头孢菌素C乙酰化酶的半理性改造及7-ACA的生物合成

doi:10.13523/j.cb.20151210

中国生物工程杂志

2015, Vol. 35

Issue (12): 65-71 DOI: 10.13523/j.cb.20151210

技术与方法

头孢菌素C乙酰化酶的半理性改造及7-ACA的生物合成

马晨露¹, 唐存多¹, 史红玲¹, 王瑞², 岳超¹, 夏敏¹, 邬敏辰², 阚云超¹

1. 南阳师范学院昆虫生物反应器河南省工程实验室南阳 473061;
2. 江南大学无锡医学院无锡 214122

Semi-rational Modification of Cephalosporin C Acylase and Biosynthesis of 7-ACA

MA Chen-lu¹, TANG Cun-duo¹, SHI Hong-ling¹, WANG Rui², YUE Chao¹, XIA Min¹, WU Min-chen², KAN Yun-chao¹

1. Henan Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University, Nanyang 473061, China;
2. Wuxi Medical School, Jiangnan University, Wuxi 214122, China

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摘要：

CPC乙酰化酶是一步酶法制备7-ACA的关键酶,针对它的研究具有重大的经济价值。为了获得对CPC具有更高催化活性的CPC乙酰化酶,以Pseudomonas sp SE 83来源的Ⅲ型CPC乙酰化酶CA Ⅲ为亲本,借助分子对接的手段确定了它与CPC结合的关键氨基酸残基,并确定将这些关键氨基酸残基突变为侧链基团更小的氨基酸残基,对CA Ⅲ的编码基因利用多点定点突变试剂盒完成定点突变后借助pET32a质粒在E. coli BL21(DE3)中实现了可溶性表达,获得了对CPC催化活性更高的重组突变体reCA Ⅲ^M,其比酶活为26.7 IU/mg,较原酶提高了3.44倍。此外,初步研究了利用reCA Ⅲ^M进行一步酶法生产7-ACA的工艺,40 IU/g CPC的加酶量、25℃的条件下反应12 h,CPC的转化率和7-ACA的得率分别可达96.3%和63.4%,表明该酶具有良好的应用前景。CPC乙酰化酶的分子改造上取得的较为理想的结果,为该酶进一步的分子改造及应用奠定了坚实的基础,也为其它酶的分子改造提供了可资借鉴的经验。

关键词： 理性设计; CPC乙酰化酶; 分子改造; 活性检测; 生物催化

Abstract:

CPC acylases play a key role in producing 7-ACA by one-step enzymatic method, so its research is very important. In order to obtain the CPC acylase with higher catalytic activity, a type Ⅲ CPC acylase from Pseudomonas sp SE 83 was taken as parent. Some key amino acid residues of CAⅢ were determined using molecular docking approach, and substituted the residues with lesser side chains for those. Then, the coding gene of CAⅢ was site-directed mutagenesis by using Multi Site-Directed Mutagenesis Kit, it was expressed in E. coli BL21(DE3) by means of pET32a plasmid. The recombinant mutant reCAⅢ^M with higher catalytic activity towards CPC, its specific activity is 26.7 IU/mg, which improved by 3.44 folds to the parent. Moreover, its application in producing 7-ACA by one-step enzymatic method were preliminarily studied. With 40 IU/g CPC of enzyme dosage at 25℃ catalyzed for 12 h, the transformation rate of CPC and yield of 7-ACA were up to 96.3% and 63.4% respectively, which demonstated that this enzyme had a promising prospect in application. The ideal results on the CPC acylase molecular modification were obtained, and it laid a solid foundation for further molecular modification and application of this enzyme. In addition, this also provided referential experience for molecular modification of other enzymes.

Key words: Biocatalysis Rational design CPC acylase Molecular modification Activity assay

收稿日期: 2015-07-06 出版日期: 2015-12-22

ZTFLH:

Q819

基金资助:

国家自然科学基金(31401989)、河南省高等学校重点科研项目计划(15A416008)资助项目

通讯作者: 邬敏辰, 阚云超 E-mail: kanyunchao@163.com;bioch@163.com

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引用本文:

马晨露, 唐存多, 史红玲, 王瑞, 岳超, 夏敏, 邬敏辰, 阚云超. 头孢菌素C乙酰化酶的半理性改造及7-ACA的生物合成[J]. 中国生物工程杂志, 2015, 35(12): 65-71.

MA Chen-lu, TANG Cun-duo, SHI Hong-ling, WANG Rui, YUE Chao, XIA Min, WU Min-chen, KAN Yun-chao. Semi-rational Modification of Cephalosporin C Acylase and Biosynthesis of 7-ACA. China Biotechnology, 2015, 35(12): 65-71.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20151210 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I12/65

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