Biodegradation of 2,4,6-Trinitrophenol in Rhodococcus opacus and Nocardioides simplex : Developing a mutagenesis system and cloning of the nitrite eliminating enzyme

Bioremediation tends to be an effective technology, used to remove pollutants and it involves relatively little capital. Microorganisms, especially bacteria, evolving into many types with exceedingly diverse metabolic capabilities, which can be applied in bioremediation. The two bacterial strains, Nocardioides simplex FJ2-1A and Rhodococcus opacus HL PM-1 are capable of biodegradation of 2,4,6-trinitrophenol (TNP, picric acid). In N. simplex, the biodegradation pathway is inducible while in R. opacus is constitutive. Some enzymes were identified in both strains and shown to be isofunctional. In this work, a molecular characterisation of the orfB from the npd gene cluster was achieved. The mutant R. opacus AR1 strain deficient in the orfB gene was constructed by a gene replacement method using the expression of sacB as the selectable marker. The gene was also cloned as the His tag fusion protein and expressed in E. coli. Theoretical pI / Mw predictions for the protein sequence were 5.47 / 42536.23 Da. SDS-PAGE analysis showed the size of the purified fusion protein, which was calculated to be 42.5 kDa. The protein was purified by Ni-NTA metal affinity chromatography. This study furthermore identified a DNA fragment, which encodes the nitrite-eliminating enzyme (NEE) activity. This was achieved by enriching the activity using the fast performance liquid chromatography (FPLC) and protein sequencing. The two internal peptide sequences were obtained: L P G D Y T D Q L L R and S G A I V G L G H A Q V D R. These were used to design the degenerate primers in order to pool a clone from nocardioform genome. Two different genomic libraries were constructed and screened using R. opacus HL PM-1 and N. simplex FJ2-1A total DNA, since the activity in crude extracts was reported with both strains. The partial DNA sequence of NEE was obtained (252 bp). The similarity with fatty acid hydroxylase from Nocardioides sp. JS6614 was detected