自诱导系统在酶促合成2’-脱氧胞苷中的应用  

Application of Auto-induction System in the Synthesis of 2'-deoxycytidine

作　　者：王玺[1] 段胜林[2] 熊舒莉 郑桂兰[1] 张贵友[1] 王洪钟[1] 

机构地区：[1]清华大学生命科学学院,北京100084 [2]中国食品发酵工业研究院,北京100015

出　　处：《生物技术通报》2014年第11期225-232,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(21176138);国家自然科学基金国家基础科学人才培养基金项目(J1103506;J1030622;J1310020)

摘　　要：旨在高效合成2’-脱氧胞苷(d Cyd)。DCyd作为一类重要的药物中间体,能够用于合成吉西他滨(d Fd C)、扎西他滨(dd C)等多种抗病毒或抗肿瘤的药物。采用ZYM-Fe-5052自诱导培养基对含有N-脱氧核糖转移酶II(NDT)的大肠杆菌BL21(DE3)进行诱导,所得完整菌体直接用于催化合成d Cyd。结果表明,自诱导系统不仅无需额外添加诱导剂,还能够达到较高的菌体密度,且与LB诱导系统所得菌体在合成d Cyd方面具有同样高效的催化活性。2’-脱氧胸苷(d Thd)和胞嘧啶的浓度比为1∶3时,产物转化率可高达86.5%。合成d Cyd的最适反应条件为:2’-脱氧胸苷(d Thd)和胞嘧啶的浓度比为1∶1.5,p H6.5的磷酸缓冲液,1 mg/m L的菌体量,终体系10 m L,30℃条件下反应1 h,产物转化率可达71.9%。此方法还可用于制备其他核苷类似物,具有广泛的适用性和应用前景。This experiment was carried out in order to synthesize 2’-deoxycytidine（dCyd）more efficiently. DCyd is a significant intermediate for many antiviral and anticancer drugs such as Gemcitabine（dFdC）, Dideoxycytidine（ddC）, etc. A convenient and efficient bioprocess was reported here to synthesize dCyd byE.coli BL21（DE3）containing gene of N-deoxyribosyltransferaseⅡ（NDT）over-expressed using ZYM-Fe-5052 auto-induction system. The results showed that auto-induction system could make the addition of inducer during cultivation unnecessary and obtain a higher cell density. The recombinant cells from auto-induction system were as efficient as that from LB system on the conversion yields of dCyd. When the ratio of 2’-deoxythymidine（dThd）and cytosine was 1∶3, the conversion yield could be 86.5%. The substrates ratio of 2’-deoxythymidine （dThd） and cytosine as 1∶1.5 and 1 mg/mL recombinant cells which were dissolved in phosphate buffer of pH6.5, then cultured at 30℃ for 1 h were selected as the suitable reaction conditions,and the conversion yield could be 71.9%. This method also has potential for the preparation of other nucleoside analogues.

关 键 词：生物催化 N-脱氧核糖转移酶 2’-脱氧胞苷 自诱导系统 核苷类似物 

分 类 号：TQ460.1[化学工程—制药化工]