S-2-氯丙酸脱卤酶的定向进化及其应用  

Directed Evolution and Application of S-2-CPA Dehalogenase

作　　者：刘鹏[1] 林春娇[1] 杨立荣[1] 徐刚[1] 吴坚平[1] 

机构地区：[1]浙江大学化学工程与生物工程学系,杭州310027

出　　处：《中国生物工程杂志》2012年第5期66-72,共7页China Biotechnology

基　　金：国家"973"计划(2011CB710805);国家自然科学基金面上项目(21076187)资助项目

摘　　要：为提高S-2-氯丙酸脱卤酶的活力,通过易错PCR的方法将源于假单胞菌(Pseudomonas sp.CGMCC 3267)的脱卤酶(DehII)进行定向进化,进化酶DehII-B2的比活提高3.9倍。同源模建两者的三维结构,并与底物进行分子对接。结果表明,突变位点为A7I,进化酶DehII-B2的结合能比原始酶降低了1.4kcal/mol,活性中心Asp10与底物α碳原子的距离缩短了0.321 6nm,因而加快了酶反应速率、提高了酶比活。目前,该酶的比活高于实验室已得酶。与原始酶相比,其最适温度和热稳定性略有增加,最适pH和pH稳定性没有明显变化。对该酶的应用做了初步的探索,结果表明在40℃,60mmol/L底物浓度下反应10h,转化率达到49.6%,ees>99.9%,因此该酶有一定的应用价值。To enhance the functionality of dehalogenase from Pseudomonas sp.CGMCC 3267,Used error-prone PCR to create mutants with improved specific activity.Through error-prone PCR and high-throughput screening method,a desirable mutant DehII-B2 was obtained.Compare to initial strain,the specific activity of DehII-B2 was increased 3.9-fold.Three-dimensional structures of DehII-S and DehII-B2 were constructed by homology modeling,and molecular docking of dehalogenase and S-2-CPA.The results suggested that the mutant site is Ala7,the binding energy of DehII-B2 is reduced 1.4kcal/mol than DehII-S,and the distance of active site Asp10 and the α carbon atom of substrate is shorten 0.321 6nm,thus accelerate the enzyme reaction rate and increased enzyme specific activity.At present,the specific activity of DehII-B2 was higher than all enzyme in our lab.According to the results,the optimum pH and pH-stability were not changed,but the optimum temperature and thermo-stability of DehII-B2 were slightly increased than those of initial strain.The preliminary application of DehII-B2 was studied,and the best result was obtained at 40℃,60mmol/L 2-CPA reacting for 10h.

关 键 词：2-氯丙酸 脱卤酶 定向进化 分子对接 

分 类 号：Q556[生物学—生物化学]