Process Control And Optimization Of Glutathione Production By Candida Utilis

Glutathione (GSH), as an active tripeptide, can be biosynthesized from L-glutamic acid, L-cysteine and glycine catalyzed consecutively byγ-glutamyl-cysteine synthase (GSH I) and glutathione synthase (GSH II) in the presence of ATP. GSH plays many important physiological roles in living cells, such as clear active oxygen species and free radical, protect integrity of cell membrane and delay oxidation of fat acids. As a result, the demand for GSH in the fields of food industry, clinical medicine, health care and antiaging is ever-increasing and expanding.Compared with other methods, fermentative GSH production is considered as an efficient and practical approach. In this paper, Candida utilis WSH 02-08, a strain that can accumulate high concentration of GSH intracellularly, was used for GSH production. According to the principles of fermentation optimization, a series of feasible approaches or strategies were achieved to maximize GSH production during the processes of optimization of C. utilis WSH 02-08 cultivation. The main contents of this dissertation are as follows:(1) Effects of different cultivation ways on cell growth and GSH production were investigated in high cell density (HCD) cultivation. Results showed that the optimal initial glucose concentration was 15g/L for batch culture, optimal specific growth rate (μ) was 0.2 h-1 for exponential feeding, and optimal rate was 5.5g/L/h for constant feeding. By applying three-stage operation mode (9 h batch fermentation stage, 12 h exponential glucose feeding and 24 h constant glucose feeding), after 45 h fermentation, cell concentration reaches 102 g/L and GSH yield is 981mg/L.(2) High cell density cultivation is preferable for increasing cell concentration, but intracellular GSH content decreased with an increase in cell density. Therefore, single-shot addition of cysteine was utilized to increase GSH production at 45 h as the glucose feeding stopped. Results indicated that as cysteine concentration reaches 50 mmol/L, GSH concentration reaches the maximum of 1534 mg/L and corresponding intracellular GSH content is 1.50% (w/w) after 60 h cultivation. However, an increase in intracellular GSH content triggered inhibition of GSH I. A strategy that combines two shots cysteine addition (25 mmol/L for each time) with low-pH stress was developed to further enhance GSH production, and the final GSH concentration reaches 1825 mg/L.(3)Based on cysteine (50mmol/L) addition in above experiment, it was found that utilization ratio of cysteine for GSH production was very low. Effects of dissolved oxygen (DO) concentration on GSH production and cysteine oxidation were investigated accordingly. It was shown that lower DO concentration favors cysteine absorption but retards GSH production. Higher DO promotes GSH production but favors cysteine oxidation in the broth. A two-step DO control strategy after cysteine addition, in which DO was controlled at 5% in the first 3 h and then at 20% in the following 12 h, was developed and compared for the potential in enhancing GSH production and cysteine absorption. As a result, cysteine addition is decreased from 50 mmol/L to 30 mmol/L, while GSH yield reaches 1734 mg/l after 60 h fermentation. So, by using two-step DO control strategy, a 40% decrease in cysteine addition and a 13% increase in GSH production are observed as compared with that without control of DO(4) Whole process for batch GSH fermentation was divided into two phases of cell growth and GSH synthesis. At cell growth phase, effects of three precursor amino acids on cell growth and GSH production were investigated separately. Glutamic acid shows an insignificant effect on promoting cell growth and enhancing GSH production; Also, cysteine increases GSH content remarkably but inhibits cell growth notably. Compared to glutamic acid and cysteine, glycine can promote cell growth as well as enhance intracellular GSH content significantly. At 2 h with 6 mmol/L glycine addition, DCW and GSH yield reach the highest value of 10.41 g/L and 125 mg/L, which are 10.1% and 34.1% higher than the control.(5) Moreover, a mixture of glutamic acid, glycine and cysteine was added at 12 h at stationary phase of cell growth. The optimum concentrations of three amino acids (glutamic acid 6 mmol/L, glycine 5.5 mmol/L, cysteine 6.5 mmol/L) were obtained by response surface methodology (RSM). With the two-step addition strategy, biomass and GSH yield reached 10.41 g/L and 277.9 mg/L in flask experiments after 30 h fermentation, increased by 10.1% and 129.8%, respectively, as compared with the control without amino acids addition. By applying this amino acids addition strategy to batch fermentation in 7 L fermentor, GSH yield reaches 328.1 mg/L mg/L, which is 118.7% higher than the control. Moreover, further applying this strategy to HCD cultivation, after 60 h fermentation, a GSH concentration of 1952 mg/L is achieved and corresponding intracellular GSH content is 1.81%(6) According to two-step amino acids addition strategy mentioned above, concentrations of intracellular ATP and three amino acids and GSH I activity were determined during the period of 45 h to 60 h. Results indicated that cessation of GSH production at 60 h was caused by ATP shortage and decrease in GSH I. However, compared with decrease in GSH I activity, ATP shortage accounts for a lion share on inhibiting GSH production. So a strategy of combining addition of amino acids addition with ATP was developed to further enhance GSH yield as its production stopped at 60 h. With 3 g/L ATP addition at 60 h, after 72 h cultivation, GSH yield reaches 2141 mg/L and corresponding intracellular GSH content is 1.98% (w/w). By further treating the cells with ionic surfactants to increase cell osmosis for ATP absorption, a GSH yield of 2153 mg/L is achieved by adding 2 g/L ATP. Moreover, by feeding suitable concentrations of glucose and (NH4)SO4 for ATP regeneration to synthesize GSH, GSH yield reaches 2163 mg/L, suggesting the application of above strategy as being feasible.(7) Effect of H2O2-induced oxidative stress on GSH production was investigated. Results indicated that H2O2 can effectively stimulate GSH accumulation but inhibit cell growth simultaneously. So a novel strategy of multiple H2O2 stresses with different concentrations (1 mmol/L at 6h, 2 mmol/L at 8h, and 4 mmol/L at 10 h) were developed to maximize GSH production. As a result, a maximal GSH yield of 188 mg/L was achieved and a corresponding intracellular GSH content was 2.05%. By applying this strategy to 7 L fermentor, GSH yield and intracellular GSH content were 258 mg/L and 2.53%. Further applying above strategy to HCD cultivation, after 60 h fermentation, a GSH concentration of 1482 mg/L is achieved and corresponding intracellular GSH content is 1.52%. Moreover, increased activities of catalase (CAT) and GSH reductase (GR) indicated that GSH and CAT were directly involved in protecting cell against oxidative stress by H2O2.(8) Also, impact of Na2SO4 stress on GSH production was studied. Results showed that Na2SO4 can enhance GSH production remarkably but inhibit cell growth at the same time. Moreover, the key substances and enzymes relative to GSH production were determined and results indicated Na2SO4 stress can lead to a significant increase in cysteine content and activities of GSHⅠand GR, explaining the reason for enhancement of GSH production by Na2SO4 stress.