Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp M231 using a computationally aided method

文献类型： 外文期刊

第一作者： Tian, Jian

作者： Tian, Jian;Wang, Ping;Huang, Lu;Chu, Xiaoyu;Wu, Ningfeng;Fan, Yunliu

作者机构：

关键词： Methyl parathion hydrolase;Site evolutionary entropy;Thermostability;Prethermut;Unfolding free energy

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

ISSN： 0175-7598

年卷期： 2013 年 97 卷 7 期

页码：

收录情况： SCI

摘要： Good protein thermostability is very important for the protein application. In this report, we propose a strategy which contained a prediction method to select residues related to protein thermal stability, but not related to protein function, and an experiment method to screen the mutants with enhanced thermostability. The prediction strategy was based on the calculated site evolutionary entropy and unfolding free energy difference between the mutant and wild-type (WT) methyl parathion hydrolase enzyme from Ochrobactrum sp. M231 [Ochr-methyl parathion hydrolase (MPH)]. As a result, seven amino acid sites within Ochr-MPH were selected and used to construct seven saturation mutagenesis libraries. The results of screening these libraries indicated that six sites could result in mutated enzymes exhibiting better thermal stability than the WT enzyme. A stepwise evolutionary approach was designed to combine these selected mutants and a mutant with four point mutations (S274Q/T183E/K197L/S192M) was selected. The T (m) and T (50) of the mutant enzyme were 11.7 and 10.2 A degrees C higher, respectively, than that of the WT enzyme. The success of this design methodology for Ochr-MPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering.

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