Cloning And Expression Of Ophc2, A New Organphosphorus Hydrolase Gene From Pseudomonas Pseudoalcaligenes

Organphosphorus (OPs) are commonly used as insecticides throughout the world and have made a great contribution to agriculture. However, it has been noticed that OPs brought about harm for human health and pollution for environment. In the early 1970s, the bacterium in natural environment with the capability of degrading OPs was found, which provided us a good way to eliminate the pollution of OPs.A bacterium with the capability of degrading organphosphorus, identified as Pseudomonas pseudoalcaligenes, is isolated from OP-treated soil. The organphosphorus hydrolase OPHC2 from this bacterium has been purified and characterized. OPHC2 with a molecular weight of 36 kD has optimum activity for the reaction at 65 C and pH 9.0 with methyl parathion as a substrate, it also shows good thermal and pH stability. Most metal ions and chemicals have no effect on the activity of OPHC2, but SDS inactivates this enzyme. The amino acid sequences of N-terminal and internal peptides of OPHC2 were determined. The partial nucleotide sequence of organphosphorus hydrolase gene ophc2 is obstained by PCR using the degenerate primers designed according to the aa sequences; The full-length ophc2 is obstained by future inverse PCR using specific primers designed according to the known nucleotide sequence. The ophc2 gene is 975 bp long with G+C content of 63%, comprising one open reading frame encoding a polypeptide of 324 amino acids which includes a signal peptide of 24 aa. The nucleotide sequence of ophc2 shows low homologies with those organphosphorus hydrolase genes deposited in GenBank, one of which exhibits the highest homology of 46.4% with ophc2. The ophc2 gene has been assigned and the GenBank accession number is AJ605330. The organphosphorus hydrolase genes with and without the signal sequences are respectively inserted into the vector pET-30a, and both expression products bore normal bioactivity in E. coli.