude enzyme samples

[0126] Collect the thalli from the above cultured culture solution, after washing, suspend the thalli in 1...

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Abstract

A mutant glucose dehydrogenase having an amino acid sequence at least 80% identical to SEQ ID NO:3 and having glucose dehydrogenase activity, wherein amino acid residues corresponding to positions 326, 365 and 472 of said amino acid sequence are replaced with glutamine, tyrosine and tyrosine, respectively, and wherein said mutant glucose dehydrogenase shows an improved substrate specificity to glucose and a reduced reactivity to disaccharides.

Description

technical field [0001] The present invention relates to mutant glucose dehydrogenases with improved substrate specificity. More specifically, the present invention relates to a glucose dehydrogenase comprising a mutant α subunit that constitutes cytochrome C-containing glucose dehydrogenase (hereinafter also referred to as CyGDH) and a gene thereof. ) into the α-subunit amino acid residues that introduce variation. The glucose dehydrogenase of the present invention can be applied to glucose sensors, glucose detection kits, and the like, and is useful in the fields of biochemistry, clinical fields, and the like. Background technique [0002] Currently, wild-type CyGDH and PQQGDH with pyrroloquinoline quinone as coenzyme are used in blood glucose self-test sensors. Wild-type CyGDH and PQQGDH have the disadvantage that they react not only with glucose but also with maltose, and therefore cannot measure accurate blood sugar levels when the concentration of maltose in the blood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12N15/53C12N1/15C12N1/19C12N1/21C12Q1/54C12Q1/32C12Q1/02
CPCC12N9/0006C12Q1/006
Inventor 早出广司小岛胜博
Owner ARKRAY INC
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