Validation of biphenyl degradation pathway by polymerase chain reaction, peptide mass fingerprinting and enzyme analysis

Our previous studies showed, bacterium Aquamicrobium sp. SK-2 could degrade biphenyl and polychlorinated biphenyls (PCBs). In the present study, proteins involved in the biphenyl degradation was evaluated using various molecular biology methods. The gene bphC present in the strain SK-2 was identified using the polymerase chain reaction method. Further the key enzyme in biphenyl degradation, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) was purified through anion exchange and gel filtration chromatography, subsequently the enzyme activity was measured. The N-terminal amino acid sequence of the purified enzyme showed 92% homology with BphC enzyme of Gram-negative bacteria (Pseudomonas sp. KKS102, Comamonas testosterone, Burkholderiaceae bacterium, Delftia acidovorans, and Achromobacter denitrificans). Fractions collected during protein purification were applied on SDS-PAGE gel. Significant bands were selected in SDS-PAGE gel, and the gel pieces were cut out to analyze the proteins using peptide mass fingerprinting (PMF) method. PMF method provided useful information about the proteins involved in biphenyl degradation. Apart from BphC, two other enzymes, benzoate dioxygenase and catechol 2,3-dioxygenase which were involved in biphenyl degradation process were identified. The results indicate that catechol can be degraded to 2-hydroxymuconic-semialdehyde and this result is in accordance with the results from our previous study. Based on all these results we can conclude that the strain SK-2 is a potential candidate for the bioremediation of biphenyl contaminated places.