AbstractA 56-kDa β-glucosidase (TthBgl) derived from Thermotoga thermarum DSM 5069 was expressed and purified from Escherichia coli BL21 (DE3). The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-β-D-glucopyranoside among the synthetic glycosides tested. The pH maximum was 5.0, and under the conditions tested, maximal activity was at 85 ºC, and pH stability occurred from 5.0 to 6.0. After being incubated at 80 ºC for 120 min, TthBgl retained 80% of its original activity. The β-glucosidase had no apparent requirement for metal ions or other co-factors, but its activity was significantly inhibited by 0.1% SDS and 1mM Cu2+, in which only 3% and 10% residual activity was maintained, respectively. The Vmax of TthBgl was 8.79 U mg-1 for p-nitrophenyl-β-D-glucopyranoside, while the Km was 2.41 mM. The Enzyme activity was gradually inhibited by the addition of glucose, but remained approximately 50% of its original value in 500 mM glucose. 789.25 mg/L glucose was released from cellobiose by the incubation of 0.2 U/mL TthBgl for 9 h at 75 ºC. According to a phylogenetic analysis, TthBgl belongs to the glycosyl hydrolase family 3 (GH3).