Study On Fermentation Conditions Of Recombinant Pichia Pastoris And Separation And Purification Of GSH

The glutathione is an important tripeptide broadly applied for the therapy of many diseases in clinic. Fermentation of microorganism and extracting GSH from the cell is the main industrial production process. The study of production and application about GSH is already quite mature at home and abroad, however, the industrialized production technology is still monopolized by a few other countries. The domestic yield and purity of GSH are relatively low, and the production is still not reached realized industrialized production, which results the price of GSH very high and constrains its application at large. In this study, the following approaches were adopted during the experiments for realizing high yield and purity of GSH and providing theory of reference for industrial production in China.Firstly, the metabolic process of sulfur containing material in the cell of Pichia pastoris was explored, and its metabolic path was then modified rationally. The optimal medium for GSH fermentation was screened as 20g/L glycerol, 20g/L peptone, 10g/L yeast extract, 0.05mol/L phosphate buffer, 13.4g/L YNB, 4×10^-4g/L Biotin, 0.05mol/L L-cys; the optimal cultural conditions were confirmed as 28℃-30℃, fully saturated OD, initial pH 6.0, under such conditions GSH output might reach as high as 6.0g/L after 5 days fermentation.Secondly, as per GSH is mainly endocellular distribution and may release through cytomembrane once its permeability changed by high temperature or organic reagent and/or the cell wall being destroyed, and isoelectric point of GSH is 5.93, under acerbic condition positive charge ion exchange adsorption was applied for separating GSH from other components. Furthermore, for higher purity GSH product ultra-filtration and nano-filtration were introduced into the purification process, and resulting the recovery rate of GSH as high as 90% and 95% purity.Conclusively, GSH separation and purification is a complicated process in general. It is more difficult while the output is lower than a threshold in the fermentation. This experiment through genetically modified Pichia pastoris and optimized conditions for fermentation and separation/ purification, realized high yield and purity of GSH, as well as simplified the process for purification, and solved the problem existed in industrial production.