Abstract
β-D-Mannosidase [EC 3.2.1.25] and β-N-acetyl-D-hexosaminidase [EC 3.2.1.30] were purified approximately 500- and 200-fold, respectively, from the cell extract of Tremella fuciformis. Both glycosidases showed single protein bands in disc gel electrophoresis and the molecular weights of β-D-mannosidase and β-N-acetyl-D-hexosaminidase were about 140, 000 and 125, 000, respectively, as estimated by Sephadex gel exclusion chromatography. The substrate specificities and kinetics of the two enzymes were tested with p-nitrophenyl glycosides and related oligosaccharides. The β-D-mannosidase hydrolyzed p-nitrophenyl β-D-mannoside, with Km 2.1mM and Vmax 0.21 μmol per min per mg protein. 4-O-β-D-Mannosyl-D-mannose was readily hydrolyzed, but β-(1→4)-linked mannotriose and mannotetraose were hydrolyzed much more slowly. Unlike other known β-D-mannosidases, its activity was not inhibited by D-manno-γ-lactone, but was strongly inhibited by p-chloromercuribenzoate. The β-D-mannosidase tended to be inactivated in the presence of oxygen but was reactivated by dithiothreitol. The β-N-acetyl-D-hexosaminidase hydrolyzed p-nitrophenyl β-N-acetyl-D-glucosaminide, with Km 0.31mM and Vmax 4.6 μmol per min per mg protein. It was active on N, N'-diacetylchitobiose and higher saccharides (DP up to 5) and liberated N-acetyl-D-glucosamine. The glycosidase preparation was also slightly active on p-nitrophenyl β-N-acetyl-D-galactosaminide (Km 0.19mM, and Vmax 0.6 μmol per min per mg protein). Both β-D-mannosidase and β-N-acetyl-D-hexosaminidase had an optimum pH of 5.0. Inhibition of both glycosidases by various metal ions was tested and their stabilities were investigated.
