Purification, Characterization, and Functional Role of a Novel Extracellular Protease from Pleurotus ostreatus

Abstract

A new extracellular protease (PoSl; Pleurotus ostreatus subtilisin-like protease) from P. ostreatus culture broth has been purified and characterized. PoSl 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 PoSl is a serine protease. The N-terminal and three tryptic peptide sequences of PoSl 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 PoSl is a subtilisin-like protease. This hypothesis is further supported by the finding that PoSl hydrolysis sites of the insulin B chain match those of subtilisin. PoSl 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, Ca 2؉ binding slows PoSl autolysis, triggering the protein to form a more compact structure. These effects have already been observed for subtilisin and other serine proteases. Moreover, PoSl protease seems to play a key role in the regulation of P. ostreatus laccase activity by degrading and/or activating different isoenzymes.White rot basidiomycetes have received extensive attention because of their lignin-degrading activity. The biochemistry of lignin degradation is a complex process involving a series of enzymatic and nonenzymatic reactions. Extracellular enzymes which catalyze oxidative reactions are lignin peroxidases, laccases, manganese peroxidases, and hydrogen peroxide-producing enzymes (3, 13). Even though their catalyzed reactions have been studied in detail, their in vivo coordination and, possibly, synergistic action are not clearly understood.Pleurotus ostreatus is a white rot basidiomycete which belongs to the subclass of ligninolytic microorganisms that produce laccases, manganese peroxidases, and veratryl alcohol oxidases but no lignin peroxidase. Among these enzymes, laccases have been the most widely studied and characterized (11,12,17). In a recent study (16), it has been demonstrated that a laccase isoenzyme (POXA1b; phenol oxidase A1b) is specifically degraded in the early phase of fungal growth by proteases present in P. ostreatus culture broth; hence, the disappearance of POXA1b seems to be correlated with the appearance of extracellular protease activity. A similar relationship was observed for lignin peroxidases in Phanerochaete chrysosporium, and, in this case, the extracellular proteases caused an almost complete disappearance of lignin peroxidase activity due to degradation of all lignin peroxidase isoenzymes (8). Furthermore, a recent report (21) suggests that both intracellular and extracellular proteases are involved in the regulation of ligninolytic activities in cultures of Trametes versicolor under nutrient limitation. In contrast, it has been reported (2) that proteases are not responsible for the decrease in peroxidase activity in Pleurotus pulmonarius ...

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