Cloning with kinetic and thermodynamic insight of a novel hyperthermostable β-glucosidase from Thermotoga naphthophila RKU-10T with excellent glucose tolerance

Abstract

With a paradigm shift in industry, moving from natural fuels to alternative renewable resource utilization, the need of efficient thermostable cellulases are expected to increase in future. β-glucosidase, an essential member of cellulases that plays a critical role in cellulosic biomass degradation and in many biological processes. Therefore, a novel β-glucosidase gene encodes a protein (BglA) of 446 amino acid, belonging to glycoside hydrolase family 1 (GH1), was cloned from a hyperthermophilic bacterium Thermotoga naphthophila RKU-10T and over-expressed in Escherichia coli BL21CodonPlus. An extracellular BglA with a molecular weight of 51.50kDa, was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography after heat treatment. Purified enzyme displayed optimal activity at pH 7.0 and 95°C. It was quite stable over a broad range of pH (6.0​–9.0) and temperature (60–90°C), fairly stable up to 8h at 80°C. Enzyme activity was stimulated by glucose concentration up to 600mM and exhibited high glucose tolerance with a Ki value of 1200mM. BglA showed great affinity toward p-nitrophenyl substrates and cellobiose. The Km, Vmax and Kcat values, against pNPG as substrate, were 1.5mM, 297mmolmg−1min−1 and 1527778s−1, respectively. Thermodynamic parameters for pNPG hydrolysis by BglA like ΔH*, ΔG* and ΔS* were calculated at 95°C as 25.7kJmol−1, 47.24kJmol−1 and −58.6Jmol−1K−1, respectively. It displayed a half-life (t1/2) of 5.21min at 97°C with denaturation parameters of enzyme including ΔH*D, ΔG*D and ΔS*D were 662.04kJmol−1, 110.10kJmol−1 and 1.491kJmol−1K−1, respectively. This is the first ever report on a highly glucose and thermotolerant β-glucosidase from T. naphthophila with high catalytic efficiency and low product inhibition, also exhibited independence of detergents and metal cations. All these significant features make BglA an appropriate candidate for biotechnological and industrial applications.