A new alkali-thermostable azoreductase from Bacillus sp strain SF

A screening for dye-decolorizing alkali-thermophilic microorganisms resulted in a Bacillus sp. strain isolated out of the wastewater drain of a textile finishing company. An NADH-dependent azoreductase of this strain, Bacillus sp. strain SF, was found to be responsible for the decolorization of azo dyes. This enzyme was purified by a combination of ammonium sulfate precipitation and anion-exchange and affinity chromatography and had a molecular mass of 61.6 kDa and an isoelectric point at pH 5.3. The pH optimum of the azoreductase depended on the substrate and was within the range of pHs 8 to 9, while the temperature maximum was reached at 80degreesC. Decolorization only took place in the absence of oxygen and was enhanced by FAD, which was not consumed during the reaction. A 26% similarity of this azoreductase to chaperonin Cpn60 from a Bacillus sp. was found by peptide mass mapping experiments. Substrate specificities of the azoreductase were studied by using synthesized model substrates based on di-sodium-(R)-benzyl-azo-2,7-dihydroxy-3,6-disulfonyl-naphthaline. Those dyes with NO2 substituents, especially in the ortho position, were degraded fastest, while analogues with a methyl substitution showed the lowest degradation rates.