Glutathione production method

A technology of glutathione and cysteine, which can be used in the biological field to solve problems such as low yield

Active Publication Date: 2015-05-13
风火轮(上海)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there are a series of GSH-producing bacterial strains in the prior art, which can usua

Method used

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Experimental program
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Embodiment 1

[0069] Embodiment 1, the construction of the single-enzyme expression strain that produces glutathione (GSH)

[0070] 1. Construction of pET24a-SAG

[0071] According to the reported gene sequence of glutathione bifunctional enzyme SAG derived from Streptococcus agalactiae (NCBI accession number: AE009948.1), the sequence was synthesized from the entire gene, and restriction sites NdeI and HindIII were designed at both ends, and subcloned into the vector pET24a (purchased from Novagen) at the corresponding site to obtain the recombinant plasmid pET24a-SAG, see the plasmid map figure 1 .

[0072] The constructed recombinant plasmid pET24a-SAG was transformed into Escherichia coli expression host BL21(DE3) by calcium chloride method to obtain BL21(DE3) / pET24a-SAG.

[0073] 2. Construction and expression of pET24a-STH

[0074] According to the reported gene sequence of the glutathione bifunctional enzyme STH derived from Streptococcus thermophilus strain SIIM B218 (NCBI access...

Embodiment 2

[0132] Embodiment 2, the construction of the combined enzyme expression strain that produces GSH

[0133] 1. Construction of STH and gshB-ack co-expression strains

[0134] (1) Construction of pMWK-gshB-ack

[0135] Using the plasmid pPIC3.5K (purchased from Invitrogen) as a template, the primers were designed as follows: Kandn: CCAACCAATTAACCAATTCTGATTAG (SEQ ID NO: 10), Kanup: CCTGCAGGGGGGGGGGGAAAGCCACGTTGTGTC (SEQ ID NO: 11), and a 0.9 kb Kan fragment was amplified by PCR. The PCR reaction system includes: 0.3μM each primer, 50ng template, 1×KODneo plus buffer, 0.2mM dNTP, 1.5mM MgSO 4 , KOD neo plus1U, add ddH2O to the total system 50μL. The temperature conditions are: 94°C for 2min; 98°C for 10s, 55°C for 30s, 68°C for 30s, repeating 30 cycles; 68°C for 10min. After the PCR reaction was completed, the agarose gel electrophoresis was used to identify the kan fragment, and the gel recovery kit recovered the kan fragment. Plasmid pMW118 (purchased from Nippon Gene, Toyam...

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Abstract

The invention relates to an improved glutathione production method. Recombination strain capable of efficiently conversing L-cysteine, L-glutamic acid and glycine into glutathione can be obtained by screening of recombinant expression host and selection of appropriate enzyme for over expression, and the recombination strain can effectively improve the yield of GSH(glutathione). The method can increase the yield of GSH by more than 50%.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to an improved method for producing glutathione. Background technique [0002] Glutathione (GSH) is an important drug that regulates physiological functions. It has been widely used clinically and has become one of the important drugs that can regulate human immune function and assist in anticancer. The antioxidant properties of GSH make its application in the food industry attract people's attention, and its role in food storage and preservation, improving product flavor, and increasing product nutritional value is becoming more and more optimistic. Glutathione is a biologically active tripeptide compound containing γ2 glutamyl and thiol, which is condensed from L-glutamic acid, L-cysteine ​​and glycine. It is used in the synthesis of proteins and DNA, and the synthesis of amino acids. It plays a direct or indirect role in important biological phenomena such...

Claims

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Application Information

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IPC IPC(8): C12P21/02C12N15/70C12N1/21C12R1/19
Inventor 杨晟陶荣盛朱傅赟沈正权沈青孙梁栋陈成
Owner 风火轮(上海)生物科技有限公司
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