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Method for producing oxidized gamma-glutamylcysteine and oxidized glutathione

A technology of glutamylcysteine ​​and glutathione, applied in biochemical equipment and methods, ligases, enzymes, etc.

Inactive Publication Date: 2017-05-10
KANEKA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Glutathione usually exists in the form of reduced GSH in the living body, and it is easy to mass-produce GSH using yeast, etc. In contrast, it has been difficult to efficiently mass-produce GSSG

Method used

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  • Method for producing oxidized gamma-glutamylcysteine and oxidized glutathione
  • Method for producing oxidized gamma-glutamylcysteine and oxidized glutathione
  • Method for producing oxidized gamma-glutamylcysteine and oxidized glutathione

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0079] The base sequence of the gene (DNA or RNA) encoding GSH I that can be used in the preparation of GSH I is not limited to the base sequence shown in SEQ ID NO: 1, and may be the amino acid sequence encoding the target GSH I, An appropriate base sequence corresponding to the type of host organism.

[0080]

[0081] Glutathione synthetase (GSH II) that can be used in the present invention has the ability to recognize γ-Glu-Cys as a substrate and catalyze the generation of γ-Glu-Cys-Gly by bonding it to Gly in the presence of ATP As long as the reactive enzyme has the activity, its source, structure, etc. are not particularly limited. In the present invention, this activity is referred to as glutathione synthetase activity. 1 U of this activity refers to the activity of producing 1 μmol of glutathione in 1 minute at 30° C., and was measured under the following measurement conditions. In the present invention, the novel knowledge that glutathione synthase also has the ab...

Embodiment

[0166]

[0167] Preparation of γ-glutamylcysteine ​​synthetase (GSH I) derived from Escherichia coli K12 strain

[0168]A DNA primer having a cleavage site and SD sequence bonded to the restriction enzyme SacI at the N-terminal portion of the GSH I gene (SEQ ID NO: 1) derived from the Escherichia coli K12 strain was prepared. A DNA primer (Primer-1: SEQ ID NO: 2) of the resulting sequence, a DNA primer (Primer-2: SEQ ID NO: 3) having a sequence in which a restriction enzyme KpnI cleavage site was bonded to the base sequence of the C-terminal part ). Using the DNA primers, the DNA between the sequences was amplified by PCR, thereby obtaining a DNA fragment containing the full length of the GSH I gene. At this time, the template used for PCR amplification is the genomic DNA of Escherichia coli K12 strain. The nucleotide sequence of the obtained DNA fragment was analyzed, and it was confirmed that it contained the full length of the GSH I gene (SEQ ID NO: 1). The obtained DN...

Embodiment 2

[0195] chemical formula 5

[0196]

[0197] Add glycine 0.19g (2.53mmol), glutathione synthase (GSH II) enzyme solution 2g prepared in experiment 2, PAP enzyme solution prepared in experiment 4 in the reaction solution after the reaction of the above-mentioned embodiment 1 for 22 hours 2g, start the reaction. At this time, the pH was adjusted to 7.5 with 0.2 g of a 15% by mass sodium hydroxide aqueous solution. The reaction liquid was analyzed after 1 hour of reaction, and production of oxidized glutathione was confirmed. The yield was 20 mol% after 1 hour of reaction and 86 mol% after 6 hours of reaction relative to the initial L-cystine. In this case, the yield of reduced glutathione after the 6-hour reaction was 1 mol% or less relative to the initial L-cystine.

[0198] It should be noted that the GSH II enzyme solution prepared in Experiment 2 and the PAP enzyme solution prepared in Experiment 4 contained ADK derived from Escherichia coli used as host cells, so no add...

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Abstract

The present invention addresses the problem of providing a method for producing oxidized glutathione (GSSG) and oxidized gamma-glutamylcysteine, which is a precursor thereof, by a simple process. This method for producing GSSG includes, as means for addressing the problem described above, a step A' for reacting L-cysteine and L-glutamic acid to generate oxidized gamma-glutamylcysteine, and a step B' for reacting oxidized gamma-glutamylcysteine and glycine to generate GSSG.

Description

technical field [0001] The present invention relates to a method for producing oxidized γ-glutamylcysteine. [0002] The present invention also relates to a method for producing oxidized glutathione. Background technique [0003] Glutathione is a peptide composed of three amino acids, L-cysteine, L-glutamic acid, and glycine. It exists not only in the human body, but also in other animals, plants, microorganisms, and other organisms. Oxygen elimination, detoxification, amino acid metabolism, etc., are important compounds for living organisms. [0004] Glutathione exists in the body as reduced glutathione (N-(N-γ-L-glutamyl-L-cysteinyl) glycine, hereinafter sometimes referred to as "GSH") and oxidized glutathione Glutathione (hereinafter sometimes referred to as “GSSG”) exists in any form of SH in which the thiol group of L-cysteine ​​residue is reduced, and the oxidized glutathione Type glutathione is a form in which the thiol group of the L-cysteine ​​residue is oxidized...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C12N9/00
CPCC12P21/02C12Y603/02003C12Y603/02002C12N9/00
Inventor 野尻增俊八十原良彦
Owner KANEKA CORP
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