TNT nitroreductase from a Pseudomonas aeruginosa strain isolated from TNT-contaminated soil

Microbial degradation and detoxification of TNT (2,4,6-trinitrotoluene) in soils ultimately depends on the presence of effective intracellular or extracellular enzymes. TNT was rapidly degraded by an enzyme obtained from a Pseudomonas aeruginosa strain isolated from TNT-contaminated soil. The nitroreductase enzyme was purified to apparent electrophoretic homogeneity by sequential chromatography on phenyl-sepharose, hydroxyapatite, and ion exchange resins. The TNT-nitroreductase catalyzed reduction of TNT to 4-hydroxylamino-4,6-dinitrotoluene (4HADNT) and 4-amino-2,6-dinitrotoluene (4ADNT). TNT reduction to 4ADNT in cell-free enzyme preparations corresponded to 2ADNT production by intact cells. The enzyme required NADH as a cosubstrate and the optimal pH for the reaction was approximately 6.5. The purified enzyme also exhibited NADH diaphorase activity and its denatured molecular weight was approximately 54 kDa.