枯草芽孢杆菌分泌表达有机磷水解酶突变体和降解沙林活性的研究  

Secretory Expression of Organophosphorus Hydrolase Mutant in Bacillus subtilis and Its Activity of Degrading Sarin

作　　者：顾恰敏 赵传欣 刘敏 陈高云 李丹 GU Qiamin;ZHAO Chuanxin;LIU Min;CHEN Gaoyun;LI Dan(Institute of NBC Defense,Beijing 102205;66072 PLA Troops,Beijing 100041;State Key Laboratory of NBC prevention for Civilian,Beijing 102205)

机构地区：[1]陆军防化学院,北京102205 [2]66072部队,北京100041 [3]国民核生化灾害防护重点实验室,北京102205

出　　处：《生物化工》2022年第1期99-103,共5页Biological Chemical Engineering

基　　金：国民核生化灾害防护国家重点实验室科研基金项目(SKLNBC2018-20)。

摘　　要：有机磷水解酶是一种能够有效水解有机磷酸酯类化合物的酯酶,天然含有机磷水解酶的生物体中分离的有机磷水解酶存在水解速率较慢、产量低、提纯复杂等问题。本文通过枯草芽孢杆菌分泌表达有机磷水解酶,提高有机磷水解酶产量。同时对有机磷水解酶基因进行点突变,构建了H254R-H257Y-L303T三位点联合突变的突变体,用联苯胺定量法比较野生型和突变体水解神经性毒剂沙林的能力。结果表明,该枯草芽孢杆菌表达体系能够稳定分泌表达有机磷水解酶,野生型有机磷水解酶水解沙林的活性为0.407 mg/(min·mg),含有突变体的活性为0.790 mg/(min·mg),较野生型有机磷水解酶提升了94.1%,水解能力显著增强。通过BIOVIA软件模拟有机磷水解酶结构,发现突变体稳定性优于野生型有机磷水解酶。本枯草芽孢杆菌表达体系能够稳定分泌表达有机磷水解酶,具有较好的生物活性,突变体比野生型有机磷水解酶结构更加稳定,水解能力得到了提升,是一个比较理想的突变方向。Organophosphorus hydrolase(OPH)is an esterase that can effectively hydrolyze organic phosphate compounds.OPH isolated from organisms containing natural OPH have some disadvantages,such as slow hydrolysis rate,low yield,complex purification and so on.OPH by Bacillus subtilis are erpressed to improve the yield of OPH and simplify the means of protein purification in this paper.Meanwhile,point mutation of OPH gene were carried out,hoping to improve the ability of OPH to hydrolyze organic phosphate compounds.Methods:Bacillus subtilis expression system was used to secretory expresses OPH and OPH mutants containing H254R-H257Y-L303T combined mutation,and their ability to hydrolyze nerve agent sarin was detected by benzidine quantitative method.Results:the expression system of Bacillus subtilis could stably secrete the expressed OPH.The activity of wild-type OPH in hydrolyzing sarin was 0.407 mg/(min·mg).The activity of hydrolyzing sarin by the organic phosphorus hydrolase mutant containing H254R-H257Y-L303T mutation site was 0.790 mg/(min·mg),which was 94.1%higher than that of the wild-type organic phosphorus hydrolase.The structure of OPH is simulated by biovia and the results show that the stability of H254R-H257Y-L303T mutant is better than that of wild-type OPH.Conclusion:The expressed OPH,which has good biological activity,can be stably secrete by the expression system of Bacillus subtilis.The OPH mutant with H254R-H257Y-L303T mutation site selected in this paper has more stable structure and improved hydrolysis ability than the wild-type OPH,which is an ideal mutation direction.

关 键 词：有机磷水解酶 分泌表达 点突变 水解 沙林 

分 类 号：S852.61[农业科学—基础兽医学]