Molecular cloning and characterization of the gene coding for azoreductasefrom Bacillus sp OY1-2 isolated from soil

Azo dyes are regarded as pollutants because they are not readily reduced un der aerobic conditions. Bacillus sp, OY1-2 transforms azo dyes into colorle ss compounds, and this reduction is mediated by a reductase activity for th e azo group in the presence of NADPH, A 1.2-kbp EcoRI fragment containing t he gene that encodes azoreductase was cloned by screening the genomic libra ry of Bacillus sp, OY1-2 with digoxigenin-labeled probe designed from the N -terminal amino acid sequence of the purified enzyme. An open reading frame encoding the azoreductase, consisting of 178 amino acids, was predicted fr om the nucleotide sequence. In addition, because only a Bacillus subtillis hypothetical protein was discovered in the public databases (with an amino acid identity of 52.8%), the gene encoding the azoreductase cloned in this study was predicted to be a member of a novel family of reductases. Souther n blot analysis revealed that the azoreductase gene exists as a single copy gene on a chromosome. Escherichia coli-expressing recombinant azoreductase gave a ten times greater reducing activity toward azo dyes than the origin al Bacillus sp, OY1-2, In addition, the expressed azoreductase purified fro m the recombinant E. coli lysate by Red-Sepharose affinity chromatography s howed a similar activity and specificity as the native enzyme. This is the first report describing the sequencing and characterization of a gene encod ing the azo dye-reducing enzyme, azoreductase, from aerobic bacteria and it s expression in E. coli.