烷基卤去卤化酶对芥子气的催化水解  

Catalytic hydrolysis of sulfur mustard by haloalkane dehalogenases

作　　者：郭楠[1] 董亮[2] 刘景全[1] 董志扬[2] 钟近艺[1] 孔令策[1] 

机构地区：[1]防化研究院,北京102205 [2]中国科学院微生物研究所,北京100101

出　　处：《环境化学》2015年第7期1363-1370,共8页Environmental Chemistry

摘　　要：本文通过密码子优化、高效表达并纯化出三种烷基卤去卤化酶DhaA、DmbA与LinB,评价了它们对芥子气的水解效率,分析并优化了酶促水解过程中需要调控的机制.通过实验,最终确定了活性成分为DhaA,缓冲剂为HEPES-NaOH,助溶剂为丙三醇的芥子气高效生物酶洗消体系,使用高效液相色谱质谱确定该体系的洗消产物为无毒硫二甘醇.酶活实验及对芥子气的消毒效果评价结果表明:该洗消体系对芥子气的最佳水解pH值为8.6,kcat/Km值为9.6s-1mmol·L-1,是国外同类水解酶的1.7倍,水解速率为自然水解速率的320倍,对10mg·mL-1芥子气的水解率从27.4%提高至93.2%,较好解决了高浓度芥子气高效水解脱毒问题.Sulfur mustard （HD） enzymatic decontaminantion has always been the difficult point in chemical agent defence. The requirement of battlefield decontamination has not been satisfied because of the low degradation efficiency of HD by enzymatic products from abroad. Herein, three haloalkane dehalogenases, including DhaA, DmbA and LinB, were codon optimized, over expressed and purificated. Using these haloalkane dehalogenases, hydrolysis efficiency of HD was evaluated. Hydrolysis mechanisms of HD catalyzed by haloalkane dehalogenase were further analyzed and optimized. Based on the experiment result, an efficient HD enzymatic decontaminantion system composed of main active component DhaA developed. HPLC-MS result illustrated that , buffer HEPES-NaOH and co-solvent glycerol was the main product was non-toxia thiodiglycol. The experiment and evaluation results showed that the optimum pH of HD enzymatic catalysis was 8.6, HD enzymatic decontaminant＇s kcat/Km was 9.6 s^-1 mmol· L^-1, which was 1.7-fold higher than the haloalkane dehalogenase developed abroad researchers. The enzymatic hydrolytic ratio rate of HD was 320 times faster than the uncatalyzed hydrolytic ratio of HD. Degradation rate of HD （ Co = 10 mg· mL^-1） was improved from 27.4% to 93.2% by the decontamination system we developed. The low degradation efficiency of high initial concentration of HD was solved by our enzymatic degradation system.

关 键 词：烷基卤去卤化酶 芥子气 催化水解. 

分 类 号：O621.251[理学—有机化学]