Characterization of Novel β-Glucosidases with Transglycosylation Properties from Trichosporon asahii

Two novel β-glucosidases from Trichosporon asahii, named BG1 and BG2, were purified to electrophoretic homogeneity using ammonium sulfate precipitation, hydrophobic interaction, ion exchange, and gelfiltration chromatography. The molecular weight of BG1 and BG2 were estimated as 160 kDa and 30 kDa, respectively. The Km, Vmax, Kcat, and Kcat/Km values of the two β-glucosidases for p-nitrophenyl-β-d-glucopyranoside were determined. Both enzymes showed relatively high affinity to p-nitrophenyl-β-d-glucopyranoside in 4-nitrophenol glycosides and gentiobiose in saccharide substrates. The enzymes exhibited optimum activity at pH 6.0 and pH 5.5, respectively. Their respective optimum temperatures were 70 and 50 °C. Metal ions and inhibitors had different effects on the enzymes activities. Circular dichroism (CD) spectroscopy demonstrated that the purified BG1 exhibited a β-sheet-rich structure and that BG2 displayed a high random coil conformation. HPLC analysis of transglycosylation and reverse hydrolysis assay...