定点突变提高D-氨甲酰水解酶的可溶性表达  

Improving D-carbamoylase Soluble Expression by Site-directed Mutagenesis

作　　者：袁野[1,2] 蔡渊恒[2] 姜世民[2] 姜卫红[2] 

机构地区：[1]吉林大学畜牧兽医学院,长春130062 [2]中科院植物生理生态研究所,上海200032

出　　处：《生物技术通报》2012年第1期94-98,共5页Biotechnology Bulletin

基　　金：中国科学院知识创新工程重要方向项目(KSCX2-YW-G-018);国家重点基础发展研究计划(2007CB707803)

摘　　要：采用定点突变的方法对皮氏伯克霍尔德氏菌(Burkholderia pickettii)来源的D-氨甲酰水解酶(D-carbamoylase,DCase)编码基因的3个位点A18、Y30、K34进行突变,并将获得的突变体基因片段构建入高表达载体pET-28b中,转化E.coliBL21(DE3),获得带有组合三突变(A18E/Y30D/K34E)的DCase-SM表达菌株BL21/pET-DCSM。当以IPTG诱导目的蛋白表达时,发现突变菌株(DCase-SM)与出发菌株(DCase)菌株相比,目的蛋白的可溶性表达显著提高,其可溶蛋白比例约为64%;与出发菌株相比,其单位菌体酶活增加427%;另外,与本实验室前期构建的高可溶性三叠加突变体菌株DCase-M3相比,单位菌体酶活亦增加7.9%。D-carbamoylase（DCase） is an important industrial enzyme involved in producing medicine intermediate D-hydroxyphenylg lycine.At present,overexpression of DCase in E.coli is the main biological approach for producing this enzyme,but one common problem during this process is that most of the target protein product accumulates in inclusion body,thus largely limiting the product yield.In this study,through in vitro simultaneous site-directed mutagenesis of three amino acid residues of Burkholderia pickettii DCase（A18,Y30,K34） as well as followed vector-dependent（pET-28b） introduction of this mutated DCase（DCase-SM） into E.coli BL21（DE3）,we obtained the mutant strain BL21/pET-DCSM.With IPTG induction,the Dcase-SM,expressed in strain BL21/pET-DCSM,showed significant improvement in solubility,which occupied 64% of total expressed target protein.Furthermore,as compared to that of the parent strain,the enzyme activity per unit microbial biomass of strain BL21/pET-DCSM increased by 427%;even compared to the previous mutant strain DCase-M3,which exhibited excellent DCase solubility,strain BL21/pET-DCSM still showed 7.9% higher enzyme activity,demonstrating a promising application prospect.

关 键 词：D-氨甲酰水解酶 定点突变 诱导表达 可溶性 酶活 

分 类 号：Q78[生物学—分子生物学]