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.

Characterization, occurrence, and molecular cloning of a lectin from Grifola frondosa: jacalin-related lectin of fungal origin.

A lectin named GFL was isolated from the fruiting body of the basidiomycete mushroom Grifola frondosa, which belongs to Aphyllophorales. The lectin had a molecular mass of 24 kDa on SDS-PAGE. The hemagglutinating activity of GFL was not inhibited by any monosaccharide, and inhibited only by porcine stomach mucin so far as tested. The occurrence of GFL was studied at three stages during fruiting body formation. The largest quantity of hemagglutinating activity was found in the fruiting body, and lesser amounts in the mycelial mat and the primordium. The 24-kDa band of GFL was found at all three stages, and the band-intensity corresponded to the level of activity in each sample. By cloning and sequencing the GFL-cDNA, the primary structure of this lectin was determined. GFL is composed of 181 amino acids, having no signal peptide. The amino acid sequence was found to be homologous to those of so-called jacalin-related plant lectins, suggesting that GFL is the first example of a jacalin-related lectin of fungal origin.