甲基对硫磷水解酶的酿酒酵母表面展示及在甲基对硫磷降解中的应用

Surface Display of Methyl Parathion Hydrolase on Saccharomyces cerevisiae and Its Application in Degradation of Methyl Parathion

PCR扩增假单胞菌WBC-3的甲基对硫磷水解酶基因,插入表面展示质粒pYD1的多克隆位点,构建pYD1-MPH重组质粒。重组质粒转化酿酒酵母EBY100,2%半乳糖诱导甲基对硫磷水解酶表达,并利用免疫荧光检测甲基对硫磷水解酶在酿酒酵母细胞表面的表达展示。研究了表面展示甲基对硫磷水解酶的酶学性质和酵母工程菌对水体中甲基对硫磷的降解效果。结果表明成功构建具有全细胞甲基对硫磷水解酶催化活性的酵母工程菌,经2%半乳糖诱导48 h,表面展示甲基对硫磷水解酶比酶活力为18.2 U/mg细胞干重。表面展示甲基对硫磷水解酶的最适作用pH为9.5,最适作用温度为30℃,在p H4.0-10.5之间和45℃以下稳定性较好,Mn2+、Co2+、Zn2+、Ca2+、Hg2+、K+、Ni2+对表面展示甲基对硫磷水解酶活性有激活作用,Na+、Fe3+、Ag+对展示酶活力有抑制作用。工程菌在1 h内对淡水中20 mg/L的甲基对硫磷的降解率在80%以上。

The methyl parathion hydrolase（MPH）gene of Pseudomonas sp.WBC-3 was amplified by PCR and cloned into the multiple cloning site of the surface display vector pYD1 to construct a recombinant plasmid p YD1-MPH. Then plasmid pYD1-MPH was transformed into Saccharomyces cerevisiae EBY100. The 2% galactose was used to induce the expression of MPH on the cell surface of EBYl00, and the display of MPH on the cell surface of S.cerevisiae was confirmed by immunofluorescence. The characteristic of the displayed MPH and degradation effect of methyl parathion in water by the engineered yeast were also investigated. The result showed that the engineered yeast strain, which have a whole cell catalytic activity of MPH, was successfully constructed. The activity of MPH displayed on the yeast cells was 18.2 U/mg of cell dry cells by the induction of 2% galactose for 48 h. The displayed MPH had the optimal pH of 9.5 and the optimal temperature of 30℃, respectively and was stable in the p H range of 4.0-10.5 and up to 45℃. The displayed MPH was stimulated by Mn2＋,Co2＋,Zn2＋,Ca2＋,Hg2＋,K＋,Ni2＋, and was inhibited by Na＋,Fe3＋,Ag＋. The engineered yeast strain could hydrolyze over 80% of 20.0 mg/L methyl parathion in tap water in 1 h.