Helvellisin, a novel alkaline protease from the wild ascomycete mushroom Helvella lacunosa.

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-cellulose, 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 5.35x10(-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 Mg(2+), Fe(2+) and Mn(2+), but was curtailed by Cu(2+), Hg(2+) and Fe(3+). It was devoid of antifungal and ribonuclease activities. 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.