Purification and characterization of a novel protease from solid substrate cultures of Phanerochaete chrysosporium.

The white-rot basidiomycete Phanerochaete chrysosporium is mostly known for its extracellular ligninolytic enzymes. In this paper, the purification and characterization of a novel extracellular protease secreted by this fungus in solid substrate cultures under ligninolytic conditions are described. The purification steps included extraction of enzymes from the wood substrate, concanavalin A-Sepharose chromatography, anion-exchange chromatography (Mono Q), and size-exclusion chromatography (Superose 12). The purified protein migrates with Mr = 40,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has an isoelectric point of 5.6 and a sharp pH optimum at 4.0. The protease is totally inhibited by Hg2+, p-hydroxymercuribenzoic acid, and N-bromosuccinimide, but is insensitive to phenylmethanesulfonyl fluoride, pepstatin A, and EDTA. Its amino acid composition and NH2-terminal sequence have also been determined. The sequence data and the binding of the protease to concanavalin A indicate that the protease is a glycoprotein. The protease differs in its physicochemical properties and its response to inhibitors from other extracellular proteases previously found in another strain of P. chrysosporium. The data suggest that it has properties of both aspartate-type and thiol-type proteases.