Pesticides are indispensable in today’s agriculture. Methyl-parathion hydrolase (MPH, E.C.3.1.8.1) could hydrolyze organophosphorus pesticides, including methyl-parathion. MPH could rehabilitate soil and water resources contaminated by organophosphorus pesticides. However, natural MPHs generally exhibited a low tolerance to high temperatures and low catalytic efficiency. In this study, we improved the catalytic efficiency toward methyl-parathion and the thermal stability of the MPH from Pseudomonas sp. WBC-3 through saturation mutagenesis and fusion with self-assembling amphipathic peptides (SAP). The experimental characterization showed that compared to the wild-type enzyme, the kcat/Km of the mutant T271S yielded by saturation mutagenesis was increased by 224.3% compared to the wild-type MPH. T50 and Tm of SAP3-MPH with an SAP fused at the N-terminus were increased by 6.2°C and 6.0°C, respectively. Compared to the wild-type enzyme, T271S fused with SAP3 at the N-terminus (SAP3-T271S) exhibited a 2.1-fold increase in kcat/Km without significantly affecting the thermal stability. The simultaneous improvement of the catalytic efficiency and thermal stability of MPH would be beneficial for its application in the degradation and detection of organophosphorus pesticides. Furthermore, our study provides a potential combination strategy for the design of the other enzyme preparations of pollutant degradation.

中文翻译：

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提高甲基对硫磷水解酶降解甲基对硫磷农药的催化活性和热稳定性

农药在当今的农业中是不可或缺的。甲基对硫磷水解酶 (MPH, EC3.1.8.1) 可以水解有机磷农药，包括甲基对硫磷。MPH可以恢复被有机磷农药污染的土壤和水资源。然而，天然MPHs通常表现出对高温的低耐受性和低催化效率。在这项研究中，我们提高了对甲基对硫磷的催化效率和来自假单胞菌的 MPH 的热稳定性。WBC-3 通过饱和诱变和与自组装两亲肽 (SAP) 融合。实验表征表明，与野生型酶相比，饱和诱变产生的突变体T271S的kcat/Km与野生型MPH相比增加了224.3%。SAP3-MPH 的 T50 和 Tm 与 N 端融合的 SAP 分别增加了 6.2°C 和 6.0°C。与野生型酶相比，在 N 端与 SAP3 融合的 T271S (SAP3-T271S) 的 kcat/Km 增加了 2.1 倍，而对热稳定性没有显着影响。同时提高MPH的催化效率和热稳定性有利于其在有机磷农药降解和检测中的应用。此外，我们的研究为其他污染物降解酶制剂的设计提供了一种潜在的组合策略。同时提高MPH的催化效率和热稳定性有利于其在有机磷农药降解和检测中的应用。此外，我们的研究为其他污染物降解酶制剂的设计提供了一种潜在的组合策略。同时提高MPH的催化效率和热稳定性有利于其在有机磷农药降解和检测中的应用。此外，我们的研究为其他污染物降解酶制剂的设计提供了一种潜在的组合策略。