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Glucose dehydrogenase

A technology of glucose dehydrogenase and glucose concentration, applied in the direction of enzyme, enzyme stabilization, oxidoreductase, etc., to achieve the effect of high-efficiency production

Active Publication Date: 2013-06-19
TOYOBO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

but there is no information yet about which part of the sequence encodes glucose dehydrogenase

Method used

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no. 1 Embodiment approach

[0197] In the first embodiment, the present invention is a eukaryotic-derived glucose dehydrogenase that catalyzes the following reaction.

[0198] D-glucose + electron transport substance (oxidized form) →

[0199] D-glucono-δ-lactone + electron transport substance (reduced form)

[0200] The present invention is characterized by high heat resistance, which is different from the known eukaryotic-derived GDH in this point. Compared with the residual activity of Aspergillus terreus-derived GDH, which is excellent in stability among known eukaryotic-derived glucose dehydrogenases, after heating at 55°C for 15 minutes (residual activity when the activity before heating treatment is 100%) rate) was 60%, whereas the residual activity of the GDH of the present invention after heat treatment at 55°C for 15 minutes was 90% or more. Furthermore, the GDH of the present invention preferably maintains GDH activity even after being treated at 60°C for 15 minutes, more preferably the resi...

no. 2 Embodiment approach

[0247] In order to achieve the above object, the present inventors discovered a gene DNA presumed to be glucose dehydrogenase using the NCBI database.

[0248] The DNA (gene) containing the nucleotide sequence described in SEQ ID NO: 7 is predicted from the NCBI database, includes DNA (gene) encoding glucose dehydrogenase derived from Aspergillus oryzae RIB40 strain, and is a genomic gene sequence without removing introns.

[0249] The DNA (gene) containing the base sequence described in SEQ ID NO: 8 is a genome gene sequence in which introns are removed from SEQ ID NO: 7.

[0250] The gene encoding a protein having the amino acid sequence described in SEQ ID NO: 9 represents the entire sequence of the glucose dehydrogenase gene predicted from the NCBI database.

[0251] The present inventors predicted that DNA (gene) encoding a protein having glucose dehydrogenase activity derived from Aspergillus oryzae can be easily identified from Non-Patent Documents 1 to 4, NCBI database...

no. 3 Embodiment approach

[0289] The method for producing GDH, one of the embodiments of the present invention, is characterized in that, in the method for recombinantly producing GDH from filamentous bacteria, by introducing a mutation into the signal peptide sequence present in its N-terminal region, compared with before introducing the mutation Increased expression of the target enzyme.

[0290] As a result of the inventors' diligent efforts to obtain a large yield of GDH protein, in order to achieve the above object, first of all, using the genome information of Aspergillus oryzae in the above invention, the gene DNA that is the source of the change was found to be glucose dehydrogenase. gene DNA.

[0291] Using the NCBI database, the present inventors discovered gene DNA presumed to be FAD-dependent glucose dehydrogenase.

[0292] The present inventors expected that identification of DNA (gene) encoding a protein having glucose dehydrogenase activity can be easily performed from Non-Patent Docume...

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Abstract

The invention provides a glucose dehydrogenase which is excellent in thermostability and substrate specificity and is not affected by dissolved oxygen. More particularly, the invention relates to the glucose dehydrogenase which is derived from a eukaryote and is characterized by maintaining 90% or more activity after a heat treatment at 55°C for 15 minutes in a liquid state compared to the activity before the heat treatment. The invention efficiently produces a glucose dehydrogenase derived from Aspergillus oryzae and provides a more practical glucose dehydrogenase. The invention provides a method for producing a filamentous Aspergillus oryzae and provides a more practical glucose dehydrogenase. The invention provides a method for producing a filamentous fungus-derived FAD-GDH with excellent substrate specificity by high expression in a recombinant, a FAD-GDH protein obtained by the method and a glucose assay reagent utilizing the protein. The invention relates to a method of improving the stability of a composition containing a soluble glucose dehydrogenase (GDH). As the soluble GDH, the filamentous fungus-derived FAD-dependent GDH is preferable, and particularly, the best effect has been observed in an A. oryzae -derived FAD-GDH or an A. terreus -derived FAD-GDH.

Description

technical field [0001] The present invention relates to a novel glucose dehydrogenase (hereinafter, sometimes abbreviated as "GDH") usable in a glucose measurement reagent and a glucose sensor. [0002] The present invention relates to a gene encoding glucose dehydrogenase derived from Aspergillus oryzae and a method for producing the glucose dehydrogenase by gene recombination. [0003] The present invention relates to a method for highly expressing glucose dehydrogenase derived from filamentous fungus using a recombinant. [0004] The present invention relates to a method of increasing the stability of a composition comprising a coenzyme-requiring soluble glucose dehydrogenase and a composition using the method. More specifically, the present invention relates to a method for improving the stability of a composition comprising soluble glucose dehydrogenase using a flavin compound or the like as a coenzyme, and a composition using the method. Background technique [0005]...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N1/15C12N1/19C12M1/40C12N1/21C12N9/04C12Q1/32G01N33/66
CPCC12Q1/54C12Y101/9901C12N9/0006C12N9/96G01N27/3271C12N1/20C12Q1/32
Inventor 北林雅夫辻裕二相场洋志川南裕岸本高英西矢芳昭
Owner TOYOBO CO LTD
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