Characterization and purification of thermostable β-glucosidase from Clostridium thermocellum

A β-glucosidase was isolated from Clostridium thermocellum; the enzyme was localized in the periplasmic space. It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing. The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity. Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.