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

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Abstract
A .beta.-glucosidase (EC 3.2.1.21) was purified to homogeneity from cell-free extracts of an extremely thermophilic anaerobic bacterium. The enzyme has an Mr 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 .beta.-glycosides and .beta.-linked disaccharides, with .beta.-galactosidase activity only slightly less than .beta.-glucosidase activity, and significant .beta.-xylosidase activity. Lineweaver-Burk plots for p-nitrophenyl .beta.-glucoside, o-nitrophenyl .beta.-glucoside and cellobiose substrates are biphasic concave-downwards. Inhibition of the .beta.-glucosidase by substrates and glucose is negligible. Thermal inactivation follows first-order kinetics, with t1/2 (65.degree.C) 45 h, t1/2 (75.degree.C) 47 min and t1/2 (85.degree.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, t1/2 (75.degree.C) is 130 min. At 75.degree.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.