南京农业大学学报2024，Vol.47Issue(4)：750-759,10.DOI:10.7685/jnau.202306013
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
摘要
Abstract
[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 Km values of mutant enzymes S2,S10 and S12 were 56.89,27.00 and 40.65 μmol·L-1,respectively,which depicted 91.7%,43.5%and 65.5%of the wild type,respectively,while the kcat/Km 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.关键词
呕吐毒素解毒酶/定向进化/催化活性/分子动力学模拟Key words
deoxynivalenol-degrading enzyme/directed evolution/catalytic activity/molecular dynamics simulations分类
轻工纺织引用本文复制引用
李越,池慧兵,牛家峰,马斌,周慧敏,朱萍,吕凤霞..基于定向进化技术提高呕吐毒素解毒酶DepA的催化活性[J].南京农业大学学报,2024,47(4):750-759,10.基金项目
国家自然科学基金项目(32272267) （32272267）
