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.
State Key Laboratory for Agrobiotechnology and Department of Microbiology, China Agricultural University, Beijing, China.
Guoqing
G
Zhang
Hexiang
H
Wang
Xiaoqing
X
Zhang
TziBun
T
Ng
D001203:Ascomycota / Q000201:enzymology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D048690:Fruiting Bodies, Fungal / Q000201:enzymology; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D057057:Serine Proteases / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D001426:Bacterial Proteins; D002364:Caseins; D005656:Fungal Proteins; D008670:Metals; D011480:Protease Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D010450:Endopeptidases; D057057:Serine Proteases; C087822:alkaline protease