Cloning, Expression And Characterization Of A Nitrobenzene Nitroreductase From Pandoraea Sp.BD-33 In Escherichia Coli

Nitroreductase family can utilize FMN or NADH or NADPH as a cofactor and catalyze reduction of a variety of nitroaromatic compounds, including nitrofuran, nitrobenzen, nitrophenol, nitrobenzoate and quinone in an obligatory two-election transfer mechanism. Nitrobenzene nitroreductase, one member of nitroreductase family, can catalyze reduction of nitrobenzene coupled with oxidation of NADPH to generate NADP+. Compared with other nitroreductases, nitrobenzene nitroreductase NN-BD-33 has many advantages such as low Km, almost no reverse reaction, wide thermostability, etc.In this dissertation, we started from homologous sequences of nitrobenzene nitroreductases, using some bacteria preserved in our laboratory as the target, cloned nitrobenzene nitroreductase gene from Pandoraea sp. BD-33 strain by molecular biology method, constructed recombinant expression plasmid of nitrobenzene nitroreductase gene, finally got the recombinant nitrobenzene nitroreductase by induced expression and affinity chromatography purification, and then we studied the detailed properties of this nitrobenzene nitroreductase.The nitrobenzene nitroreductase gene consists of 792 bp nucleotides encoding a peptide of 263 amino acid residues. Recombinant nitrobenzene nitroreductase was purfied by Ni-NTA affinity chromatography. The purified nitrobenzene nitroreductase displayed a single band in SDS-PAGE. Compared with the standard protein marker, molecular weight of the recombinant enzyme was calculated as 29.24 kDa.The properties of this recombinant nitrobenzene nitroreductase were intensively studied. The results showed that:the optimum temperature was 40℃; after treating at different temperatures at pH 7.0 in MOPS buffer for 30min, residual enzyme activity was measured and we found that the recombinant enzyme was stable below 45℃; its optimal pH value was 7.5, after treated at different pH at 25℃for 1 h, we measured the residual enzyme activity and found that the enzyme was the most stable at pH 7.5; the Km and Kcat for nitrobenzene of the recombinant enzyme were 1.154±0.495μM and 2.626±0.034 S-1, the Km and Kcat for NADPH were 833.78±89.5μM and 21.066±1.528 S-1, respectively.These studies have provided a reliable data for environmental protection, and have laid the foundation for further study of the catalytic mechanism and three-dimensional structure of this enzyme.