Cloning And Characterization Of Two Genes Involved In The Catabolism Of 4-nitrophenol Of Pseudomonas Sp. Strain 1-7

4-Nitrophenol (4-NP), a persistent organic pollutant, had entered the environment through industrial releases and degradation of parathion-based pesticides. Microorganisms play an important role in transforming these recalcitrant contaminants. The elimination of the nitroaromatic compound in environment by specific microorganisms has become a promising technology and attracted more attention from the researchers.In this study, we collected some activated sludge from a pesticide factory. The degenerate primers were designed according to the conservative domains of hydroxyquinol 1, 2-dioxygenase and were used to amplify the partial hydroxyquinol 1, 2-dioxygenase genes from the activated sludge. As a result, a total of 68 target sequences were sequenced and analyzed. There were 12 unique hydroxyquinol dioxygenase fragments in the 68 sequences. T11 was the most dominant sequence which represented 55.9% of the total sequences and shared the highest sequence identity(90%) with 1,2,4-benzenetriol 1,2-dioxygenase of Pseudomonas sp. PNP5. These results showed that there were many bacteriums and multiple genes related to the degradation of 4-Nitrophenol in the acivated sludge. A bacterium 1-7 with the capability of degrading both methyl parathion and 4-Nitrophenol was isolated by soil enrichment method from OP-polluted activated sludge and identified as Pseudomonas sp. by 16SrDNA ananlysis. The partial hydroxyquinol 1, 2-dioxygenase gene of Pseudomonas sp.1-7 was amplified using the degenerate primers of hydroxyquinol 1, 2-dioxygenase. Then the full-length sequence of hydroxyquinol 1, 2-dioxygenase gene dio1 and maleylacetate reductase gene mal were obtained by TAIL-PCR. The dio1 gene is 873bp long, comprising one open reading frame encoding a polypeptide of 290 amino acids. The mal gene is 1068bp long, comprising one open reading frame encoding a polypeptide of 355 amino acids. The genes of dio1 and mal have been deposited in the GenBank under accession number FJ821776 and FJ821777 respectively.In order to confirm the function of these two genes, dio1 and mal were overexpressed in E.coli BL21 respectively. The recombinant protein Dio1 and Mal were further purified with Ni-NTA affinity chromatography. During the enzyme assay, the absorbance peak at 289 nm was gradually depleted and a new peak at 243 nm appeared due to the transformation of hydroxyquinol to maleylacetate. This result indicated that the hydroxyquinol 1, 2-dioxygenase Dio1 had the catalytic activity. The maleylacetate was then transformed to p-ketoadipate by maleylacetate reductase. Purification of maleylacetate reductase Mal resulted in a yield of 58.9%, and a specific activity of 193.04U/mg.In this study, the mechanism of 4-NP degradation pathway of Pseudomonas sp. 1-7 has been discussed preliminarily. This study will enhance our understanding of the genetic and biochemical diversity of microbial 4-NP degradation.