头孢菌素酰化酶高β-折叠区保守氨基酸附近位点突变研究  

Amino acid substitution for the vicinity of many conserved amino acids in theβ-sheet region of the cephalosporin acylase

作　　者：任羽 常瑞 王继莲 马刘峰 王东 REN Yu;CHANG Rui;WANG Ji-lian;MA Liu-feng;WANG Dong(Key Laboratory of Ecological and Biological Resources Research of the Yarkand Oasis,Kashi University,Kashi 844000,China)

机构地区：[1]喀什大学叶尔羌绿洲生态与生物资源研究重点实验室,喀什844000

出　　处：《生物学杂志》2020年第5期19-23,共5页Journal of Biology

基　　金：新疆维吾尔自治区自然科学基金(201442137-10);新疆维吾尔自治区优秀科技人才项目(2017Q031);新疆维吾尔自治区科研创新团队(2014751002);喀什大学高层次人才引进计划项目(GCC14ZK-001)。

摘　　要：头孢菌素酰化酶(Cephalosporin acylase)能够一步催化头孢菌素C(Cephalosporin C,简称CPC)生成7-氨基头孢烷酸(7-Aminocephalosporanic acid,简称7-ACA),后者是重要的医药中间体。但头孢菌素酰化酶对CPC的一步催化活性低,远未达到工业生产的要求。研究以来源于Pseudomonas sp.SE83头孢菌素酰化酶(CPCase)蛋白序列为模板,对其基因进行全局优化设计,人工合成目的基因,并克隆至表达载体pET28a,实现了CPCase的高效表达,命名为WT。将其与来自Pseudomonas SY-77、Pseudomonas N176等菌株的头孢菌素酰化酶氨基酸序列进行相似性比较,在保守氨基酸附近选择了25个突变位点,采用定点突变的方法分别突变为Ala,获得了25个突变酶。结果表明,突变体蛋白F311A对CPC的催化活性上升50%,k cat/K m提高到原来的1.7倍,比酶活达到4.302 U/mg,其他突变体对CPC催化活性均有不同程度的下降。结构分析显示,第311的Phe突变为Ala,能够使结合口袋变大,减少空间位阻,使酶与底物CPC的结合更加牢固,提高催化效率。Cephalosporin acylase can catalyze cephalosporin C(CPC)to produce 7-amino cephalosporanic acid(7-ACA)which is the important pharmaceutical intermediate.However,the cephalosporin C acylase has low catalytic activity for CPC and is far from the requirements of industrial production.The gene of cephalosporin acylase(CPCase)from Pseudomonas sp.SE83 was synthesized,optimized and cloned into the vector pET28a to achieve high expression of CPCase,named WT.By comparing WT with the sequences of cephalosporin acylase from Pseudomonas SY-77,Pseudomonas N176 and CAD,25 sites were selected near the conserved amino acids and 25 mutants were obtained by the site-directed mutagenesis.The results from enzyme activity showed that the catalytic activity of the mutant F311 was increased by 50%and others had decreased activity to CPC.The specific activity of F311 was 4.302 U/mg and the k cat/K m was 1.7 times to that of the WT.The structural analysis revealed that the F311A mutant made the binding pocket larger,the binding to the substrate CPC was stronger,and the catalytic efficiency was improved.

关 键 词：头孢菌素酰化酶 7-氨基头孢烷酸 定点突变 头孢菌素C 

分 类 号：Q517[生物学—生物化学] TQ465.9[化学工程—制药化工]