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

Publication date: Available online 10 December 2015 Source:Journal of Molecular Catalysis B: Enzymatic Author(s): Fatima Akram, Ikram ul Haq, Mahmood Ali Khan, Zahid Hussain, Hamid Mukhtar, Kaleem Iqbal 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 51.50 kDa, 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 towards p-nitrophenyl substrates and cellobiose. The K m, V max and K cat values, against pNPG as substrate, were 1.5mM, 297 mmol mg−1min−1 and 1527778 s−1, respective...
Source: Journal of Molecular Catalysis B: Enzymatic - Category: Biochemistry Source Type: research