Abstract:

Methyl parathion hydrolase can biodegrade organophosphorus pesticides. Due to its special application value, researchers paid more and more attension. Wild-type MPH-Och from Ochrobactrum sp. could not be secreted in Pichia pastoris. In order to increase the secretion level, the MPH-S274Q mutant, which thermal stability was increased, was expressed in Pichia pastoris. After recombinant strain was induced by shaking culture flask for 120 h, the enzyme activity of culture supernatant reached 0.7 U/mL. It was 14 times of that in wild type MPH-Och. The SDS-PAGE electrophoresis showed that the culture supernatant of mutant MPH-S274Q had a distinct protein band, which further proved the improvement of secretion efficiency. This study indicated that improving protein thermal stability can be an effective way to increase secretion efficiency of exogenous protein in Pichia pastoris.

Key words: methyl-parathion hydrolase, secretion, thermal stability, Pichia pastoris

摘要：

甲基对硫磷水解酶可生物降解有机磷农药，由于其特殊的应用价值，越来越受到人们的关注。来源于苍白杆菌的甲基对硫磷水解酶MPH-Och在毕赤酵母系统中不能有效地分泌表达。将热稳定性提高的突变体MPH-S274Q在毕赤酵母中表达，发现其毕赤酵母重组菌株经摇瓶诱导培养120 h后，培养上清的酶活力达到0.7 U/mL，蛋白分泌量是野生型的14倍，SDS-PAGE电泳图中培养上清有明显的蛋白质条带，进一步证明了分泌效率提高,该结果表明提高蛋白质的热稳定性可以作为提高外源蛋白在毕赤酵母中的分泌效率的一条有效途径。

关键词: 甲基对硫磷水解酶;分泌;热稳定性;毕赤酵母