产朊假丝酵母生物转化生成CDP-choline的研究

【作者】 陆佩丽；

【导师】 钱秀萍；

【作者基本信息】 上海师范大学 ， 微生物学， 2005， 硕士

【摘要】 CDP-choline 是磷脂代谢的重要前体,广泛用于治疗各种脑血管疾病。本文选育了生物转化CDP-choline 的菌株产朊假丝酵母candida utilis,研究了发酵条件及发酵培养基,探索了其酶反应体系的优化条件。利用放线菌酮和TTC 作为一种筛选压力,应用紫外线及N⁺注入等方法对candida utilisYL 进行了诱变筛选。结果表明,突变株candida utilis YL-2-24 的CDP-choline 比出发菌株candida utilis YL 提高2.01 倍。对candida utilis YL-2-24 的发酵工艺进行了优化。通过培养基的单因子试验及正交试验,最终确定最佳发酵培养基配方为蔗糖4%、蛋白胨1.0%、NaNO₃2.0%、K₂HPO₄0.5%、MgSO₄·7H₂O 0.5%、KH₂PO₄ 0.1%;摇瓶发酵条件为初始pH5.5,接种量10%,装液量25ml/250ml , 温度32℃, 培养时间24h , 转速230 r/min , 生物转化生成18.63mg/gCDP-choline。candida utilis YL-2-24 细胞转化的酶促反应条件为CMP10mM、磷酸盐浓度300mM、磷酸胆碱50mM、葡糖糖1000mM、34℃、PH7.4、湿细胞的加量为600mg/ml、反应时间为10h,生物转化生成CDP-choline 的得率为28.63%,转化率达到84.4%。更多还原

【Abstract】 CDP-choline is an important precursor in the biosynthesis of phospholipids and widelyemployed as an effective drug for various kinds of brain injuries.In this paper,TTC andantidione were used in strain screening afrer the candida utilis was treated by UV irradiationand N+ions injection.After the mutation,candida utilis YL-2-24 was obtained.Compared withthe candida utilis YL,the yields of CDP-choline was enhanced 2.01 times.The optimun culture medium ingredients was 4%sucrose, 1.0%peptone, 2.0%NaNO₃,0.5%K₂HPO4,0.5%MgSO₄·7H₂O,0.1%KH₂PO₄.The fermentation process wasoptimied and it was 32℃,starting pH 5.5,rotating speed 230 r/min,incubation for24h,10 % seed culture,250ml flask with 25ml liquid.The transformation ofCDP-choline was 18.90mg/g.When the enzymatic reaction was carried with 10mM CMP,300mM phosphates,50mMcholine phosphate,1000mM Glucose,pH7.4,600mg/ml wet cells,and itwas incubated at34℃for 10h.The transformation of CDP-choline was 18.90mg/g and the rate oftransformation reached 84.4%.更多还原