Gene cloning, characterization and thermodynamic analysis of a novel multidomain hyperthermophilic GH family 3 β-glucosidase (TnBglB) from Thermotoga naphthophila RKU-10~T

摘要

The gene of a multidomain glycoside hydrolase family 3 beta-glucosidase (TnEglB) was cloned from a hyperthermophilic Thermotoga naphthophila RKU-10(T) and overexpressed in Escherichia coli BL21 CodonPlus (DE3)-RIPL. Extracellular TnBglB was purified to homogeneity using heat precipitation, followed by anion-exchange and hydrophobic interaction chromatography, with a molecular weight of 81 kDa on SDS-PAGE. TnBglB exhibited great affinity towards p-nitrophenyl and cellobiose substrates. The enzyme was optimally active at 85 degrees C and pH 5.0, and showed great thermostability over a broad range of temperature (60-85 degrees C) mainly at pH 6.0-7.5 for 540 min. TnBglB activity was stimulated with Ca2+ addition. The K-i value was 150 mm and 200 mM for glucose and xylose inhibition, respectively. K-m, V-max, k(cat) and k(cat) K-m(-1) values were 0.45 mM, 153 mmolmg(-1) min(-1), 1214285 s(-1) and 2698413 mM(-1)s(-1), respectively using pNPG as substrate. Thermodynamic parameters as Delta H*, Delta G* and Delta S* for pNPG hydrolysis at 85 degrees C were 24.09 kJ mol(-1), 46.55 kJ mol(-1) and -62.74 Jmol(-1)K(-1), respectively. TnBglB displayed a half-life (t(1/2)) of 4.44 min at 94 degrees C with denaturation parameters including Delta H-D*, Delta G(D)* and Delta S-D* were 283.78 kJ mol(-1), 108.69 kJ mol(-1) and 0.477 kJ mol(-1)K(-1), respectively. This hyperthermostable multimodular beta-glucosidase exhibited noteworthy properties, which make it a favorable candidate for various industrial applications.