A 33.5-kDa serine protease designated as helvellisin was isolated from dried fruiting bodies of the wild ascomycete mushroom Helvella lacunosa. it was purified by using a procedure which entailed ion exchange chromatography on DEAE-cellullose, CM-Sepharose, Q-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was characterized by unique N-terminal amino acid sequence, thermostability and pH stability. The protease exhibited a pH optimum of 11.0 and a temperature optimum of 65 degrees C, with about 40% activity remaining at 87 degrees C and pH 5 and 13. Helvellisin demonstrated a protease activity of 14600 U/mg toward casein. The K-m of the purified protease for casein was 3.81 mg/ml at pH 11.0 and 37 degrees C. The V-max was 5.35 x 10(-2) mg ml(-1) min(-1). It was adversely affected by phenylmethylsulfonyl fluoride, suggesting that it is serine protease. The activity of the protease was enhanced by Mg2+, Fe2+ and Mn2+, but was curtailed by Cu2+, Hg2+ and Fe3+. It was devoid of antifungal and ribonuclease activities. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.