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Bifunctional glutathione synthetase and method for producing glutathione by using same

A technology of glutathione and synthetic enzymes, applied in the direction of microorganism-based methods, biochemical equipment and methods, enzymes, etc., to achieve the effect of improving production levels and improving production levels

Inactive Publication Date: 2011-05-25
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

γ-GCS-GS derived from Streptococcus agalactiae is not sensitive to the feedback inhibition of GSH, and GSH has no inhibitory effect on the activity of γ-GCS and GS, which is different from previous reports of glutathione synthase in organisms of

Method used

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  • Bifunctional glutathione synthetase and method for producing glutathione by using same
  • Bifunctional glutathione synthetase and method for producing glutathione by using same
  • Bifunctional glutathione synthetase and method for producing glutathione by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 utilizes actinobacillus succinic acid to synthesize glutathione

[0042] 1. Culture of Actinobacillus succinogenes

[0043] Put Actinobacillus succinogenes 130Z (CICC 11014) from the glycerol tube stored at -20°C into the fermentation medium at an inoculum size of 1%. Na 2 HPO 4 12H 2 O 0.5g, NaH 2 PO 4 2H 2 O 0.5g, pH 6.5, 37°C, 220rpm, shaking culture for 24 hours.

[0044] 2. Cell pretreatment

[0045] The cultivated Actinobacillus succinici culture solution was centrifuged at 8000rpm for 5min to collect the cells, washed and centrifuged three times with 0.05mol / L, pH7.0 phosphate buffer under the same conditions. Cells were frozen at -20°C for 2 hours and permeabilized.

[0046] 3. Glutathione synthesis reaction

[0047] Weigh 2g of permeabilized wet bacteria and add to 20mL reaction solution, the reaction solution is 0.2mol / L (pH7.0) potassium phosphate buffer solution, containing 40mmol / L L-glutamic acid, L-cysteine 20mmol / L, glycine 40mmol...

Embodiment 2

[0048] Embodiment 2 utilizes bacillus cereus to synthesize glutathione

[0049] Put Bacillus cereus (CGMCC 1.932) from a glycerol tube stored at -20°C into the fermentation medium at an inoculum size of 1%. 220rpm, shaking culture for 12 hours, permeabilization treatment and glutathione synthesis reaction were carried out on Bacillus cereus according to the method described in Example 1, after 8 hours of reaction, glutathione could accumulate 163mg / L.

Embodiment 3

[0050] Example 3 Construction of Recombinant Escherichia coli JM109 (pTrc99a-gshFap)

[0051] 1. Extraction of Actinobacillus pleuropneumonia genomic DNA

[0052] Put Actinobacillus pleuropneumonia (CVCC 259) from the glycerol tube stored at -20°C into the fermentation medium at an inoculum size of 1%. The specific components are (1L contains): peptone 10.0g, yeast extract 5g, NaCl 10g, NAD 0.2 g. pH 7.4, 37°C, static culture for 24 hours, centrifuge at 13400×g, 4°C for 2 minutes, and collect the bacteria. The whole genome DNA of Actinobacillus pleuropneumonia was extracted by BBI Whole Genome DNA Extraction Kit.

[0053] 2. Cloning of the gshF gene of Actinobacillus pleuropneumoniae

[0054] The upstream primer is: 5'-GCGC GGATCC ATGAAATTACAACAAC-3', the underlined part is the restriction site of BamH I.

[0055] The downstream primer is 5'-GAT GTC GAC TTAAGGCAGTTCTGGGAA-3', the underlined part is the Sal I restriction site.

[0056] Using the whole genome of Actinoba...

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Abstract

The invention discloses a method for producing glutathione by using bifunctional glutathione synthetase contained in actinobacillus pleuropneumonia, actinobacillus succinogenes, bacillus cereus, streptococcus sanguis, streptococcus gordonii, streptococcus uberis and streptococcus thermophilus. The method comprises heterologous expression by using the seven microbes or enzymes of the microbes in other microbes. The glutathione synthesized by using the method has the structure of natural glutathione, and has high response rate and high output and yield of the glutathione.

Description

technical field [0001] The present invention relates to a kind of method for producing glutathione, specifically, is to utilize a kind of γ-glutamylcysteine ​​synthetase (γ-GCS) and glutathione synthetase (GS) two kinds Functional enzymatic method for producing glutathione. Background technique [0002] Glutathione (GSH) is a thiol-containing compound with important physiological functions, which is composed of L-glutamic acid (L-Glu), L-cysteine ​​(L-Cys) and glycine ( Gly) is a tripeptide consisting of three amino acids. Glutathione is widely distributed in animals, plants, and microorganisms. It participates in the synthesis of proteins and ribonucleic acids, the transport of oxygen and nutrients, the maintenance of endogenous enzymes, the tricarboxylic acid cycle and sugar metabolism in the body, and the removal of glutathione. Functions such as excessive free radicals in the body, have a wide range of applications. Clinically, glutathione is used for anti-radiation, ...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12P21/02C12R1/01C12R1/085C12R1/46C12R1/19C12R1/125C12R1/38C12R1/865C12R1/84C12R1/78C12R1/685C12R1/69
Inventor 叶勤李志敏李娓
Owner EAST CHINA UNIV OF SCI & TECH
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