Purification and properties of a stable β-glucosidase from an extremely thermophilic anaerobic bacterium

Abstract

A β-glucosidase (EC 3.2.1.21) was purified to homogeneity from cell-free extracts of an extremely thermophilic anaerobic bacterium. The enzyme has an M(r) of 43,000 as determined by molecular-exclusion chromatography, has a pI of 4.55 and shows optimum activity at pH 6.2. The enzyme is active against a wide range of aryl β-glycosides and β-linked disaccharides, with β-galactosidase activity only slightly less than β-glucosidase activity, and significant β-xylosidase activity. Lineweaver-Burk plots for p-nitrophenyl β-glucoside, o-nitrophenyl β-glucoside and cellobiose substrates are biphasic concave-downwards. Inhibition of the β-glucosidase by substrates and glucose is negligible. Thermal inactivation follows first-order kinetics, with t( 1/2 ) (65°C) 45 h, t( 1/2 ) (75 °C) 47 min and t( 1/2 ) (85°C) 1.4 min and a deactivation energy of 380 kJ/mol at pH 6.2. At pH 7.0, which is the optimum pH for thermostability, t( 1/2 ) (75°C) is 130 min. At 75°C, at pH 6.2, the thermostability is enhanced about 8-fold by 10% (w/v) glycerol, about 6-fold by 0.2 M-cellobiose and about 3-fold by 5 mM-dithiothreitol and 5 mM-2-mercaptoethanol.