Pseudomonas sp.1-7中甲基对硫磷水解酶基因的克隆及酶学性质研究

Gene cloning and properties of a methyl parathion hydrolase from Pseudomonas sp.1-7

为了研究和分离有机磷降解酶及其编码基因,从农药厂污水处理池的活性污泥中分离出一株可以高效降解甲基对硫磷的细菌1-7,经16S rDNA鉴定为假单胞菌Pseudomonas sp.。通过构建基因组文库的方法克隆了1-7的甲基对硫磷水解酶基因ophc3,该基因全长为975 bp,编码324个氨基酸,其中前24个氨基酸残基可能为信号肽序列。将其在大肠杆菌中表达,并对重组甲基对硫磷水解酶(OPHC3)进行纯化和酶学性质的研究结果表明,其酶促反应最适pH值为8.0,在pH 6.0～10.0的范围内放置30 min酶的相对活性均在70%以上;最适反应温度为45℃,但该酶不耐高温,60℃下保温10 min,相对活性降至46.77%。

In order to isolate organophosphorus hydrolases and their encoding genes,a bacterium with the capability of degrading methyl parathion,identified as Pseudomonas sp.by 16S rDNA analysis,was isolated from active sludge in organophosphate-treated aeration basin,and designated as Pseudomonas sp.1-7.The full-length of methyl parathion hydrolase（MPH） gene,ophc3,from this bacterium was cloned by constructing genomic library.The ophc3 gene is 975 bp long,encoding a polypeptide of 324 amino acids that includes a signal peptide of 24 aa.The gene ophc3 was expressed in E.coli.The recombinant methyl parathion hydrolase OPHC3 was purified,and the enzymatic properties were studied.The optimum temperature and pH for enzyme activity are 45 ℃ and pH 8.0,respectively.It also shows good pH stability and poor temperature stability.Over 70% of the relative activities were retained from pH 6.0-10.0.About 46.77% of the activity was retained after heating the enzyme at 60 ℃ for 10 min.