Cloning, purification, and characterization of a thermostable β-Glucosidase from Thermotoga thermarum DSM 5069

Abstract

A 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 °C, and pH stability occurred from 5.0 to 6.0. After being incubated at 80 °C 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 °C. According to a phylogenetic analysis, TthBgl belongs to the glycosyl hydrolase family 3 (GH3).