MOLECULAR CHARACTERISATION OF BACTERIA MONOOXYGENASE AND DEHYDROGENASE GENES INVOLVED IN PAHs REMEDIATION

Abstract

Bacterial catabolic genes (alkB, alkH, C12O, and C23O) are a good biomarker for choosing the choice of the organism for polycyclic aromatic hydrocarbon (PAH) degradation. Low molecular weight (LMW) and high molecular weight (HMW) PAHs metabolism can be made possible by monooxygenase and dehydrogenase enzymes which code for the catabolic genes. In this study, the monooxygenase and dehydrogenase genes were characterized from the bacterial population isolated from motor mechanic workshop soils and landfill soil artificially polluted with waste engine oil (WEO). Standard microbiological methods were followed for the isolation and characterization of the bacterial population.  The PCR cycling for alkB and alkH followed initial denaturation at 94^oC for 5 minutes, followed by 35 cycles of denaturation at 95^oC for 1 minute, annealing at the correct temperature (alkB 49^oC, alkH 72^oC). PCR cycling for C12O and C23O genes followed initial denaturation at 95^oC for 5 minutes, 35 cycles of denaturation at 94^oC for   20 s, annealing at 63^oC for 30 s, extension at 60^oC for 45 s, with final extension for 5 minutes at 72^oC. Final elongation step for all the catabolic genes at 72^oC for 10 minutes and holding temperature at 10^oC forever. Amplified fragments were visualized on safe view-stained 1.5% agarose gel electrophoresis. The result of the characterization revealed base pair sizes of the genes; alkB (100 to 300 bp), alkH (< 700 bp), C12O (>250 bp), and C23O (<80 pb). All the bacterial populations invested in this study expressed the monooxygenase and dehydrogenase genes. Monooxygenase and dehydrogenase genes are coding for the enzymes responsible for hydroxylation and intradiol or extradiol ring-cleaving of PAHs.