头孢菌素C酰化酶突变位点的研究

doi:10.13523/j.cb.20150209

中国生物工程杂志

2015, Vol. 35

Issue (2): 59-65 DOI: 10.13523/j.cb.20150209

技术与方法

头孢菌素C酰化酶突变位点的研究

徐雪丽^1,2, 张伟¹, 刘艳¹, 谢丽萍¹, 胡又佳¹

1. 中国医药工业研究总院张江分院上海 201203;
2. 上海医药工业研究院上海 200437

Study on Mutations of Cephalosporin C Acylase

XU Xue-li^1,2, ZHANG Wei¹, LIU Yan¹, XIE Li-ping¹, HU You-jia¹

1. China State Institute of Pharmaceutical Industry Zhangjiang Institute, Shanghai 201203, China;
2. Shanghai Institute of Pharmaceutical Industry, Shanghai 200437, China

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摘要：

头孢菌素C酰化酶能直接将头孢菌素C(CPC)转化为7-氨基头孢烷酸(7-ACA),此一步酶法具有很大的经济价值,所以得到很多研究者的关注,特别是提高CPC酰化酶活性及专一性的研究。以CPC酰化酶基因 ecs50 为基础,利用重叠延伸PCR,对文献报道的活性提高的突变体酶S12的6个位点分别进行突变,将得到的6个突变体V122A、G140S、F297N、I314T、I415V、S710L进行诱导纯化后,检测酶活,以此来研究不同突变位点对酶活力的影响。结果V122A的比活为106U/mg,它的转化效率比初始酶提高23.7%。

关键词： CPC酰化酶; 重叠延伸PCR; 酶活; 头孢菌素C; 7-氨基头孢烷酸

Abstract:

Cephalosporin C acylase can directly convert cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA), and this one-step enzymatic process has great economic value. A lot of researchers pay attention on it, especially on how to improve the activity and specificity of CPC acylase. The reported CPC acylase mutant S12 has an improved activity that contains 6 point mutations. On the basis of ecs50 gene, six individual mutations (V122A,G140S,F297N,I314T,I415V,S710L) were obtained by using the overlap PCR. Then recombinant proteins were purified after induction, enzymatic activities were tested to study the relationship between different mutation site and the activity of CPC acylase. The result showed that mutant V122A had an increase activity by 23.7% compared to ECS50.

Key words: CPC acylase Overlap extension PCR Enzyme activity Cephalosporin C 7-aminocephalosporanic acid

收稿日期: 2014-11-11 出版日期: 2015-02-25

ZTFLH:

Q819

基金资助:

国家"十二五"重大新药科技专项(2011ZX09203-001-06)、上海市科委科技支撑项目(1343100204)资助项目

通讯作者: 胡又佳 E-mail: bebydou@126.com

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引用本文:

徐雪丽, 张伟, 刘艳, 谢丽萍, 胡又佳. 头孢菌素C酰化酶突变位点的研究[J]. 中国生物工程杂志, 2015, 35(2): 59-65.

XU Xue-li, ZHANG Wei, LIU Yan, XIE Li-ping, HU You-jia. Study on Mutations of Cephalosporin C Acylase. China Biotechnology, 2015, 35(2): 59-65.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150209 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I2/59

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