关键词: 甲基对硫磷；4-硝基酚；假单胞菌；TAIL-PCR；偏苯三酚1,2-双加氧酶

Abstract:

A bacterium 1-7 with the capability of degrading both methyl parathion and 4-nitrophenol, identified as Pseudomonas sp. by 16S rDNA, was isolated by enrichment method from OP-polluted activated sludge. The degenerate primers were designed according to the conservative domains of hydroxyquinol 1, 2-dioxygenase to amplify the partial hydroxyquinol 1, 2-dioxygenase gene. The complete enzyme gene dio1 was obtained by TAIL-PCR subsequently and confirmed by DNA sequence analysis. The dio1 gene is 873 bp long comprising one open reading frame encoding a polypeptide of 290 amino acids with a molecular weight of 32.8 kDa. The dio1 gene was then over-expressed in E.coli BL21, and the recombinant protein Dio1 was further purified with Ni-NTA affinity chromatography. The hydroxyquinol 1, 2-dioxygenase Dio1 was proved to have catalytic activity. It was suggested that 4-NP was degraded via hydroxyquinol by Pseudomonas sp.1-7. 4-NP degradation pathway was preliminarily discussed in this research.

Key words: methyl parathion;4-nitrophenol;Pseudomonas sp.;TAIL-PCR, hydroxyquinol 1,2-dioxygenase