Properties of Purified Orange II Azoreductase, the Enzyme Initiating Azo Dye Degradation by Pseudomonas KF46

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Abstract
Orange II azoreductase [NAD(P)H: 1-(4′-sulfophenylazo)-2-naphthol oxidoreductase], an enzyme catalyzing the reductive cleavage of the azo bridge of Orange II and related dyes, was purified to electrophoretic homogeneity from Pseudomonas species, strain KF46. This organism utilized carboxy-Orange II [1-(4′-carboxyphenylazo)-2-naphthol] but not Orange II as the sole source of carbon, energy, and nitrogen. Orange II azoreductase was induced 80-fold by both Orange II and carboxy-Orange II. With two successive runs of affinity chromatography using two chromatographic media with different triazinyl dyes as ligands, the enzyme was purified 120-fold with 43% yield. The purified enzyme is a monomer with a molecular weight of 30000. Its Km values were 1.5 μM for both Orange II and carboxy-Orange II. 5 μM for NADPH, and 180 μM for NADH. A survey of the efficiency of various Orange dyes as substrates for Orange II azoreductase showed that: (a) a hydroxy group in the 2-position of the naphthol ring is required: (b) charged groups in proximity to the azo group hinder the reaction; (c) a second polar substituent on the dye molecule impedes the reaction; (d) electron-withdrawing groups on the phenyl ring accelerate the reaction.