米曲霉LY-128广谱有机磷农药水解酶的纯化和鉴定*

米曲霉LY-128的培养物经硫酸铵分级沉淀,Sephadex G-100 凝胶过滤, DEAE-Sephrose CL-6B 和Sephadex G-100层析手段,获得了电泳纯的广谱有机磷农药水解酶。通过SDS-PAGE 和IEF电泳测得其分子量为62 kDa, 等电点为pH 5.2。该酶的最适反应温度为45℃,最适 pH 6.8, 在50℃以下及pH6.0～9.5 范围内活性稳定。Hg2+、Fe3+、对氯高汞苯甲酸、碘乙酸和N-乙基马来酰亚胺对该酶有强烈的抑制作用,而Cu2+、 巯基乙醇、二硫苏糖醇、二硫赤藓糖醇、谷光甘肽和去污剂对酶有不同程度的激活作用。底物的专一性实验表明,该酶不仅可以作用于含P-O键的有机磷农药;而且也能水解含P-S键的有机磷农药。以甲基对硫磷和内吸磷为底物的Km值分别为52祄ol、236 祄ol; Vmax分别为317祄ol min-1 mg-1、179 祄ol min-1 mg-1;Kcat分别为1152 s-1、650 s-1。

PURIFICATION AND CHARACTERIZATION OF A BROAD-SPECTRUM ORGANOPHOSPHORUS PESTICIDE HYDROLASE FROM ASPERGILLUS ORYZAE LY-128

A electrophoretic homogeneity broad-spectrum organophosphorus pesticide hydrolase from Aspergillus oryzae LY-128 mycelia was purified by four steps of ammonium sulfate precipitation, Sephadex G-100, DEAE-Sepharose CL-6B and Sephadex G-100 chromatography. Its molecular mass was estimated to be about 62 kDa by SDS-PAGE, and 61 kDa by Sephadex G-100, and the isoelectric point was pH5.2. The optimal temperature and pH of the enzyme activity were 45℃ and pH 6.8 respectively. The enzyme was stable in the range of pH 6.0~9.5 and below 50℃. The activities were strongly inhibited by Hg2+, Fe3+,ρ-chloromercuribenzoate, iodoacetic acid and N-ethylmaleimide, while Cu2+,β-mercaptoethanol, dithiothreitol, dithioerythritol, glutathione and detergents slightly activated the enzyme. The result showed that the enzyme can not only split organophosphorus pesticides possessing P-O linkage such as methyl parathion, parathion, paraoxon, coumaphos, but also act organophosphorus pesticide containing P-S bond including demeton-S, phosmet, and malathion. The Km, Vmax, and Kcat was 52 祄ol, 317祄ol min-1 mg-1 and 1152 s-1 when methyl-parathion was as substrate, and the parameters were 236 祄ol, 179 祄ol min-1 mg-1 and 650 s-1 respectively, when demeton-S as substrate.