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Mutants of bifunctional glutathione synthetase and application of mutants in glutathione synthesis

A glutathione and synthetase technology, applied in the direction of peptides, enzymes, ligases, etc., can solve the problems of low product tolerance, insufficient stability, low synthetase activity, etc., to achieve increased activity, reduced production costs, The effect of increasing product concentration

Active Publication Date: 2019-04-09
湛江五洲生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In view of the deficiencies in the current prior art, the purpose of the present invention is to provide a mutant of glutathione synthetase to solve the problem of low activity of existing glutathione synthase, insufficient stability and excessive tolerance to products. low level problem

Method used

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  • Mutants of bifunctional glutathione synthetase and application of mutants in glutathione synthesis
  • Mutants of bifunctional glutathione synthetase and application of mutants in glutathione synthesis
  • Mutants of bifunctional glutathione synthetase and application of mutants in glutathione synthesis

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1, the construction of wild-type gdhF expression vector

[0042] According to the coding sequence of Bacillus cereus bifunctional glutathione synthetase in the GenBank database (GenBank: ACLS01000213.1, SEQ NO.2), Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to synthesize the gene fragment and named it gshFbc gene. In order to facilitate subsequent cloning, two bases of CC were added to the 5' end of the gene fragment to form a NcoI restriction site, and six bases of GGATCC were added to the 3' end to form a BamHI site.

[0043] The synthesized gshFbc gene and pET28a vector were double-digested with NcoI and BamHI, respectively, and the fragments were recovered with DNA gel kits (purchased from AXYGEN Company), respectively. After ligation with T4 DNA ligase, E. coli DH5α was transformed, and Kana Mycin-resistant LB plate screening to obtain clones.

[0044] Pick 8 clones, use a plasmid extraction kit (purchased from AXYGEN) to extract the plasmids...

Embodiment 2

[0045] Embodiment 2, the expression of GshFbc and the preparation of enzyme

[0046] The expression vector pET28a-gshFbc obtained in Example 1 was transformed into Escherichia coli BL21(DE3) to obtain a genetically engineered Escherichia coli strain capable of highly expressing glutathione synthetase.

[0047] The engineering bacteria highly expressing glutathione synthase were fermented and cultivated in shake flasks according to conventional methods, and when the OD600 of the fermentation broth reached 0.6-0.8, adding a final concentration of 1 mM isopropylthiogalactoside (IPTG) induced , lower the temperature to 28 degrees and continue to cultivate for about 16 hours, centrifuge at 4000 rpm for 20 minutes, collect the bacteria, wash twice with 50mM PBS buffer solution of pH7.4, and obtain the bacteria containing glutathione synthase for subsequent catalysis Research and Enzyme Activity Assays.

Embodiment 3

[0048] Embodiment 3, the mensuration of glutathione synthetase activity

[0049] Weigh 10 g of the collected thalli containing glutathione synthase, and add cetyltrimethylammonium bromide (CTAB) with a final concentration of 0.5% to permeabilize the cells, add 100 mM PBS with pH 8.0 Buffer to a final volume of 50mL to obtain glutathione synthase crude enzyme solution

[0050] In the 5mL reaction system, add L-glutamic acid, L-cysteine, L-glycine and ATP to a final concentration of 50mM, add magnesium ions to a final concentration of 10mM, add 500uL of crude enzyme solution, and add 100mM PBS with pH8.0 buffer to a final volume of 5 mL to obtain a reaction solution.

[0051] The reaction solution was reacted at 37°C for 30 minutes, terminated in a boiling water bath for 5 minutes, centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected for subsequent detection.

[0052] Take 250uL of the supernatant sample, add 750uL of 0.2M sodium hydroxide solution, and th...

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Abstract

The invention discloses a series of mutants of bifunctional glutathione synthetase and an application of the mutants in glutathione synthesis. The series of mutants are obtained by single-point mutation or combined mutation on amino acids at 29th, 440th, 442nd, 483rd, 488th, 573rd and 576th sites of bacillus cereus bifunctional glutathione synthetase, wherein activity of the M4 mutant with the highest activity is 26.6 times higher than that of wild glutathione synthetase, glutathione synthesized by the M4 mutant under enzymatic synthesis has the highest concentration reaching 24.8 g / L, whichis 5.76 times higher than that of the wild glutathione synthetase, the production cost is greatly reduced, and the mutants have great application value.

Description

technical field [0001] The invention belongs to the field of biocatalysis, and relates to a novel bifunctional glutathione synthetase mutant and its application in glutathione synthesis. Background technique [0002] Glutathione is a non-protein sulfhydryl short peptide that widely exists in organisms, and generally exists in two forms: oxidized glutathione (GSSG) and reduced glutathione (L-glutathione, GSH). Participate in a series of physiological reactions of the body and have various physiological functions. In particular, the reduced glutathione has a strong affinity, can combine with various metabolites in the body, can remove oxygen free radicals in the body, and has a series of functions such as anti-oxidation and enhancing immunity. Carcinogens, heavy metals, toxic compounds, etc. in the body, prompt them to be excreted from the body, and play a role in detoxification. [0003] Due to its important physiological functions, GSH is widely used in many fields, especi...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12P21/02
CPCC07K5/0215C12N9/93C12P21/02C12Y603/02003
Inventor 陈远才陈雪松张敬松王聿修王立序黎炎桃
Owner 湛江五洲生物工程有限公司
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