PURIFICATION AND CHARACTERIZATION OF INTRACELLULAR PROTEINASES IN PLEUROTUS-OSTREATUS FRUITING BODIES

A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.3.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL. ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn2+-depleted ProB can regain the activity with Zn2+, Co2+, or Mn2+. Specific cleavage of Pro(29)-Lys(30) in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the alpha-amino group of lysyl residues. ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn2+ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.