Purification, characterization, and functional role of a novel extracellular protease from Pleurotus ostreatus

A new extracellular protease (PoSI; Pleurotus ostreatus subtilisin-like protease) from P, ostreatus culture broth has been purified and characterized. PoSI is a monomeric glycoprotein with a molecular mass of 75 kDa, a pi of 4.5, and an optimum pH in the alkaline range. The inhibitory profile indicates that PoSI is a serine protease, The N-terminal and three tryptic peptide sequences of PoSI have been determined. The homology of one internal peptide with conserved sequence around the Asp residue of the catalytic triad in the subtilase family suggests that PoSI is a subtilisin-like protease, This hypothesis is further supported by the finding that PoSI hydrolysis sites of the insulin B chain match those of subtilisin, PoSI activity is positively affected by calcium. A 10-fold decrease in the K-m value in the presence of calcium ions can reflect an induced structural change in the substrate recognition site region. Furthermore, Ca2+ binding slows PoSI autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases, Moreover, PoSI protease seems to play a key role in the regulation of P, ostreatus laccase activity by degrading and/or activating different isoenzymes.