An oxygen insensitive intracellular azoreductase has been purified from Pseudomonas aeruginosa by chromatographic methods including ion exchange and Gel filtration chromatography. The enzyme was purified 53-fold with the recovery of 41% and the specific activity of the purified enzyme was 23 U. The enzyme gave a single band on native PAGE and SDS-PAGE with a molecular mass of 29,000 Da. Zymogram also revealed one clear zone of azoreductase activity that corresponded to the band obtained with native PAGE and SDS-AGE. The enzyme had an optimum pH of 7.0 with maximal activity at 35 degreesC. The enzyme was almost completely inhibited by Fe2+ and considerably by Cu2+ and Hg2+. The affinity of the enzyme for different azo dyes studied varies and had high affinity for Navitan fast blue S5R with a K-m value of 0.0625 mM for this substrate. (C) 2004 Elsevier Inc. All rights reserved.