Taiwanofungus camphorata nitroreductase: cDNA cloning and biochemical characterisation

Abstract

Nitroreductases (Nrs) play important roles in redox system via NADPH or NADH as a reductant. A TcNr cDNA encoding a putative Nr was cloned from Taiwanofungus camphorata. A 3-D structural model of the TcNr has been created based on the known structure of BcNr (Bacillus cereus). To characterise the TcNr, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His(6)-tagged TcNr was purified by Ni affinity chromatography. The purified enzyme showed a single band at molecular mass of approximately 25 kDa on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme exhibited Nr activity via ferricyanide assay. The Michaelis constant (K(M)) value for ferricyanide was 0.86 mM. The enzyme(')s half-life of deactivation at 45°C was 12.3 min. The enzyme was most active at pH 6. The enzyme's preferred substrate is 1-chloro-2, 4-dinitrobenzene.