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Bifunctional glutathione synthetase and method for producing glutathione by using same

A glutathione and synthetase technology, applied in the direction of microbial-based methods, biochemical equipment and methods, bulk chemical production, etc., to achieve the effect of improving production levels and improving production levels

Inactive Publication Date: 2013-04-10
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] Example 1 Synthesis of glutathione using Actinobacillus succinate

[0042] 1. Cultivation of Actinobacillus succinogenes

[0043] Connect Actinobacillus succinogenes 130Z (CICC 11014) stored at -20°C into the fermentation medium at a 1% inoculum. The specific ingredients are (contained in 1L): 5g xylose, 5g yeast extract, 5g corn steep liquor, Na 2 HPO 4 ·12H 2 O 0.5g, NaH 2 PO 4 ·2H 2 O 0.5g, pH 6.5, 37°C, 220 rpm, shaking culture for 24 hours.

[0044] 2. Cell pretreatment

[0045] The cultured Actinobacillus succinate culture solution was centrifuged at 8000 rpm for 5 min to collect the cells, washed and centrifuged three times with 0.05 mol / L, pH 7.0 phosphate buffer under the same conditions. The cells were frozen at -20°C for 2 hours for permeabilization.

[0046] 3. Glutathione synthesis reaction

[0047] Weigh 2g of the permeabilized wet bacteria into 20mL reaction solution, the reaction solution is 0.2mol / L (pH7.0) potassium phosphate buffer, containing 40mmol / L L-gluta...

Embodiment 2

[0048] Example 2 Synthesis of Glutathione by Bacillus cereus

[0049] The glycerol tube of Bacillus cereus (CGMCC 1.932) stored at -20°C was inserted into the fermentation medium at an inoculum of 1%. The specific ingredients (contained in 1L): tryptone 10g, yeast extract 5g, NaCl10g, 37°C, 220 rpm, shaking culture for 12 hours, permeabilizing Bacillus cereus and glutathione synthesis reaction according to the method described in Example 1. After 8 hours of reaction, glutathione can accumulate 163 mg / L.

Embodiment 3

[0050] Example 3 Construction of recombinant E. coli JM109 (pTrc99a-gshFap)

[0051] 1. Extraction of genomic DNA of Actinobacillus pleuropneumonia

[0052] The glycerol tube of Actinobacillus pleuropneumonia (CVCC 259) stored at -20°C was inserted into the fermentation medium at an inoculum of 1%. The specific ingredients (contained in 1L): 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 to collect the bacteria. The whole genome DNA of Actinobacillus pleuropneumonia was extracted using BBI whole genome DNA extraction kit.

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

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

[0055] Downstream primer is 5’-GAT GTCGAC TTAAGGCAGTTCTGGGAA-3', the underlined part is the Sal I restriction site.

[0056] Using the whole genome of Actinobacillus pleuropneumonia as a t...

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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 method for producing glutathione. Specifically, it utilizes two kinds of glutathione synthetase (γ-GCS) and glutathione synthase (GS). Functional enzyme production method of glutathione. Background technique [0002] Glutathione (Glutathione, GSH for short) is a sulfhydryl-containing compound with important physiological functions. It is composed of L-glutamic acid (L-Glu), L-cysteine ​​(L-Cys) and glycine ( Gly) A tripeptide composed of three amino acids. Glutathione is widely distributed in animals, plants, and microorganisms. It participates in the synthesis of protein and ribonucleic acid, the transport of oxygen and nutrients, maintains the vitality of endogenous enzymes, the cycle of tricarboxylic acid in the body and the metabolism of sugar, and it is eliminated. Excessive free radicals and other functions in the body have a wide range of applications. Clinically, glutathione is used for anti-radiation, anti-tumo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N15/52C12P21/02C12R1/01C12R1/085C12R1/46C12R1/19C12R1/125C12R1/38C12R1/865C12R1/84C12R1/78C12R1/685C12R1/69
CPCY02P20/52
Inventor 叶勤李志敏李娓
Owner EAST CHINA UNIV OF SCI & TECH
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