Purification and characterization of an oxygen insensitive azoreductase from Pseudomonas aeruginosa

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 °C. 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 Km value of 0.0625 mM for this substrate.