组合突变提高甲基对硫磷水解酶MPH热稳定性的研究

Studies on Enhancing Thermostability of Methyl Parathion Hydrolase by Combinatorial Mutagenesis

来源于苍白杆菌Ochrobactrum sp.M231的甲基对硫磷水解酶MPH-Och具有优良的催化水解甲基对硫磷农药的能力,针对其热稳定性较差的缺点,通过组合3种提高蛋白热稳定性的突变策略,最终获得热稳定性改良的突变酶MPHM7,其T50达到65℃,在之前研究热稳定性提高的四点突变酶(S274Q/T183E/K197L/S192M)的基础上又提高了5℃。同时,该突变酶的催化效率也获得了一定的提高,其kcat/Km值是四点突变酶的3.8倍。热稳定性改良突变酶的获得证实了这些提高热稳定性的方法是有效的,并且其作用具有加合性,这为分子设计改良酶蛋白热稳定性提供了新的研究思路。

The methyl parathion hydrolase（ MPH-Och） from Ochrobactrum sp. M231 exhibits better catalytic activity than other MPHs. In order to increase enzyme thermostability,3 different strategies were used with combinatorial mutagenesis. As a result,the T50 of the mutant enzyme MPHM7 was approximately 65℃. The temperature was 5℃higher than that of MPHM4,the mutant with 4 point mutations（ S274 Q / T183 E / K197 L / S192M）. At the same time,the catalytic capacity（ kcat/ Km） of MPHM7 was up to 3. 8-fold of MPHM4. The success of these design methodology for MPH suggested that it was an efficient strategy for enhancing thermostability and it had additivity property. This study had provided a new thought for molecular designing to improve enzyme protein thermostability.