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Method for production of glutathione or gamma-glutamylcysteine

A technology of glutamyl cysteine ​​and glutathione, applied in chemical instruments and methods, peptide sources, bacterial peptides, etc.

Active Publication Date: 2010-05-26
KYOWA HAKKO BIO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] As a method for producing γ-glutamylcysteine, a method for producing γ-glutamylcysteine ​​using a yeast mutant strain in which the glutathione synthase gene has been destroyed or weakened has been reported (Non-Patent Documents 16 to 18), but in this fermentation method γ-Glutamylcysteine ​​accumulates in the bacteria

Method used

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Examples

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Effect test

preparation example Construction

[0177] 2. Preparation of microorganisms used in the present invention

[0178] (1) Preparation of microorganisms with higher protein activity than parent strains with glutathione transport activity

[0179] Among the microorganisms in which the activity of the protein having glutathione transporting activity is higher than that of the parent strain, the microorganism having a specific activity higher than that of the protein having glutathione transporting activity of the parent strain can be obtained as follows: by encoding The DNA of the active protein is subjected to mutagenesis treatment using a mutagen in vitro or error-prone PCR (Error-prone PCR), etc., and a mutation is introduced into the DNA, and then a known method [Proc.Natl.Acad.Sci.USA ., 97 , 6640 (2000)], the mutant DNA was substituted for the DNA encoding a protein having glutathione transport activity before the introduction of the mutation, which existed on the chromosomal DNA of the parent strain, thereby p...

Embodiment 1

[0269] Construction of γ-glutamylcysteine ​​synthetase or glutathione synthetase expression plasmid

[0270] Using the Model 8905 DNA Synthesizer manufactured by Perseptive Biosystems, based on the base sequence shown in SEQ ID NO: 49, the code Escherichia coli The γ-glutamylcysteine ​​synthetase gene ( wxya gene), synthesized DNA having the nucleotide sequences shown in SEQ ID NO: 59 and 60, and based on the nucleotide sequence shown in SEQ ID NO: 50, encoding Escherichia coli The glutathione synthase gene ( wxya gene) to synthesize DNAs having the base sequences shown in SEQ ID NOs: 61 and 62.

[0271] by Escherichia coli The chromosomal DNA of the W3110 strain was used as a template, and PCR was performed using DNAs having the nucleotide sequences shown in SEQ ID NOs: 59 and 60, and SEQ ID NOs: 61 and 62, respectively, as primer sets. PCR was carried out as follows: prepare 0.1 μg of chromosomal DNA, 0.5 μmol / L of each primer, 2.5 units Pfu DNA polymerase, 4 μL ...

Embodiment 2

[0283] Construction of expression plasmids for genes encoding proteins with glutathione transport activity

[0284] (1) cydDC Construction of gene expression plasmids

[0285] Similar to the above-mentioned Example 1, using a DNA synthesizer, based on the base sequence shown in SEQ ID NO: 27 Escherichia coli of cyd gene and cydC gene (hereinafter referred to as cyd - cydC gene) to synthesize DNAs having the base sequences shown in SEQ ID NOs: 63 and 64, respectively.

[0286] by Escherichia coli PCR was performed using the chromosomal DNA of the W3110 strain as a template, and DNA having the nucleotide sequences shown in SEQ ID NOs: 63 and 64 as a primer set. PCR was carried out as follows: prepare 0.1 μg of chromosomal DNA, 0.5 μmol / L of each primer, 2.5 units PfuDNA polymerase, 4 μL Pfu DNA polymerase x10 buffer solution, 40 μL of 200 μmol / L reaction solution of each dNTP, 1 minute at 94°C, 2 minutes at 55°C, and 4 minutes at 72°C were repeated 30 times. ...

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Abstract

According to the present invention, the following process for producing glutathione or gamma-glutamylcysteine is provided. A process for producing glutathione or gamma-glutamylcysteine by culturing in a medium a microorganism with a higher activity of one of the following proteins having an activity to transport intracellular glutathione to the outside of cells, and a higher activity of a protein involved in glutathione or -glutamylcysteine biosynthesis, compared with that of the parent strain, forming and accumulating glutathione or -glutamylcysteine in the medium, and recovering the glutathione or -glutamylcysteine from the culture. [1] A protein having the amino acid sequence shown by any of SEQ ID NOs: 1 to 26 [2] A protein consisting of an amino acid sequence wherein one or more amino acids have been deleted, substituted or added in the amino acid sequence shown by any of SEQ ID NOs: 1 to 26, and having glutathione transporting activity [3] A protein having 80% or more homology to the amino acid sequence shown by any of SEQ ID NOs: 1 to 26, and having glutathione transporting activity.

Description

technical field [0001] The present invention relates to a method for producing glutathione and γ-glutamylcysteine. Background technique [0002] Glutathione (γ-L-glutamyl-L-cysteinyl-glycine) is known to be widely present in the living body, and it is known to have a detoxification action in the liver in addition to acting as a coenzyme. Therefore, glutathione is widely used as products, raw materials, or intermediates of pharmaceuticals, health foods, and cosmetics. [0003] In addition, γ-glutamylcysteine ​​(γ-L-glutamyl-L-cysteine) is an intermediate in the biosynthesis of glutathione as described above, and is expected to have One of the thiol compounds that have the same food flavor-improving effect as amino acid and are used as a therapeutic agent for lifestyle diseases and Alzheimer's disease. [0004] It is known that glutathione is passed in Escherichia coli ( Escherichia coli ), use γ-glutamylcysteine ​​synthetase (GSHI) to convert L-glutamic acid and L-cysteine...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N15/09
CPCC07K14/195C12P13/005C12P21/02
Inventor 田畑和彦米谷良之
Owner KYOWA HAKKO BIO CO LTD
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