Transformant transfected with flavin adenine dinucleotide-binding glucose dehydrogenase gene and method for producing flavin adenine dinucleotide-binding glucose dehydrogenase using the same

A technology of glucose dehydrogenase and flavin adenine, which is applied in biochemical equipment and methods, oxidoreductase, microbial measurement/inspection, etc. It can solve the problems of low glucose selectivity, difficult extraction and separation operations, and measurement of dissolved oxygen. The results have an impact, etc.

Inactive Publication Date: 2010-11-17
AMANO ENZYME INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The method using GO has been pointed out to have the following problems: It is easily affected by the dissolved oxygen in the measured sample, and dissolved oxygen has an influence on the measured results
However, in PQQ-GDH, there are the following problems: (1) PQQ is easily dissociated from the enzyme, (2) the selectivity for glucose is low, and (3) since it generally exists in the membrane fraction, it is accompanied by its extraction, Difficulties in separation operations, etc.
However, it is difficult to say that the production of these FAD-GDHs is sufficient for industrial use

Method used

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  • Transformant transfected with flavin adenine dinucleotide-binding glucose dehydrogenase gene and method for producing flavin adenine dinucleotide-binding glucose dehydrogenase using the same
  • Transformant transfected with flavin adenine dinucleotide-binding glucose dehydrogenase gene and method for producing flavin adenine dinucleotide-binding glucose dehydrogenase using the same
  • Transformant transfected with flavin adenine dinucleotide-binding glucose dehydrogenase gene and method for producing flavin adenine dinucleotide-binding glucose dehydrogenase using the same

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preparation example Construction

[0081] (Preparation method of FAD-GDH)

[0082] The second aspect of the present invention is to provide a method for producing FAD-GDH using the above transformant. In the production method of the present invention, first, the above-mentioned transformant is cultured under conditions that produce the protein encoded by the gene introduced therein (step (1)). The culture method and culture conditions are not particularly limited as long as the desired enzyme can be produced. That is, on the condition that the present enzyme is produced, the method and culture conditions for culturing the transformant to be used can be appropriately set. Hereinafter, as culture conditions, medium, culture temperature, and culture time are exemplified.

[0083] Any medium may be used as the medium in which the transformant to be used can grow. For example, carbon sources to which glucose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, etc. are added, as well...

Embodiment 1

[0093]

[0094]According to the following procedure, Aspergillus oryzae BB-56 was cultured to produce FAD-GDH. 50 mL of a pre-cultured medium having the following composition was dispensed into a 300-mL Erlenmeyer flask, and sterilized at 121° C. and 0.12 MPa for 20 minutes. After cooling the medium to room temperature, Aspergillus oryzae BB-56 was inoculated, and cultured at 30° C. and 200 rpm for 3 days.

[0095] (Composition of pre-culture medium)

[0096] Yeast extract (Becton, Dickinson Company) 0.2% (w / v)

[0097] Soy peptone (DMV company) 1.0% (w / v)

[0098] Glucose (Wako Pure Chemical Industries, Ltd.) 2.0% (w / v)

[0099] K H 2 PO 4 (Wako Pure Chemical Industries, Ltd.) 0.1% (w / v)

[0100] MgSO 4 ·7H 2 O (Sigma-Aldrich Japan Co.) 0.05% (w / v)

[0101] Medium pH5.7

[0102] Production of FAD-GDH was carried out in a medium for main culture comprising the following composition. 50 mL of this main culture medium was divided into Erlenmeyer flasks with a volume...

Embodiment 2

[0112]

[0113] Purification of FAD-GDH was performed under the following conditions. The culture solution obtained as described above was filtered through celite to remove bacterial cells and other solid components in the culture solution. The clarified liquid obtained by this operation was desalted and concentrated with an ultrafiltration membrane. The concentrated solution is salted out with 90% saturated ammonium sulfate, and the centrifuged supernatant is desalted and concentrated with an ultrafiltration membrane. The desalted and concentrated solution was used in CM Sepharose Fast Flow (Amersham Biosciences) parallelized with 10 mM McIlvaine buffer pH 5.5 to adsorb FAD-GDH on the column. After the column was washed with 10 mM McIlvaine buffer pH 5.5, FAD-GDH was eluted with 10 mM McIlvaine buffer pH 5.5 containing 0.1 M NaCl, and the active fraction of FAD-GDH was recovered. The recovered active fraction was concentrated with an ultrafiltration membrane, and the conc...

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Abstract

It is intended to highly efficiently produce a large amount of novel FAD-GDH capable of more accurately measuring a glucose level. A transformant transfected with a DNA encoding a flavin adenine dinucleotide-binding glucose dehydrogenase selected from the group consisting of (a) a DNA encoding an amino acid sequence represented by SEQ ID NO: 20; (b) a DNA composed of a base sequence represented by SEQ ID NO: 19; (c) a DNA having a base sequence homologous to the base sequence represented by SEQ ID NO: 19 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenaseactivity; (d) a DNA encoding an amino acid sequence represented by SEQ ID NO: 34; (e) a DNA composed of a base sequence represented by SEQ ID NO: 33; and (f) a DNA having a base sequence homologous to the base sequence represented by SEQ ID NO: 33 and encoding a protein having a flavin adenine dinucleotide-binding glucose dehydrogenase activity is provided. Further, a method for producing a flavin adenine dinucleotide-binding glucose dehydrogenase using the transformant is provided.

Description

technical field [0001] The present invention relates to a transformant introduced with a flavin adenine dinucleotide-binding glucose dehydrogenase (also referred to as "FAD-GDH" in this specification) gene, and a method for producing FAD-GDH using the transformant . Background technique [0002] In recent years, simple self-blood glucose measuring devices using electrochemical biosensors have been widely used. A biosensor is a sensor in which electrodes and an enzyme reaction layer are formed on an insulating substrate. Examples of the enzyme used here include glucose dehydrogenase (GDH), glucose oxidase (GO), and the like. The method using GO has been pointed out to be susceptible to the influence of dissolved oxygen in the measurement sample, and the dissolved oxygen has an influence on the measurement results. [0003] On the other hand, GDH (PQQ-GDH) using pyrroloquinoline quinone (PQQ) as a coenzyme is known as an enzyme that is not affected by dissolved oxygen and a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N1/15C12N9/04C12N15/09C12P21/02C12Q1/32C12Q1/54
CPCC12Q1/006C12Q1/54C12Q1/32C12N9/0006
Inventor 结城健介中西雄二
Owner AMANO ENZYME INC
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