Journal of Proteomics & Bioinformatics
Open Access

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

2^nd International Conference on Genetic & Protein Engineering

November 14-16, 2016 Atlanta, Georgia, USA

Fatima Akram

Government College University, Pakistan

Posters & Accepted Abstracts: J Proteomics Bioinform

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. �?²-glucosidase, an essential member of cellulases that plays a critical role in cellulosic biomass degradation and in many biological processes. Therefore, a novel �?²-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 BL21 CodonPlus. An extracellular BglA with a molecular weight of 51.50 kDa, was purified to homogeneityby 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 8 h at 80�?°C. Enzyme activity was stimulated by glucose concentration up to 600 mM and exhibited high glucose tolerance with a Ki value of 1200 mM.BglA showed great affinity towardsp-nitrophenyl substrates and cellobiose. The Km, Vmax and Kcat values, against pNPG as substrate, were 1.5 mM, 297 mmol mg-1min-1 and 1527778 s-1, respectively.Thermodynamic parameters for pNPG hydrolysis by BglA like �?�?H*, �?�?G* and �?�?S* were calculated at 95�?°C as 25.7 kJ mol-1, 47.24 kJ mol-1 and -58.6 J mol-1 K-1, respectively. It displayed a half-life (t1/2) of 5.21 min at 97�?°C with denaturation parameters of enzyme including �?�?H*D, �?�?G*D and �?�?S*D were 662.04 kJ mol-1, 110.10 kJ mol-1 and 1.491 kJ mol-1 K-1, respectively. This is the first ever report on a highly glucose and thermotolerant �?²-glucosidase from Thermotoga 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.

Biography :

Email: fatima_iib@yahoo.com