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Mutant enzyme and application thereof

A technology for mutating enzymes and glucose oxidases, applied in the fields of enzymes, applications, oxidoreductases, etc.

Active Publication Date: 2012-11-07
AMANO ENZYME INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Under such measurement conditions, temperature correction is required and measurement errors are prone to occur

Method used

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  • Mutant enzyme and application thereof
  • Mutant enzyme and application thereof
  • Mutant enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0231] (Preparation method of mutant enzyme)

[0232]Another aspect of the present invention relates to a method for producing a mutant enzyme. In one embodiment of the method for preparing a mutant enzyme of the present invention, the mutant GO successfully obtained by the inventors of the present invention is prepared by genetic engineering. In the case of this mode, a nucleic acid encoding an arbitrary amino acid sequence of SEQ ID NO: 7 to 10 is prepared (step (I)). Here, the "nucleic acid encoding a specific amino acid sequence" is a nucleic acid that can be expressed to obtain a polypeptide having the amino acid sequence, and of course may be a nucleic acid composed of a base sequence corresponding to the amino acid sequence, or Redundant sequences (which may or may not encode amino acid sequences) may be added to such nucleic acids. In addition, codon degeneracy is taken into account. "Nucleic acid encoding any amino acid sequence of sequence number 7-10" refers to t...

Embodiment

[0243] With the goal of creating a highly practical GDH, we will search for new methods to replace conventional methods (methods of modifying existing GDHs, methods centered on screening). First, focus on glucose oxidase (GO), which does not have the problems unique to FAD-GDH (relatively high reactivity to xylose, high optimum temperature). It is known that the homology of the amino acid sequences of GO and FAD-GDH is relatively high. A novel method was adopted in which GDH activity is imparted to GO, that is, GDHylation of GO by modification, while paying attention to this homology.

[0244] 1. Comparison of GO and FAD-GDH

[0245] From the comparison of GO from Aspergillus niger and the FAD-GDH from Aspergillus oryzae, Aspergillus terreus, Penicillium italicum, and Penicillium lenticulae with known amino acid sequences, and the stereostructure of GO from Aspergillus niger with clear stereostructure See, amino acids near the active center of GO are conserved (high commonal...

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Abstract

Disclosed is a novel enzyme that exhibits glucose dehydrogenase activity. Furthermore, disclosed is a novel method pertaining to enzyme modification. The mutant enzyme comprises an amino acid sequence wherein one or at least two amino acids selected from the group consisting of (1)-(13) below are replaced with another amino acid in the amino acid sequence of a microorganism-derived glucose oxidase: (1) the amino acid corresponding to the amino acid at position 115 of the amino acid sequence indicated by SEQ ID NO: 1; (2) the amino acid corresponding to the amino acid at position 131 of the amino acid sequence indicated by SEQ ID NO: 1; (3) the amino acid corresponding to the amino acid at position 132 of the amino acid sequence indicated by SEQ ID NO: 1; (4) the amino acid corresponding to the amino acid at position 193 of the amino acid sequence indicated by SEQ ID NO: 1; (5) the amino acid corresponding to the amino acid at position 353 of the amino acid sequence indicated by SEQ ID NO: 1; (6) the amino acid corresponding to the amino acid at position 436 of the amino acid sequence indicated by SEQ ID NO: 1; (7) the amino acid corresponding to the amino acid at position 446 of the amino acid sequence indicated by SEQ ID NO: 1; (8) the amino acid corresponding to the amino acid at position 472 of the amino acid sequence indicated by SEQ ID NO: 1; (9) the amino acid corresponding to the amino acid at position 511 of the amino acid sequence indicated by SEQ ID NO: 1; (10) the amino acid corresponding to the amino acid at position 535 of the amino acid sequence indicated by SEQ ID NO: 1; (11) the amino acid corresponding to the amino acid at position 537 of the amino acid sequence indicated by SEQ ID NO: 1; (12) the amino acid corresponding to the amino acid at position 582 of the amino acid sequence indicated by SEQ ID NO: 1; (13) the amino acid corresponding to the amino acid at position 583 of the amino acid sequence indicated by SEQ ID NO: 1.

Description

technical field [0001] The invention relates to a mutant enzyme and a method for modifying the enzyme, and provides dehydrogenase-formed glucose oxidase and a preparation method thereof. This application claims priority based on Japanese Patent Application No. 2009-277096 filed on December 5, 2009, the entire content of which is incorporated by reference. Background technique [0002] Simple self-blood glucose monitors using electrochemical biosensors are widely used. Glucose oxidase (hereinafter abbreviated as "GO") and glucose dehydrogenase (hereinafter abbreviated as "GDH"), which are enzymes that use glucose as a substrate, are used in this biosensor. GO has the advantages of high specificity for glucose and excellent thermal stability, but on the other hand, it has been pointed out that the measurement using GO is easily affected by dissolved oxygen in the measurement sample, and dissolved oxygen affects the measurement results. . [0003] On the other hand, GDH usin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00C12N1/15C12N1/19C12N1/21C12N5/10C12N9/04C12N15/09C12Q1/26
CPCC12Y101/03004C07K2319/21C12Y101/9901C12N9/0006C12Q1/26C12Q1/54C12N15/52C12Q1/32
Inventor 西尾享一小池田聪
Owner AMANO ENZYME INC
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