Streptomyces antibioticus tyrosinase was purified using PEG-8000/phosphate phase partitioning and ammonium sulfate fractionation between 0 and 60%, rendering a clear and stable enzyme compared with the black one that is usually obtained when only ammonium sulphate fractionation is used, and with a 93% of recovery. This enzyme shows both monophenolase (also termed cresolase) and diphenolase (catecholase) activities, confirming that it is a real tyrosinase. The monophenolase activity was characterized by a lag period, whose duration depends on the substrate concentration, the pH, and the presence of catalytic amounts of o-diphenol. The enzyme showed substrate inhibition for p-cresol, with a K-M of 0.97 rnM and a K-si of 23 mM. By increasing the concentration of o-diphenols, it was possible to evaluate the enzyme activation constant, K-act, which showed a value of 0.2 mu M. The diphenolase activity was kinetically characterized with 4-methylcathechol, showed an optimal pH at 6.5 and a K-M of 1.3 mM. (c) 2006 Elsevier Inc. All rights reserved.