基于结构B因子分析指导的头孢菌素C酰化酶动力学稳定性改造

摘要

[目的]筛选Pseudomonas sp.SE83 acyⅡ定点饱和突变库,获得动力学稳定性提高的头孢菌素C (CPC)酰化酶突变体,并对突变酶进行初步的结构-功能关系分析.[方法]靶标酶Pseudomonas sp.SE83 acyⅡ与Pseudomonas diminuta N176具有较高的同源性,通过分析N176的结构B因子,构建CPC酰化酶SE83定点饱和突变库;基于pH指示剂显色法,采用BiomekFXP自动工作站建立CPC酰化酶高通量筛选方法,获得优良突变酶,对其活性、稳定性等酶学性质进行表征;利用SWISS-MODEL对突变体进行同源建模,探讨突变体结构与功能的关系.[结果]通过B因子分析和同源结构比对,共找出9个靶标位点;经过3轮筛选,发现R218及K226位点突变显著提高酶的热稳定性,其中最显著的R218Q和K226V在40℃的半衰期分别为野生型的3.77和2.77倍,催化效率Kcat/Km分别为野生型的1.8和3.1倍.同源建模分析表明氢键作用和疏水相互作用的增加可能是突变体稳定性提高的原因.[结论]B因子指导的酶分子改造是一种高效可靠的动力学稳定性改造策略,突变体R218Q和K226V均可提高CPC酰化酶的稳定性和催化效率,对进一步的CPC酰化酶分子改造具有一定的参考价值和指导意义.%[Objective] The aim of this study was to obtain a more stable cephalosporin C (CPC) acylase by screening site-directed saturation mutation libraries of Pseudomonas sp.SE83 acyⅡ and to characterize the structure and function of mutant enzyme.[Methods] We computated B factor based on the structure ofPseudomonas diminuta N176,a homologue ofPseudomonas sp.SE83 acyⅡ,and constructed site-directed saturation mutation libraries of SE83 acyⅡ.Combined with pH indicator assay,we used a Biomek FXP automation workstation high-throughput screening method to screen more stable CPC acylase.The enzyme properties were further defined by comparison with the wild type enzyme.The relationship between structure and function of the positive mutants was studied by homology modeling,using SWISS-MODEL software.[Results] We found 9 targeting sites by B factor homologous structure alignment and computation.After 3 rounds of screening,we found that the mutations occurring at residues R218 and K226 could enhance the thermostability of acylase,and the most stable mutants were identified as R218Q and K226V.Their half-lives at 40 ℃ were approximately 3.77-and 2.77-fold of the wild type enzyme,respectively.The catalytic efficiency kcat/Km was also approximately 1.8-and 3.1-fold of the wild type,respectively.The possible mechanism for the enhanced stability might be the increments of hydrogen bonds and hydrophobic interaction,which were analyzed by homology modeling.[Conclusion] It proved to be an efficient and reliable strategy for improving enzymatic stability based on B factor,and the positive mutant R218Q and K226V could enhance the stability and catalytic efficiency of CPC acylase.Thus,this study may serve as a useful reference for further improving enzyme properties.