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 pnitrophenyl-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 Cu 2+ , in which only 3% and 10% residual activity was maintained, respectively. The Vmax of TthBgl was 8.79 U mg -1 for pnitrophenyl-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).

A glucose tolerant β-glucosidase from a newly isolated Neofusicoccum parvum strain F7: production, purification, and characterization

“…These findings were also reported by Qin et al 11 . The highest β -glucosidase activity was in the presence of FeSO 4, and the lowest activity was in the presence of ZnSO 4, CoCl 2, KH 2 PO 4 NaCl, similar results were also reported in other studies also where β -glucosidase activity declined in the presence of MnSO 4, ZnSO 4 and CoCl 2 11 , 18 .…”

“…The effects of eight metal ions (Ca 2+ , Co 2+ , Fe 2+ , Mg 2+ , Mn 2+ , Zn 2+ , K + , and Na + ) on β -glucosidase activity were determined at a final concentration of 4 mM at the optimal pH and temperature (4.0 and 65 °C) (Table 8 ). β -Glucosidases were either inhibited or activated by the metal ions 18 . All eight metal ions reduced β -glucosidase activity in Bgl3 by 50% or more in this study.…”

“…The OVAT technique allows for the optimization of one factor at a time, however, the disadvantage of this method is that it is laborious, time-consuming and does not allow one to study the interaction of variables thus making it impossible to detect the true optimum when multiple different variables come together 17 . Therefore, statistical methods such as PBD and BBD are used to eliminate the limitations of the OVAT optimization process 18 . PBD is a screening technique used to screen media components in shake flasks reducing the total number of experiments, thus determining the most important factors 19 .…”

Highly Efficient Biotransformation of Astragaloside IV to Cycloastragenol by Sugar-Stimulated ¥ é-Glucosidase and é é-Xylosidase from Dictyoglomus thermophilum

“…Although many β-glucosidases and β-xylosidases have been cloned and characterized from plant, yeast and bacteria, only a few studies about highly thermotolerant and sugar-tolerant β-glucosidases and β-xylosidases have been reported. Thermophilic bacteria, such as Thermotoga thermarum, Thermotoga petrophila and Thermoanaerobacterium saccharolyticum, are well known to produce plentiful glycoside hydrolases with competitive advantages, such as great catalytic ability and excellent pH and temperature stability [39][40][41]. In the previous study, we identified βglucosidase of GH3, Dth3, and β-xylosidase with GH39, Xln-DT, from Dictyoglomus thermophilum, a kind of thermophilic bacteria.…”