Method for producing glutathione based on enzymic method in vitro

A technology of glutathione and bifunctional enzyme, which is applied in the direction of fermentation, etc., can solve the problems of not being able to meet the market demand well, not easy to separate the product later, and difficult to increase the product yield.

Inactive Publication Date: 2016-01-06
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the glutathione synthesis bifunctional enzymes that have been reported for the purpose of producing glutathione all have obvious product inhibition effects, which makes it

Method used

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  • Method for producing glutathione based on enzymic method in vitro
  • Method for producing glutathione based on enzymic method in vitro
  • Method for producing glutathione based on enzymic method in vitro

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Experimental program
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Embodiment 1

[0034] 1 Cloning of glutathione synthesis bifunctional enzyme gene gs of Pasteurella multocida strain and construction of recombinant vector

[0035] 1.1 Cloning of GS gene of Pasteurella multocida strain

[0036] The DNA of glutathione synthesizing bifunctional enzyme GS gene in Pasteurellamultocida strain was synthesized by whole gene synthesis method. Using the gene DNA as a template, primers were designed according to the GenBank sequence, and the corresponding enzyme cutting sites (NdeI and EcoRI) and protective bases were added.

[0037] The primers used for PCR amplification of glutathione synthesis bifunctional enzyme GS gene are as follows:

[0038] Upstream primer: 5'-GCATATGATGGCCAAGAAGGAC-3'

[0039]Downstream primer: 5'-GCTCGAGTTACTTGGCGAG-3'

[0040] The PCR system reaction system contains 0.2 μL of genomic DNA, 0.5 μL of upstream and downstream primers, 10 μL of Extaq mixture, and added double distilled water to make up to 20 μL. The reaction conditions were...

Embodiment 2

[0044] 1 Cloning of ppk polyphosphate kinase gene in Corynebacterium glutamicum strain and construction of recombinant vector

[0045] The ppk polyphosphokinase gene in Escherichia coli strain was synthesized by the method of total gene synthesis. Using the whole gene of the strain as a template, primers were designed according to the GenBank sequence, and corresponding enzyme cutting sites (NcoI and EcoRI) and protective bases were added.

[0046] The primers used for PCR amplification of polyphosphate kinase gene ppk are as follows:

[0047] Upstream primer: 5'-GCATATGATGACCGCCACCGATTC-3'

[0048] Downstream primer: 5'-GAAGCTTCCGGTTGGTAGCTGTGAC-3'

[0049] The PCR system reaction system contains 0.2 μL of genomic DNA, 0.5 μL of upstream and downstream primers, 10 μL of Extaq mixture, and added double distilled water to make up to 20 μL. The reaction conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at ...

Embodiment 3

[0053] Transform the constructed expression vector pET22b-gs plasmid into BL21(DE3) Escherichia coli, use LB medium, grow at 37°C until the OD600 is about 0.4-1.0, induce with 0.1mM-1mM IPTG, at 30 Intracellular expression was carried out in culture at ℃. Transform the control empty vector pET22b into BL21(DE3) Escherichia coli, induce expression under the same conditions as above, and conduct protein electrophoresis to verify the expression result of the expressed recombinant protein. The protein electrophoresis is as follows: Figure 5 shown.

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Abstract

The invention relates to a method for producing glutathione based on enzymic method in vitro. Through expression and catalysis of glutathione compound difunctional enzymes and polyphosphoric acid kinases, tetraphosphate and hexametaphosphate serve as phosphoric acid donors to regenerate ATP (adenosine triphosphate), and the glutathione is generated under the catalytic action of the glutathione compound difunctional enzymes by taking glutamic acid, glycine and cysteine as substrates. Compared with existing glutathione production methods, the method has the advantages that inhibition effect of the glutathione compound difunctional enzymes is low, and the content of the glutathione obtained from a reaction system is high; sodium and kali salt of phosphoric acid donors, such as the tetraphosphate and the hexametaphosphate, used in the reaction process is low in cost and easy to get; the phosphoric acid donors serve as substrates of the polyphosphoric acid to generate the ATP, and accordingly, production cost is reduced greatly; the amino acid ratio of Gly:Gys:Gly is lowered to 2:1:2, the content of amino acid in the system is low, and later product separation is facilitated.

Description

technical field [0001] The invention belongs to the field of biochemical industry, in particular to a method for in vitro enzymatic production of glutathione. Background technique [0002] Glutathione is a compound with multiple biological functions, which is obtained by condensation of three amino acids L-cysteine, L-glutamic acid and glycine through peptide bonds. It can not only scavenge free radicals in the body, but also protect the liver and improve the body's immunity. Therefore, glutathione is widely used in the fields of biomedicine and food, and the demand for glutathione in domestic and foreign pharmaceutical markets is also increasing. [0003] Until now, there are four main methods for industrial production of glutathione: fermentation, enzyme-catalyzed synthesis, chemical synthesis and biological extraction. The product separation and post-treatment in the fermentation process are cumbersome, the chemical synthesis method has high cost, complicated operation, ...

Claims

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Application Information

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IPC IPC(8): C12P21/02
Inventor 刘珞李成成王峥
Owner BEIJING UNIV OF CHEM TECH
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