Azoreductase, an enzyme catalyzing the reductive cleavage of azo and nitro groups of various compounds, was purified from Bacillus badius an alkaline strain. This bacterial strain isolated from pristine alkaline Crater Lake of Lonar (Maharashtra State), India and identified by 16S rRNA method. The strain could decolorize azo dyes up to 1000 mg/l in 24 h under aerobic conditions. Cell extracts from the strain have demonstrated NADH dependent oxygen-insensitive azoreductase activity. The optimal pH and temperature were found to be 7.4 and 60 degrees C, respectively. The K, values for both NADH and amaranth azo dye were 0.8 and 1.33 mM, respectively. The maximal velocity (V-max) was 16.66 and 100 U/mg of protein per min for NADH and amaranth, respectively. Decolorization of dyes with different molecular structures was performed to compare their degradability by purified enzyme. This enzyme was purified by a combination of ammonium sulfate precipitation, ion exchange and molecular exclusion chromatography and 8 fold purification was achieved. The molecular mass was found to be 43 kDa. Thermal stability of purified azoreductase is up to 85 degrees C. (C) 2011 Elsevier Ltd. All rights reserved.