Properties of purified Orange II azoreductase, the enzyme initiating azo dye degradation by Pseudomonas KF46

Eur J Biochem. 1982 Dec;129(1):197-203. doi: 10.1111/j.1432-1033.1982.tb07040.x.


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 30,000. Its Km values were 1.5 microM for both Orange II and carboxy-Orange II, 5 microM for NADPH, and 180 microM 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Azo Compounds / metabolism*
  • Chemical Phenomena
  • Chemistry
  • Coloring Agents / metabolism*
  • Kinetics
  • NADH, NADPH Oxidoreductases / metabolism*
  • Pseudomonas / enzymology*
  • Substrate Specificity


  • Azo Compounds
  • Coloring Agents
  • NADH, NADPH Oxidoreductases
  • orange II azoreductase