Cytosolic and Membrane-bound Chitinases of Two Mucoraceous Fungi: a Comparative Study

Chitinases isolated from membrane and cytosolic fractions of two mucoraceous fungi, Choanephora cucurbitarum and Phascolomyces articulosus, were investigated. The membrane-bound chitinase was isolated by Bio-Gel P-100 and DEAE Bio-Gel A chromatographic techniques. On SDS-PAGE the chitinase from both fungi migrated as a single band of M(r) 66 kDa. The cytosolic chitinase from the mycelial extracts of these fungi was separated by heat treatment, ammonium sulphate precipitation, and by affinity chromatography with regenerated chitin. SDS-PAGE showed two bands for each fungus with M(r) of 69.5 and 55 kDa in C. cucurbitarum and M(r) 69.5 and 53 kDa in Ph. articulosus. Chitinases, membrane bound or cytosolic, hydrolyzed regenerated chitin, colloidal chitin, glycol chitin, N,N'-diacetylchitobiose, and N,N',N"-triacetylchitotriose. Heavy metals, inhibitors, and N-acetylglucosamine inhibited chitinase activity, whereas trypsin and an acid protease enhanced its activity. Chitinase preparations showed lysozyme activity that was inhibited by histamine but not by N-acetylglucosamine. There was no N-acetylglucosamanidase activity, but beta-1,3 glucanase activity was found in cytosolic preparations only. Despite slight differences in their molecular mass, both the membrane-bound and cytosolic chitinases showed similarities in substrate utilization, response to inhibitors, and activation by trypsin and acid protease; pH and temperature optima also were similar.