摘要
以化学合成的2-脱氧核糖-1-磷酸二环己胺盐和5-氟尿嘧啶为底物,在芽孢杆菌核苷磷酸化酶的作用下合成产物2'-脱氧-5-氟尿苷。考察了诱导剂种类及浓度对菌体核苷磷酸化酶活力的影响,同时又考察了底物比例、底物浓度、菌体量、反应温度、缓冲液浓度及p H值等参数,得到最佳的条件为核糖浓度35 mmol/L,5-氟尿嘧啶为105 mmol/L,磷酸钾缓冲液45 mmol/L(p H 8.0),游离芽孢杆菌12%(湿重)在55℃下反应3 h,转化率可达79.36%,产量可达6.83 g/L。采用该法合成2'-脱氧-5-氟尿苷,反应条件温和,同时能够一定程度地解决酶法合成脱氧核苷中脱氧核糖供体来源少、价格高的问题,具有一定的工业化价值。
In this study,5-fluoro-2'-deoxyuridine( FUdR) is enzymaticly synthesized using bacillus whole-cell with high nucleoside phosphorylase activity as catalyst. Firstly,2-deoxyribose-1-phosphate dicyclohexylamine salt was presynthesized as one of the substrates of nucleoside phosphorylase by chemical method through three steps,and it was finally collected in the form of precipitate.In next step it was used with 5-fluorouracil to synthesize the product 5-fluoro-2'-deoxyuridine by nucleoside phosphorylase from Bacillus. s2308. In order to improve the substrate conversion rate,relevant parameters are optimized. Four kinds of inducers like adenosine,uridine,are used to investigate their effects on nucleoside phosphorylase activity,and the substrate concentration,bacteria amount,temperature,buffer concentration and p H value of the transformation system are also examined to study their effects on reaction conversion rate. As a result,It was found that by adding 15 mmol / L cytidine in the medium,the activity of nucleoside phosphorylase has been significantly improved after inducing for 27 h,and the optimum conditions for convertion were obtained 35 mmol / L2-deoxyribose-1-phosphate dicyclohexylamine salt,105 mmol / L 5-fluorouracil,phosphate potassium buffer 45 mmol / L( p H 8. 0),intact cell 12%( wet weight) at 55 ℃ for 3 h,the conversion rate can reach up to 79. 36%,while the relative yield can be up to6. 83 g / L. This combined chemical-biologic method of nucleoside synthesis can effectively solve the problems such as the lack of deoxyribose donor source,low conversion rate and product poor stereoselectivity,and it shows a certain value for industrialization.
出处
《药物生物技术》
CAS
2015年第1期9-13,共5页
Pharmaceutical Biotechnology
基金
浙江省科技计划项目(No.2009C13033-1)
