自诱导系统在酶促合成2’-脱氧胞苷中的应用

生物技术通报 ›› 2014, Vol. 0 ›› Issue (11): 225-232.

• 研究报告 • 上一篇

自诱导系统在酶促合成2’-脱氧胞苷中的应用

王玺¹,段胜林²,熊舒莉¹,郑桂兰¹,张贵友¹,王洪钟¹

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

收稿日期:2014-03-31 出版日期:2014-11-07 发布日期:2014-11-07
作者简介:王玺,女,硕士,研究方向：生物转化及生物催化
基金资助:
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）

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

Wang Xi¹,Duan Shenglin²,Xiong Shuli¹,Zheng Guilan¹,Zhang Guiyou¹,Wang Hongzhong¹

1. School of Life Sciences, Tsinghua University, Beijing 100084；2. China National Research Institute of Food and Fermentation Industries, Beijing 100015

Received:2014-03-31 Published:2014-11-07 Online:2014-11-07

摘要/Abstract

摘要： 旨在高效合成2’-脱氧胞苷（dCyd）。DCyd作为一类重要的药物中间体,能够用于合成吉西他滨（dFdC）、扎西他滨（ddC）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dCyd。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dCyd方面具有同样高效的催化活性。2’-脱氧胸苷（dThd）和胞嘧啶的浓度比为1∶3时,产物转化率可高达86.5%。合成dCyd的最适反应条件为：2’-脱氧胸苷（dThd）和胞嘧啶的浓度比为1∶1.5,pH6.5的磷酸缓冲液,1 mg/mL的菌体量,终体系10 mL,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。

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

Abstract: 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 by E.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.

Key words: Biocatalysis , N-deoxyribosyltransferase , 2’-deoxycytidine , Auto-induction system, Nucleoside analogues

王玺,段胜林,熊舒莉,郑桂兰,张贵友,王洪钟. 自诱导系统在酶促合成2’-脱氧胞苷中的应用[J]. 生物技术通报, 2014, 0(11): 225-232.

Wang Xi,Duan Shenglin,Xiong Shuli,Zheng Guilan,Zhang Guiyou,Wang Hongzhong. Application of Auto-induction System in the Synthesis of 2’-deoxycytidine[J]. Biotechnology Bulletin, 2014, 0(11): 225-232.

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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

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