[Characterization of a p-nitrophenol degrading bacterium Pseudomonas sp. PDS-7 and cloning of degradation relevant genes]

Abstract

Objective: The aim of this study was to (i) isolate and characterize bacteria capable of degrading p-nitrophenol (PNP); (ii) determine the kinetics of biodegradation, (iii) clone and express the PNP-degrading related genes.

Methods: Enrichment method and serial dilution spread-plate method were employed to isolate PNP-degrading strain. Morphological, physiological & biochemical tests and 16S rDNA sequence analysis were used to identify the isolate. Degradation kinetics was studied by flask test. PNP-degrading related genes were cloned by SEFA-PCR method. Hydroxyquinol 1,2-dioxygenase encoding gene pnpC was cloned into pET29a to construct the recombinant plasmid pETpnpC and expressed in E. coli BL21 (DE3). The activity of the expressed product was determined by spectrophotometric method.

Results: Strain PDS-7 capable of utilizing PNP as the sole carbon, nitrogen and energy source was isolated and identified as Pseudomonas sp. It could tolerate the PNP concentration up to 80 mg/L, the optimal temperature for degradation was about 30 degrees C and alkaline pH benefited PNP degradation. Hydroxyquinol 1,2-dioxygenase and maleylacetate reductase encoding gene pnpC and pnpD were cloned and sequenced respectively, the sequence was deposited in GenBank with the accession number EU233791. pnpC was expressed in E. coli BL21 (DE3), the expressed product in cell-free crude extracts showed ortho ring cleavage activity to hydroxyquinol and catechol, with the special activity 0.45 U/mg protein and 0.37 U/mg protein, respectively, indicating pnpC gene encoding hydroxyquinol 1,2-dioxygenase was actively expressed.

Conclusion: One PNP-degrading strain Pseudomonas sp. PDS-7 was isolated and identified. Its degradation kinetics was studied. Its degradation relevant genes were cloned and expressed.