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

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.