基于定向进化技术提高呕吐毒素解毒酶DepA的催化活性

Enhanced catalytic activity of deoxynivalenol-degrading enzyme DepA by directed evolution

[目的]呕吐毒素(deoxynivalenol,DON)C3位羟基的异构化是生物脱毒的关键部位,其中DepA作为DON生物转化中第一步关键限速酶,存在催化活性低的问题,限制了其在食品及饲料加工领域中的应用。本文旨在通过定向进化技术对DepA进行分子改造,以期获得催化活性显著提高的突变酶,提高其工业化应用的潜力。[方法]通过易错PCR和DNA shuffling相结合的方法,构建突变文库,经高通量筛选后,对催化活性明显提高的突变体进行酶学性质表征。同时,采用分子动力学模拟和三维结构模拟分析,探究突变酶催化活性提高的分子机制。[结果]成功筛选到突变酶S2、S10和S12,与野生型相比,其比活力分别提高207%、293%和258%。动力学参数结果显示,突变酶S2、S10和S12的K m值分别为56.89、27.00和40.65μmol·L^(-1),是野生型的91.7%、43.5%和65.5%;k_(cat)/K_(m)值分别为327.30、576.67和380.07 L·mmol^(-1)·s^(-1),是野生型的1.25、2.20和1.45倍,相比野生型,突变酶亲和力和催化效率均显著提高。此外,分子动力学模拟和三维结构模拟分析结果表明,柔性增加、底物DON的进攻距离缩短、氢键增加、疏水性增加和远端效应是导致其催化活性提高的主要原因。[结论]本研究通过易错PCR和DNA shuffling相结合的定向进化策略有效提高了呕吐毒素解毒酶DepA的催化活性,为其工业化应用提供试验依据。

[Objectives]Deoxynivalenol(DON)can be biotoxified by hydroxyl isomerization at the C3 position.DepA,a rate-limiting enzyme,plays a critical role in the initial step of DON biotransformation;however,its low enzymatic activity restricts its potential for food and feed processing industrial application.To enhance its catalytic activity and improve its application potential,a directed evolution approach to molecularly alter DepA and obtain mutants with highly improved activity was utilized.[Methods]The mutant library was created using a mixture of error-prone PCR and DNA shuffling.After high-throughput screening,mutants with significantly increased enzyme activity were described in terms of their enzymatic properties.Additionally,molecular dynamics simulations and three-dimensional structure simulations were conducted in order to study the molecular mechanism behind the improved catalytic activity of the mutant enzymes.[Results]The mutant enzymes S2,S10 and S12 were successfully screened,which increased the specific activity by 207%,293%and 258%compared with the wild type.The kinetic parameters results displayed that the K m values of mutant enzymes S2,S10 and S12 were 56.89,27.00 and 40.65μmol·L^(-1),respectively,which depicted 91.7%,43.5%and 65.5%of the wild type,respectively,while the k_(cat)/K_(m) values were 327.30,576.67 and 380.07 L·mmol^(-1)·s^(-1),which were 1.25,2.20 and 1.45 folds higher than those of the wild type,and affinity and catalytic efficiency of the mutant enzyme were significantly improved compared with the wild type.Meanwhile,the results of molecular dynamics simulations and three-dimensional structure simulations showed that the increased flexibility,shorter distance of the substrate DON and Asp304,increased hydrogen bond,increased hydrophobicity and distal effects were the primary factors contributing to the increased enzymatic activity.[Conclusions]The current investigation successfully enhanced the catalytic activity of deoxynivalenol-degrading enzyme DepA by employing a directed evolution strategy with error-prone PCR and DNA shuffling,thus forming a foundation for potential industrial application.