We have investigated a specific enzymatic biosensor for detecting target pollutant 3,5-dinitro-trifluoromethylbenzene (3,5-DNBTF). The predicted enzyme is a nitroreductase that catalyzes the total nitroreduction of 3,5-DNBTF to its corresponding diamine. The photo-activation of this diamine offers a large panel of detection tools. After broad screening of microorganisms, only the strains belonging to the genus Bacillus were able to reduce the two nitro groups of 3,5-DNBTF. Among them, Bacillus LMA, isolated from explosives-polluted effluents, was the most efficient in reducing this compound. The involved nitroreductase was identified by 2D gel electrophoresis coupled to mass spectrometry, as the Bacillus subtilis oxygen-insensitive nitroreductase NfrA. The enzyme was purified by mono-P chromatofocusing. (c) 2006 Elsevier Inc. All rights reserved.