Purification and characterization of the NAD(P)H-nitroreductase for the catabolism of 2,4,6-trinitrotoluene (TNT) in<Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6

The NAD(P)H-nitroreductase of thePseudomonas sp. HK-6 which is capable of catabolizing 2,4,6-trinitrotoluene (TNT), was purified and biochemically characterized. The specific activity of the purified TNT nitroreductase was approximately 1.47 units/mg, and was concentrated to 10.1-fold compared to the crude extract. The optimal temperature and pH of the highest nitroreductase activity was 30°C and 7.5, respectively. The substrate specificity test revealed that the nitroreductase exhibited the highest enzyme activity for the TNT substrate of the nitroaromatic compounds tested in this study. Moreover, the molecular weight of the TNT nitroreductase was approximately 27 kDa on the SDS-PAGE. The N-terminal amino acid sequence of the purified protein was 5′-MDTVSLAKRRYTTKAYDASR, which is identical topnrB ofPseudomonas putida JLR11, and is capable of TNT reduction. The molecular analysis of the approximately 650-bp PCR product, orginating from the HK-6, revealed that the oxygen-insensitive NAD(P)H-nitroreductase gene, which transforms TNT in strain HK-6 with five unique amino acid sequences and diverges from the nitroreductases identified so far inPseudomonas, Burkholderia, andRalstonia, is frequently found amidst the powerful degraders of aromatic compounds.