Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China.
Jiangnan University (Rugao) Food Biotechnology Research Institute, Rugao 226500, China.
Molecules. 2020 Oct 19;25(20):4806. doi: 10.3390/molecules25204806.
High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from JCM3095 (NHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature () of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type NHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.
高温稳定性和催化活性是腈水合酶（NHase，EC 4.2.1.84）作为一种工业化成熟的催化剂的关键特性。在这项研究中，通过结合 FireProt 服务器预测和分子动力学（MD）模拟，对来自 JCM3095（NHase）的 NHase 的热稳定性进行了合理设计。随后对合理设计提供的非催化残基进行了定点突变。阳性多点突变体，即 M10（αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V），被获得并进一步分析。M10 突变体的熔点（）升高了 3.2°C，并且在高温下观察到酶的残余活性也有显著提高。此外，与野生型 NHase 相比，M10 突变体的催化活性也提高了 2.1 倍。分子对接和 MD 模拟表明，突变体具有更好的底物亲和力和提高的热稳定性。
