Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35degreesC. This activity was NADH dependant. The K,, values for both NADH and MR were 58.9 and 29.4 muM, respectively. The maximal velocity (V-max) was 9.2 mumol of NADH min(-1) mg(-1). The purified enzyme is inhibited by several metal ions including Fe2+ and Cd2+ (C) 2003 Elsevier Science (USA). All rights reserved.