Studyon The Properties And Application Of The Keyenzyme In Saccharomyces Cerevisiae Glutathione Synthetase By Cloning And Expression

In vivo, Glutathione (GSH) has various physiological functions such as antioxidant,detoxification and immune regulation. It has paid close attention to the development andutilization, as people gradually discovered and recognized the physiological functionsand market prospects of GSH, which has been applied in the food, pharmaceutical andcosmetic industries. In this paper, at first two engineering E. coli were constructed aboutγ-glutamylcysteine synthetase gene (GSH1) and glutathione synthetase gene (GSH2) inGSH metabolism of S. cerevisiae, using genetic engineering methods, with S. cerevisiaeCCCG of high producing GSH as the research object. Then, GSH1and GSH2recombinant protein were expressed, purified, and characterized about their enzymaticproperties. Finally the optimized fermentation conditions were studied about producingGSH by S. cerevisiae CCCG based on GSH1and GSH2enzymatic properties.Specifically main contents and results are as follows:1. GSH1and GSH2were cloned by PCR in S. cerevisiae CCCG with S. cerevisiaeCCCG as the research object. GSH1and GSH2engineering bacteria were successfullyconstructed with the use of the expression vector pGEX-4T-2with GST tag andexpression strain E. coli BL21. They were E. coli BL21(pGEX-GSH1) and E. coliBL21(pGEX-GSH2), respectively.2. Some conditions were optimized in expressing soluble GSH1and GSH2recombinant protein with the engineering strain as the research object, which weretemperature, time and the concentration of added IPTG. These result showed that whentwo engineering strain was cultured at37oC, at the shake speed of250r/min to the cellconcentration of OD600=0.4~0.6, followed by adding IPTG to a final concentration of0.02mmol/L IPTG, then cultured for12h at20oC, at the shake speed of250r/min.2.5mg/L GSH1recombinant protein, soluble GSH1and GSH2recombinant protein wereobtained, respectively. The GSH1or GSH2recombinant protein without GST-tag wasobtained by sonication, affinity chromatography with GST-Resin, removing GST-tagwith thrombin, gel chromatography with Sephadex G-75from engineered bacteria.It was studied about the effect of pH, temperature, metal ions and surfactants onGSH1and GSH2recombinant protein without GST tag activity by the single-factormethod. These results showed that the optimum pH of GSH1recombinant protein wasabout8.5, the optimum temperature was about35oC, Mg2+, Mn2+can promote itsactivity, SDS, TritonX-100, Span-20, Tween-80surfactant all promoted GSH1activity,SDS had the best effect as its optimal concentration is about0.08%. While the optimumpH of GSH2recombinant protein was about8.0, the optimum temperature is about35oC, Mg2+, Mn2+can promote its activity, SDS, TritonX-100, Span-20, Tween-80surfactant also promoted GSH2activity, SDS had the best effect as its optimalconcentration is about0.08%.3. Some conditions were optimized in producing GSH by S. cerevisiae CCCG in fed-batch fermentation, combined with those enzymatic properties of GSH1and GSH2,which was obtained previous experiment. They were pH, temperature and SDS addition.The optimum conditions were showed that pH was maintained about5.5within0h to24h, then pH was adjusted to about6.5to the end of culture after24h, the temperaturewas controlled at30oC within0h to36h, and then adjusted to32oC to the end ofculture after36h, and SDS was added to a final concentration of0.04%at36h. Thebiomass reached45.1g/L, intracellular GSH content was22.9mg/g, and GSH yield was1037.4mg/L at culture for48h. Compared with the initial culture, intracellular GSHcontent increased by18.0%, the biomass increased by10.0%, GSH yield increased by29.4%.