Combination Of Genetic Engineered Enzymatic Glutathione Production With ATP Recycle System

Glutathione is widely used in the fields of medicine, food and cosmetic. There are many reseach about production methods of glutathione(GSH), but for the limit of Glutathione Synthetase, GSH's production can not be improved effectively. so,it still remainds a problem to be sloved.Recently, the vitro enzymatic reaction was used as a simple and effective method for the production of GSH. Suppling of energy is an important limitation of this method. The known PPKs accept only long chain polyP or show unfavorable preference. The short chain polyP (trimer or tetermer) only commercial available and we wish to screened out a PPK, which is able to use the short chain polyP to regenerate ATP, however, with low efficiency.In this context, our study focus on two limitations above-mentioned about GSH's production, and we have already get the perfect results. we use polyphosphate kinase coupling with bifunctional r-Glutamate-cysteine ligase to catalyze the production of GSH. And get the targets of produce GSH high efficiency and low-cost ultimately. The sources of bifunctional r-Glutamate-cysteine ligase and polyphosphate kinase are the key factors for this reaserch.The main achievements are as follows:(1) Depending on the characteristic of the enzyme, take comparison of a variety of PPK and GS, then select the appropriate ppk and gs gene correspondingly. Clone and construct recombinant vector of gene ppk and gs. Then resereach conditions of heterologous expression of GS and PPK. Finally, we use SDS-PAGE protein electrophoresis to confirm PPK and GS has been expressed successfully.(2) Use bifunctional r-Glutamate-cysteine ligase coupling with polyphosphate kinase to catalyze the production of GSH. To enhance the GSH production, we optimized the coupling reaction conditions. The optimized ratio of three amino acids was Glu:Cys:Gly-2:1:2; Then the concentration of ATP, Mg2+ and hexametaphosphate were optimized. Optimization results show that glutathione-generating system can be expanded from 30 mM to 50 mM. Under the optimal reaction conditions, GSH production was increased from 8 g·L-1 to 13 g·L-1, and the maximum conversion reached to 83.8%. At the same time, the amount of added exogenous ATP in coupling reaction system is 20% of system demand. In short, compared with previous results, this study glutathione production double previous production, while the cost reduction of 67.9%.(3) On the basis of the laboratory studies above, the coupling reaction volume was amplified from 10 ml to 250 mL. First, high density fermentation to produce a large number of proteins, then high-pressure homogenizer lysing to get a lot of crude enzyme. Crude enzyme catalyzed in coupling reaction and production of GSH concentration reached to 12.32 g·L-1, the conversion rate was 80.24%. This is a major breakthrough in industrial production of glutathione.We have get a PPK, which is able to use the short chain polyP to regenerate ATP. In the bifunctional r-Glutamate-cysteine ligase and PPK coupling reaction to produce GSH, PPK catalyzed ATP renewable up to 4 times, this is a breakthrough to energy needs reaction. The r-Glutamate-cysteine ligase mentioned above allows the catalytic reaction concentration to increase up to 50 mM. This is a breakthrough in the field of production of GSH.