Comparison of two bacterial azoreductases acquired during adaptation to growth on azo dyes

Selection for utilization of carboxy-Orange I [1-(4'-carboxyphenylazo)-4-naphthol] in the chemostat yielded Pseudomonas strain K24 which was unable to grow on carboxy-Orange II [1-(4'-carboxyphenylazo)-2-naphthol] while selection for growth on carboxy-Orange II had previously led to strain KF46 which did not utilize carboxy-Orange I. Orange I azoreductase of strain K24, the key enzyme of dye degradation, was purified 80-fold with 17% yield to electrophoretic homogeneity and compared to the previously purified Orange II azoreductase of strain KF46. Common properties of the two enzymes were their monomeric structure, their specificity for NADPH and NADH as cosubstrates, the range of their Km values for substrates and cosubstrates as well as their reactivity towards a series of substrate analogs. They differed from each other with respect to molecular weight (21,000 and 30,000) and in the absolute requirement of Orange I azoreductase for a hydroxy group in the 4'position of the naphthol ring of the substrate molecule as compared to the requirement for substrates with a 2-naphthol moiety by Orange II azoreductase. The pure enzymes did not exhibit immunological cross-reaction with each other. Crude extracts of strains K24 and KF46 and of azoreductase-negative strains isolated at different stages of the adaptation experiments, however, contained material which cross-reacted (CRM) with both anti Orange I azoreductase serum and anti Orange II azoreductase serum. The CRM may represent a common precursor protein of the azoreductases in strains K24 and KF46.