2-卤代酸脱卤酶的表达优化  

Optimization of 2-haloacid dehalogenase expression

作　　者：王亚月 董超群 靳志远 张萌萌 裴冬丽 WANG Yayue;DONG Chaoqun;Jin Zhiyuan;ZHANG Mengmeng;PEI Dongli(College of Biology and Food,Shangqiu Normal University,Shangqiu 476000,China)

机构地区：[1]商丘师范学院生物与食品学院,河南商丘476000

出　　处：《商丘师范学院学报》2022年第9期46-49,共4页Journal of Shangqiu Normal University

基　　金：河南省高等学校重点科研项目(20A180024);商丘师范学院校级科研启动基金(700144)资助;博士后科研启动基金(50026003);河南省高校科技创新团队(21IRTSTHN025)。

摘　　要：2-卤代酸脱卤酶是催化2-卤代酸脱卤产生相应2-羟基酸的一类酶,其水解脱卤能力和对映体选择性使其在环境修复与光学纯手性化合物绿色合成领域具有广阔应用前景.然而天然2-卤代酸脱卤酶产量通常很低,本文以分离自Pseudomonas putida PP3菌株的DL-2-卤代酸脱卤酶DehI为研究对象,构建DehI-pET28a重组质粒,在大肠杆菌中进行原核表达,并对其表达条件进行优化.经优化获得其最佳表达条件:当细胞OD_(600)nm为0.8,IPTG为0.2 mM,30℃诱导9 h时,活性蛋白量最高,为1.36 U/mL,与常用诱导条件相比提高7.5倍.本研究为2-卤代酸脱卤酶构效关系及其改造研究奠定基础.2-Haloacid dehalogenases catalyze the dehalogenation of the 2-haloacids,releasing halogen ions and producing corresponding 2-hydroxyacids.The enzyme shows hydrolytic dehalogenation and enatioselectivity which makes them promising in the environmental remediation and green synthesis of optically pure chiral compounds.However,the production of natural enzyme is very low.In this study,DehI which is isolated from Pseudomonas putida PP3 strain is selected.The constructed DehI-pET28 a recombinant plasmid is transferred into Escherichia coli competent cells.And the protein expression condition is optimized.The optimum induction conditions is determined to be:induction at OD_(600) 0.8,30 ℃ using IPTG concentration of 0.2 mmol/L,the induction time is 9 h and the rotation speed is 180 r/min.The highest catalytic activity is obtained with 1.36 U/mL crude enzyme,which is 7.5 times higher than the usual induction conditions.It provids the foundation for investigating the structure and function of 2-haloacid dehalogenase and its modification.

关 键 词：2-卤代酸脱卤酶 异源表达 优化 

分 类 号：Q55[生物学—生物化学]