Purification and properties of pendimethalin nitroreductase from Bacillus circulans

Abstract

A pendimethalin nitroreductase was purified from Bacillus circulans to 52 fold increase with a recovery of 21.1% and had a specific activity of 4.16 U/mg. The molecular weight was 27 kDa. The optimum pH and temperature were 7.5 and 35°C, respectively. Fe 2+ , Ca 2+ , Mg 2+ and Mn 2+ did not cause any effect on the enzyme activity, whereas Ag + , Hg 2+ significantly inhibited it. The pendimethalin nitroreductase from B. circulans required NADPH as cofactor. It was not affected by GSH, DTT, L Cys and β mercaptoethanol but inhibited by p hydroxymercuribenzoate, N ethylmaleimide, and iodoacetamide. EDTA, 1,10 phenan throline and α,α' dipyridyl had a moderate effect on the enzyme activity. The nitroreductase reduced all the tested nitroaromatic compounds but was more active towards pendimethalin and trifluralin. The K M and V max for the enzyme reaction using pendimethalin as a substrate were 4.35 μM and 620 μmoles mg -1 min -1 , respectively. The broad substrate specificity towards dinitroaniline herbicides suggested the enzyme to be used as a potent reducing agent of these toxic compounds. This is the first report for the purification and charac terization of pendimethalin nitroreductase from any bacterial or fungal species.

Biodegradation and Abiotic Degradation of Trifluralin: A Commonly Used Herbicide with a Poorly Understood Environmental Fate

“…The enzymes reduced both nitro groups on trifluralin, oryzalin, and butyralin, but only reduced one nitro group on pendimethalin. A prior study 77 purified a similar nitroreductase enzyme from Bacillus circulans which was also active on several dinitroanilines, although in this case, the corresponding gene was not identified, so it is hard to know to what extent this enzyme was related in sequence to the B. subtilis Y3 enzyme. Reduction of the nitro-groups of trifluralin has also been reported for Pseudomonas f luorescens isolates; 78 this study also reported dealkylated metabolites, but unfortunately the metabolite identifications were tentative and the enzymes involved were not identified.…”

2D Strategy for the Construction of an Enzyme-Activated NIR Fluorophore Suitable for the Visual Sensing and Profiling of Homologous Nitroreductases from Various Bacterial Species

“…Nitroreductases (NTRs) are biological enzymes of the flavin enzyme family that reduce nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives using nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) as an electron donor. − The hypoxic environment of the tumor tissue results in the overexpression of NTRs in tumor cells, highlighting the importance of NTR monitoring for clinical diagnosis and tumor therapy. , Compared with the role of NTRs in mammalian cells, bacterial NTRs are thought to play a vital role in the antibacterial activity of nitroimidazole antibiotics, such as chloramphenicol. − In bacterial cells, intracellular reduction of the nitro groups of 5-nitroimidazole drugs (metronidazole, tinidazole, and ornidazole) can be mediated by endogenous NTRs along with the production of active radical intermediates, which inhibits bacterial colonization through the inhibition of DNA synthesis. Clinically, emerging problems of resistance to 5-nitroimidazole drugs make the treatment of bacterial infections a growing challenge .…”