High Density Cell Culture And Improvement Of Glutathione Biosynthesis By Candida Utilis WSH 02-08

As one of the major non-protein thiol compounds, glutathione (GSH), is widely distributed in the nature and plays many important physiological roles in living cells, such as enhancing immunity of the body promptly and maintaining the normal redox environment of cells as an antioxidant. Since GSH is widely used in the fields such as clinical medicine, food industry, athletic sports and biological research, the demand of GSH has being expanding.GSH is an intracellular product. Microbial synthesis of GSH by fermentation is one of the most common methods for GSH production at present. In this study, C. utilis WSH 02-08, a strain which could accumulate high concentration of GSH intracellularly was selected for GSH production. A series of feasible approaches or strategies were carried out to obtain high yield of cell and GSH. The main contents involved in this thesis are described as follows:(1) The increase of dry cell weight (DCW) could improve GSH concentration because GSH is an intracellular product. The effects of different fed-batch culture modes with such as constant feeding rate and exponential feeding rate on GSH production were also studied. Higher dry cell weight and GSH yield were realized in glucose fed-batch culture. As a whole, exponential feeding rate culture was more suitable approach to attain not only high concentrations but also high productivities of cell and GSH in the cultivation of C. utilis WSH 02-08. With 48 h of cultivation by glucose feeding at exponential rate, the maximum DCW, GSH and intracellular GSH content were achieved as high as 69 g·L-1, 835 mg·L-1 and 1.41 %, respectively.(2) The roles of three constituent amino acids of GSH such as L-glutamate, L-cysteine and glycine on GSH production, respectively, in flask culture were investigated. By the addition of 8 mmol·L-1 L-cysteine, GSH concentration and intracellular GSH content were improved by 51 % and 82 % compared to the control. It is indicated that L-cysteine was the key amino acid in the synthesis of GSH.(3) In flask culture, the addition of three amino acids at the late stage phase of cultivation greatly improved GSH concentration and intracellular GSH content. GSH concentration and intracellular GSH content with the addition of 8 mmol·L-1 mixed amino acids increased by 91 % and 121 % than the control.(4) As to the cultivation of C. utilis WSH 02-08 by fed-batch process in a 7 L stirred fermentor, the addition of L-cysteine at the end of cell growth phase also had positive effects on the enhancement of both GSH production and intracellular GSH content. When L-cysteine (40 mmol·L-1) was added at 45 h, the maximum GSH and intracellular GSH...