头孢菌素C乙酰化酶的半理性改造及7-ACA的生物合成  

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

作　　者：马晨露 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] 

机构地区：[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

出　　处：《中国生物工程杂志》2015年第12期65-71,共7页China Biotechnology

基　　金：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

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p ET32a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。CPC acylases play a key role is very important. In order to obtain the CPC Pseudomonas sp SE 83 was taken as parent in producing 7-ACA by one-step enzymatic method, so its research acylase with higher catalytic activity, a type III cPc acylase from 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 reCAIIIM 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.

关 键 词：CPC乙酰化酶 生物催化 分子改造 理性设计 活性检测 

分 类 号：Q819[生物学—生物工程]