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Thermostable difunctional glutathione synthetase mutant and application thereof

A technology of glutathione and mutants, which is applied in the field of bioengineering, can solve the problems of few large-scale practical applications, and achieve the effects of increasing production, synthesis efficiency, and improving thermal stability

Active Publication Date: 2021-05-11
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the limited activity and stability of enzymes, the large-scale practical application of enzymatic methods in industry is seldom

Method used

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  • Thermostable difunctional glutathione synthetase mutant and application thereof
  • Thermostable difunctional glutathione synthetase mutant and application thereof
  • Thermostable difunctional glutathione synthetase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: DNA recombination of bifunctional glutathione synthetase from different sources, construction of recombinant expression plasmids and engineering bacteria

[0020] In this embodiment, the bifunctional glutathione synthase gene (shown in sequence table SEQ ID NO.1 and SEQ ID NO.2) derived from Streptococcus sanguinis (gshFss) and S.thermophilus (gshFst) was selected. The first gene was selected from SEQ ID NO.4 and SEQ ID NO.7 in Chinese patent CN102071171B, and the gshFsa whose crystal structure had been reported was used as a template for homologous sequence comparison to find the corresponding γ-GCS, γ-GS and intermediate The functional domains of the linker domains were combined in different ways to obtain mutants that can improve thermal stability. The specific primer sequences are shown in Table 1, and the sequences were sent to Shanghai Bioengineering Co., Ltd. for synthesis.

[0021] Table 1 Sequence list of DNA recombination mutation primers

[0022] ...

Embodiment 2

[0026] Example 2: Site-directed mutagenesis of bifunctional glutathione synthetase, recombinant expression plasmid and construction of engineering bacteria

[0027] Through the calculation of ΔΔG, some potential mutation points that can improve the thermal stability of the enzyme can be screened. In the present invention, the ΔΔG of different mutation points is calculated by software such as ROSETTA and FoldX, and amino acid sites with lower ΔΔG values ​​(usually unstable amino acid positions) are selected for mutation by comparison. The specific primer sequences are shown in the table 2. The sequence was sent to Shanghai Bioengineering Co., Ltd. for synthesis.

[0028] Table 2 Design of primers for site-directed mutagenesis

[0029]

[0030]Preparation of linearized carrier: the method is the same as that of the linearized carrier in Example 1

[0031] Site-directed mutagenesis: use the recombinant as a template and perform amplification with the corresponding primers in...

Embodiment 3

[0034] Example 3: Protein expression and protein purification of different mutant strains

[0035] The different recombinant bacteria obtained in Example 1 and Example 2 were inoculated into 50ml LB containing 50μg / ml Kanamycin, 37°C, 220rpm shaking culture for 6-8h, and then the bacteria were transferred to 100ml LB, 37°C, Cultivate to OD at 220rpm 600 =0.6-0.8, add IPTG with a final concentration of 0.2mM and induce at 18°C ​​and 220rpm for 16h. Centrifuge at 6000rpm for 10min to collect the bacteria.

[0036] The protein was purified by Ni-NTA nickel column affinity chromatography. Resuspend the bacteria with 50ml of buffer solution (20mM Tris-HCl; 250mMNaCl; 10mM imidazole), then crush it with a pressure breaker, centrifuge at 8000rpm at 4°C for 20min, take the supernatant and load it on nickel that has been equilibrated with Tris-HCl buffer On the column, the adsorbed protein was gradiently eluted with loading buffer containing different concentrations of imidazole (0-...

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Abstract

The invention discloses a mutant of difunctional glutathione synthetase. The mutant is an amino acid sequence as shown in SEQ ID NO.3 or a mutant thereof obtained by recombining amino acid sequences as shown in SEQ ID NO.2 and SEQ ID NO.1 and / or carrying out site-directed mutagenesis on a recombinant. The mutant has the advantages that the thermal stability of the mutant is remarkably improved, and the half-life period t1 / 2 is 10-60 times that of wild enzyme. The half-life period t1 / 2 of the optimal mutant is 163.3 times that of the wild enzyme, and the specific enzyme activity is improved by 24% compared with that of the wild enzyme. Compared with the temperature of the wild enzyme, the optimal temperature of the optimal mutant is increased from 45 DEG C to 50 DEG C. Tests such as enzyme catalysis, intracellular self-assembly whole-cell catalysis and fermentation prove that the finally obtained mutant effectively improves the yield and synthesis efficiency of GSH.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to DNA recombination, site-directed mutation and multi-site mutation on bifunctional glutathione synthetase genes from different sources, and the application of recombinant mutants in glutathione biosynthesis. Background technique [0002] Glutathione (GSH) is a bioactive tripeptide compound containing a sulfhydryl group formed by condensation of L-glutamic acid, L-cysteine ​​and glycine. In living organisms, glutathione has many important physiological functions. Glutathione can participate in the oxidation-reduction process in the body, combine peroxides and free radicals to resist the damage of sulfhydryl groups by antioxidants, and at the same time resist the damage of free radicals to important organs. In addition, glutathione eliminates free radicals in the human body, improves human immunity, and resists aging. The effect of glutathione on the retarded cells of the elderly is g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12P21/02C07K5/037C12N15/70C12R1/19
CPCC12N9/93C12Y603/02002C12Y603/02003C12P21/02C07K5/0215C12N15/70
Inventor 李志敏崔向伟
Owner EAST CHINA UNIV OF SCI & TECH
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