Construction Of Double-enzymatic Production System Of Glutathione

Glutathione has a variety of important physiological functions in the human body and is widely used in clinical medicine,health care products,beauty and other fields.As the main method for industrial production of glutathione at present,microbial fermentation has problems such as low yield,complicated downstream processes,and intracellular inhibition of the product.Enzymatic production of glutathione has high conversion efficiency and is expected to achieve high concentration accumulation of glutathione,but the energy supply substance ATP necessary for the reaction is expensive,and enzymes usually have limitations such as poor stability and poor reusability.Therefore,selecting a suitable glutathione synthase system,constructing an efficient ATP regeneration system and introducing an appropriate double-enzyme immobilization method are urgent problems to be solved at present.In this paper,an economical,efficient and stable double-enzyme coupling system was constructed to catalyze the synthesis of glutathione,and bifunctional glutathione synthetase(Gsh F)was used to generate glutathione,polyphosphate kinase(PPK)was introduced to recycle ATP.The expression conditions of Gsh F and PPK,the double-enzyme catalytic process and immobilization technology were studied respectively.First,the recombinant E.coli expression system was constructed,and Gsh F and PPK with enzymatic activity were successfully expressed.Secondly,the expression conditions and enzymatic properties were studied.The optimal expression conditions were:0.2 m M IPTG,cultured at 20 °C for 16 h,Gsh F enzyme activity was 1.68U/m L,0.34 times higher than before,PPK enzyme activity was 114.06 U/m L,1.22 times higher than before.After purification by Ni-NTA affinity chromatography,the Gsh F enzyme activity reached 11.67 U/m L,which was6.48 times higher than that of the crude enzyme,and the thermal stability was also improved.Then,the parameters and conditions in the double-enzyme catalytic system were optimized and explored to better coordinate the production of glutathione and the circulation of ATP,and to improve the catalytic efficiency of double-enzyme and the yield of glutathione.Using sodium hexametaphosphate as the low-cost phosphate donor for ATP regeneration,the study found that the continuous synthesis of glutathione can be driven by the addition of ATP to 0.25% of the required amount for the reaction,and the double-enzyme catalytic system can achieve 248 times of ATP regeneration.After optimization of the conditions,on the basis of adding 120 m M substrate,the GSH yield reached 31.92 g/L at 3 h,and the conversion rate was 86.56%.Finally,a double-enzyme immobilization method based on microbial transglutaminase(MTG)cross-linking technology was developed.By fusing peptide tags to the N-terminal of Gsh F and PPK,reactive residues and specific cross-linking sites are provided for MTG.In addition,BSA was introduced as a protective protein to participate in the cross-linking system,and the cross-linked enzyme CLEs-BSA with high enzymatic activity retention was prepared,and the thermal stability,catalytic efficiency and operational stability of the cross-linked enzyme were significantly better than those of the free enzyme.CLEs-BSA can be used stably for 7 times,and the conversion rate of the substrate can reach 95.56% in the GSH synthesis system,and the conversion rate is still 84.67% after being reused 5 times.