Grifolisin, a member of the sedolisin family produced by the fungus Grifola frondosa

The pepstatin-insensitive carboxyl proteinase grifolisin was purified from fruiting bodies of the fungus Grifola frondosa, a maitake mushroom. The enzyme had an optimum pH of 3.0 for the digestion of hemoglobin and 2.8 for milk casein digestion. Its molecular mass was determined to be 43kDa by SDS-PAGE and 40kDa by gel chromatography on Superose 12, and its isoelectric point was found to be 4.6 by isoelectric focusing. The enzyme hydrolyzed four major bonds in the oxidized insulin B-chain: Phe1-Val2, Ala14-Leu15, Gly20-Glu21 and Phe24-Phe25 at pH 3.0. The first 15 amino acid residues in the N-terminal region were AVPSSCASTITPACL, and the coding region of the grifolisin gene (gfrF) has a 1960-base pair cDNA. The predicted mature grifolisin protein consisted of 365 residues and was 26% identical to that of sedolisin from Pseudomonas sp. 101 and 34% identical to that of aorsin from Aspergillus oryzae. Grifolisin is a member of the sedolisin S53 family and is not inhibited by pepstatin.