Overexpression and characterization of TnCel12B, a hyperthermophilic GH12 endo-1,4-β-glucanase cloned from Thermotoga naphthophila RKU-10T

Abstract

A putative cellulolytic gene (825 bp) from Thermotoga naphthophila RKU-10T was overexpressed as an active soluble endo-1,4-β-glucanase (TnCel12B), belongs to glycoside hydrolase family 12 (GH12), in a mesophilic expression host. Heterologous expression and engineered bacterial cell mass was improved through specific strategies (induction and cultivation). Hence, intracellular activity of TnCel12B was enhanced in ZYBM9 modified medium (pH 7.0) by 8.38 and 6.25 fold with lactose (200 mM) and IPTG (0.5 mM) induction, respectively; and 6.95 fold was increased in ZYP-5052 auto-inducing medium after 8 h incubation at 26 °C (200 rev min−1). Purified TnCel12B with a molecular weight of ~32 kDa, was optimally active at 90 °C and pH 6.0; and exhibited prodigious stability over a wide range of temperature (50–85 °C) and pH (5.0–9.0) for 8 h TnCel12B displayed great resistance towards different chemical modulators, though activity was improved by Mg2+, Zn2+, Pb2+ and Ca2+. Purified TnCel12B had affinity with various substrates but peak activity was observed toward barley β-glucan (1664 U mg−1) and carboxymethyl cellulose (736 U mg−1). The values of Km, Vmax, kcat, and kcatKm−1 were found to be 4.63 mg mL−1, 916 μmol mg−1min−1, 1326.7 s−1 and 286.54 mL mg−1 s−1, respectively using CMC substrate. All noteworthy features of TnCel12B make it an appropriate industrial candidate for bioethanol production and various other potential applications.