Extracellular tyrosinase from Auricularia auricula RF201 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 12.6 kDa on SDS-PAGE. The optimum pH for tyrosinase activity was 7, and the enzyme was stable between pH 6 and 9. Tyrosinase has optimal activity at 40 degrees C and retained most of its activity between 4 and 50 degrees C. A. auricula tyrosinase could oxidize L-tyrosine, L-DOPA, catechol, and caffeic acid and displayed dark brown or peach color. However, the enzyme was unable to catalyze L-phenylalanine and ferulic acid. In comparison with other substrates, L-tyrosine displayed the highest affinity (K-m of 0.11 mM) and the maximal reaction velocity (V-max of 102.58 mu mol/min). Tyrosinase activity was reduced in the presence of numerous tested compounds. Particularly SDS, it significantly inhibited enzyme activity. CuSO4 and NaCl showed an activation effect on enzyme activity, with the maximum activation found in the presence of CuSO4.