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

A new extracellular protease (PoSI; Pleurotus ostreatus subtilisin-like pro tease) 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 indica tes that PoSI is a serine protease, The N-terminal and three tryptic peptid e sequences of PoSI have been determined. The homology of one internal pept ide with conserved sequence around the Asp residue of the catalytic triad i n the subtilase family suggests that PoSI is a subtilisin-like protease, Th is hypothesis is further supported by the finding that PoSI hydrolysis site s of the insulin B chain match those of subtilisin, PoSI activity is positi vely affected by calcium. A 10-fold decrease in the K-m value in the presen ce of calcium ions can reflect an induced structural change in the substrat e 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 l accase activity by degrading and/or activating different isoenzymes.