Mixed cultivation technology of recombinant escherichia coli for producing glutathione and application of mixed cultivation technology

Abstract

The invention discloses a mixed cultivation technology of recombinant escherichia coli for producing glutathione and application of the mixed cultivation technology. According to the mixed cultivationtechnology, a DshF gene and a ppk2 gene are correspondingly integrated on a plasmid vector, escherichia coli containing the GshF gene and escherichia coli containing the ppk2 gene are obtained, and mixedly cultivated in the proportion of 5 to 4-5 to 2 to obtain a mixed bacteria cell. Under the condition of the mixed bacteria cell at37-45 DEG C with the pH of 6-7.5, reduced glutathione is catalyzed and synthesized through inducible expression. In the presence of low-cost sodium hexametaphosphate, the phosphorylation of ADP and AMP can be catalyzed, and high-energy phosphorylation bond breakingof a catalysate provides energy for the synthesis of the glutathione. The cost is saved, the problem of placement of remaining cells due to the high reaction efficiency is avoided, operation is easy,convenient and easy to control, and good application prospects are achieved.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a mixed culture process of recombinant Escherichia coli for producing glutathione and its application. Background technique [0002] Glutathione (γ-L-glutamyl-cysteinyl-glycine, GSH) is a small molecular polypeptide obtained by condensation of three amino acids, L-glutamic acid, L-cysteine ​​and glycine. In organisms, it is the most important small molecular peptide containing sulfhydryl groups in microorganisms and plant biological cells. Glutathione is an important antioxidant in the body. Reduced glutathione has many important functions in living tissues, and plays an important role in liver injury, eye diseases, substance metabolism, and regulation of cell apoptosis. In addition, it also plays an equally important role in the field of food preservation, so the demand for glutathione in the domestic and foreign pharmaceutical markets, food, and cosmetics fie...

AI Technical Summary

Problems solved by technology

However, in the prior art, there are still various problems hindering the industrial application of this technology. Patent CN201710543648 has obtained GshF-4 mutants with different amino acid sequences, and its synthetic activity, specific activity and optimum temperature have been improved, but there are The output is low; ATP consumption is large and the cost is high, which is not conducive to industrial production

[0005] Enzymatic synthesis of glutathione requires adenosine triphosphate (ATP) as an energy donor, but the price of ATP will greatly increase production costs, so the establishment of an ATP regeneration system is a key part of glutathione synthesis

Patent CN201310538982 overexpresses (exogenous) glutathione bifunctional enzyme (STH) and acetate kinase (ack) in E. coli cells to obtain recombinant expression cells. Although the purpose of ATP regeneration is achieved, Rosetta (DE3) is stable The sensitivity is not high, the cost of the required substrate dilithium acetyl phosphate is high, and the yield of the product is low, the broken enzyme solution increases the cost of separation, in addition, there are problems that the plasmid may be lost, and the use of antibiotics is not friendly to the environment It is difficult to carry out industrial promotion; patent CN201710452240 uses GshF enzyme, ATP regeneration enzyme (PPK enzyme and PAP enzyme) and AK enzyme to generate glutathione in the reactor, and uses adenosine to replace ATP or AMP, but the ATP in it is regenerated The enzyme system includes PPK enzyme, PAP enzyme and AK enzyme. There are problems of high cost caused by separate culture of multiple enzymes, cumbersome immobilization and separation steps, high energy consumption, and the possibility of loss of plasmids. The use of antibiotics is harmful to the environment. Unfriendly problems, etc., eventually lead to its failure to be well promoted in the industry

Images