Cloning, purification and possible use of a Bacillus nitroreductase in biotechnological applications

Nitroreductases (NRs) can reduce nitroaromatic compounds, which are toxic, mutagenic or carcinogenic, to nitrite, amino group or hydroxylamine groups. We have utilised a cloned nitroreductase from a moderate thermophilic Bacillus species in E. coli BL21. Purification of the enzyme, followed by electrophoresis and western blotting, revealed approximate molecular weight of 30 kDa. The optimum temperature and pH of the enzyme was found as 40 °C and 8.0, respectively. Conversion of CB1954 to the metabolic products and nitrofurazone reduction by enzyme in the presence NADPH as co-factor was studied. Km, kcat and kcat/Km values were determined as 42.5 (µM), 5.07 (s^- 1) and 0.1194 (s^- 1.µM^- 1), respectively from the data using CB1954 as substrate and NADPH as cofactor. Meanwhile, the values obtained for nitrofurazone substrate were 52.7 (µM), 0.626 (s^- 1) and 0.0119 (s^- 1.µM^- 1), respectively. The enzymatic reduction of CB1954 analyzed by LC-ESI-MS gave prominent molecular ions at the expected m/z values of CB1954 ([M + H]⁺: m/z 253), primary hydroxylamine intermediates ([M + H]⁺: m/z 239), and amino end-products ([M + H]⁺: m/z 223). The purified enzyme was also found to biotransform TNT to the product 2-amino 4,6-DNT. In addition, a proof-of-concept electrochemical detection platform based on immobilized nitroreductase produced a measurable cathodic response toward nitrofurazone, demonstrating the enzyme's electrochemical applicability. ANOVA identified significant differences in activity levels among multiple groups, while regression analysis enabled prediction of enzyme responses and characterization of the underlying kinetic patterns in statistical analysis.