分批培养产朊假丝酵母合成谷胱甘肽的前体氨基酸优化策略

ENHANCED GLUTATHIONE PRODUCTION BY OPTIMIZING PRESURSOR AMINO ACIDS ADDITION IN BATCH CULTURE CANDIDA UTILIS

通过分批培养产朊假丝酵母(Candida utilis),优化前体氨基酸(谷氨酸、甘氨酸和半胱氨酸)添加促进谷胱甘肽(GSH)合成。首先分析C.utilis培养时胞内3种氨基酸自身合成动态;菌体生长阶段,考察前体氨基酸添加对GSH合成及细胞生长的影响;GSH合成阶段,通过响应面优化混合氨基酸添加配比;最后提出菌体生长阶段添加甘氨酸及GSH合成阶段添加3种混合氨基酸的两阶段优化策略。摇瓶结果表明,菌体培养第2小时添加甘氨酸(6 mmol/L),第10小时添加混合氨基酸(谷氨酸6 mmol/L、甘氨酸5.5 mmol/L、半胱甘酸6.5 mmol/L),菌体培养30 h后,细胞质量浓度和GSH产率为10.41 g/L和278 mg/L,比对照增加10.5%和116%;应用于5 L发酵罐中培养,发酵结束后,细胞质量浓度达12.81 g/L,GSH产率为328 mg/L。

Glutathione(GSH) has the ability of antioxidant,immune function regulation,promoting carbohydrates,fat and protein metabolism in life activities,and is widely used in medicine,health care,skin care,beauty,food additives and other industries. Glutathione(GSH) production was optimized by precursor amino acids addition in batch cultivation of Candida utilis in the study. Initially,the dynamic changes of glutamic acid,glycine and cysteine in batch culture process were analyzed. Then,the effect of addition times and amounts of the three precursor amino acids on cell concentration and GSH production at bacterial growth stage was investigated.Finally, the two-stage addition optimization strategy of adding glycine at cell growth stage and mixed three kinds of amino acids at GSH production stage was developed. The results showed that glycine(6 mmol/L) was added at2 h of C. utilis culture,stage and the mixed amino acids(glutamic acid of 6 mmol/L; glycine of 5.5 mmol/L;cysteine glycine of 6.5 mmol/L) were added at 10 h. After C. utilis culturing for 30 h, the cell dry weight and the yield of GSH was 10.41 g/L and 278 mg/L, respectively, which increased by 10.5% and 116% compared with control without amino acids addition. When the obtained two-stage amino acid addition strategy was applied in 5 L fermentor cultivation, the cell concentration was reached to 12.81 g/L and the yield of GSH was 328 mg/L after fermentation, which indicating the feasibility of adopted strategy.