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Applied and Environmental Microbiology, February 2004, p. 837-844, Vol. 70, No. 2
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.2.837-844.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

A New Alkali-Thermostable Azoreductase from Bacillus sp. Strain SF

Department of Environmental Biotechnology, Graz University of Technology, A-8010 Graz, Austria,¹ Department of Textile Engineering, University of Minho, 4800 Guimarães, Portugal²

Received 14 July 2003/ Accepted 11 November 2003

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 80°C. 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.

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