Highly active proteolytic enzymes are found in the fruiting bodies of Grifola frondosa. The general properties and substrate specificities of these proteases from G. frondosa (ProGF) were studied. The optimal pH for ProGF activity was pH 3 or 7 using hemoglobin or Hammersten casein as a substrate, respectively. The ProGF exhibited over 70% of maximal activity within the pH range of 4.5-8.5. In terms of temperature, the ProGF were maximally active at 55 degrees C, while over 80% of maximal activity was observed within the range of 50-75 degrees C. These proteases were substrate-specific, mainly cleaving at Ala(14)-Leu(15), Tyr(16)-Leu(17), and Pro(28)-Lys(29) bonds, with occasional cleavage of Phe(24)-Phe(25) bonds in the oxidized insulin B-chain. The ProGF also liberated hydrophobic amino acids, such as valine, leucine, and phenylalanine, using the oxidized insulin B-chain as a substrate. When soy protein was used as a substrate, valine, leucine, phenylalanine, and tyrosine were selectively released from the hydrolysate. Thus, over the time course of incubation, the peptide concentration increased as the average peptide chain length decreased. These results indicate that the ProGF include both endopeptidases recognizing leucine, phenylalanine, and lysine at the P1' position, and aminopeptidases preferentially releasing hydrophobic and aromatic amino acids such as valine, leucine, phenylalanine, and tyrosine.