Modified flavine-adenine-dinucleotide-dependent glucose dehydrogenase

A glucose dehydrogenase and flavin adenine technology, applied in the field of modified glucose dehydrogenase, can solve the problem of destroying the accuracy of the measured value

Active Publication Date: 2009-09-16
TOYO TOYOBO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the activity of this enzyme on xylose is about 10% of the activity on glucose, the accuracy of the me

Method used

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  • Modified flavine-adenine-dinucleotide-dependent glucose dehydrogenase
  • Modified flavine-adenine-dinucleotide-dependent glucose dehydrogenase
  • Modified flavine-adenine-dinucleotide-dependent glucose dehydrogenase

Examples

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experiment example 1

[0165]In order to obtain the GDH gene derived from Aspergillus oryzae, attempts were made to purify GDH from the culture supernatant of Aspergillus oryzae TI strain in our company using various chromatography, but it was difficult to obtain high-purity GDH, and it had to be abandoned as one of the common methods for gene acquisition. One is the use of partial amino acid sequence cloning. However, we found that Penicillium lilacinoechinulatum NBRC6231 strain produced GDH, and successfully determined a partial amino acid sequence using the purified enzyme. Next, based on the determined amino acid sequence, a part of the GDH gene derived from Penicillium palpinatus NBRC6231 was obtained by using the PCR method, thereby determining the base sequence (1356bp). Finally, based on the base sequence, the Aspergillus oryzae GDH gene was deduced and obtained. This is briefly shown in the following .

[0166]

[0167] [Deduction of Glucose Dehydrogenase (hereinafter also referred to ...

experiment example 2

[0179] [Acquisition of glucose dehydrogenase gene derived from Aspergillus oryzae and introduction into Escherichia coli]

[0180] In order to obtain the AOGDH gene, mRNA was prepared from the cells of Aspergillus oryzae TI strain, and cDNA was synthesized. Two kinds of oligo DNAs shown in SEQ ID NOs: 39 and 40 were synthesized, and AOGDH gene was amplified using KOD Plus DNA polymerase (manufactured by Toyobo Co., Ltd.) using the prepared cDNA as a template. Treat the DNA fragment with restriction endonucleases NdeI and BamHI, and insert the NdeI-BamHI site into pBluescript (corresponding to the LacZ translation initiation codon atg, so that the atg of the Ndel recognition sequence is introduced into the NdeI site in a form corresponding to it) inserted into the recombinant plasmid. Using this recombinant plasmid, Escherichia coli DH5α (manufactured by Toyobo Co., Ltd.) was transformed. Using the transformant, the plasmid was extracted according to a conventional method, an...

experiment example 3

[0182] [Introduction of Aspergillus oryzae-derived glucose dehydrogenase (hereinafter referred to as AOGDH) gene into Escherichia coli]

[0183] In the case of using FAD-GDH after cleavage of the monopeptide as mFAD-GDH, a polypeptide in which only M is added to the N-terminus of mFAD-GDH so that the N-terminus of mFAD-GDH protrudes by 1 amino acid is expressed as S2 .

[0184] In S2, use the oligonucleotide of sequence number 43 as the N-terminal side primer, and carry out PCR in combination with the primer of sequence number 44, and use the same sequence to construct a recombinant plasmid with the DNA sequence (sequence number 1) encoding S2, similarly obtain transformants.

[0185] Wherein, it was confirmed by DNA sequencing that the plasmid having the DNA sequence of modified FAD-GDH has no sequence error.

[0186] The transformant was liquid-cultured for 1 to 2 days in a 10 L fermenter using a TB medium. After the bacterial cells in each culture period were collected, ...

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Abstract

Disclosed is a modified flavine-adenine-dinucleotide-dependent glucose dehydrogenase (FADGDH) having an improved thermal stability compared to a wild-type FADGDH, which is preferably derived from a eukaryote, more preferably derived from a filamentous fungus, still more preferably derived from a bacterium belonging to the genus Aspergillus. For example, the modified FADGDH has a primary structure having the substitution, deletion, insertion or addition of at least one amino acid residue in the amino acid sequence for FADGDH depicted in SEQ ID NO:2 or 46 in the Sequence Listing.

Description

technical field [0001] The present invention relates to a modified glucose dehydrogenase (GDH) with modified thermal stability. In addition, the present invention relates to a modified FADGDH-dependent glucose dehydrogenase using flavin adenine dinucleotide (FAD) as a coenzyme (FADGDH), method for its manufacture, and glucose sensor. Background technique [0002] Self-testing of blood sugar is very important for diabetics, since it is helpful for diabetics to know their own blood sugar level at ordinary times. Sensors used in self-measurement of blood glucose utilize enzymes that use glucose as a substrate. As an example of such an enzyme, glucose oxidase (EC 1.1.3.4) is mentioned, for example. Glucose oxidase has the advantages of high specificity for glucose and excellent thermostability, so it has been used as an enzyme for blood glucose sensors for a long time, and its first disclosure actually dates back to about 40 years ago. In a blood glucose sensor using glucose ...

Claims

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Application Information

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IPC IPC(8): C12N15/00C12N1/15C12N1/19C12N1/21C12N5/10C12N9/04C12Q1/32G01N27/327G01N27/416
Inventor 川南裕辻裕二北林雅夫西矢芳昭
Owner TOYO TOYOBO CO LTD
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