Cloning and biochemical characterization of β-glucosidases

Sinha, Sushant Kumar (2015) Cloning and biochemical characterization of β-glucosidases. Masters thesis, Indian Institute Science Education and Research Kolkata.

PDF (MS dissertation of Sushant Kumar Sinha (13IP022)) Sushant_Kumar_Sinha_13IP022.pdf - Submitted Version Restricted to Repository staff only Download (1MB)

Abstract

An important step in the bioconversion of lignocellulosic biomass into biofuels is the hydrolysis of the biomass into monomeric sugars by cellulases. β-glucosidases (EC 3.2.1.21) is a cellulase that cleaves β-glucosidic linkages in disaccharide or glucose-substituted molecules and produces fermentable glucose which can then be used for biofuel production. Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification and increases the need for more enzymes during the saccharification reaction. In order to understand the molecular basis of glucose inhibition, two enzymes were characterized. The first one is from a halophilic bacteria (B8CYA8, Halothermothrix orenii) and the second from a thermophilic archaea (O08324, Thermococcus sp.). Both were cloned in Escherichia coli and the proteins were recombinantly over-expressed and purified by Ni2+-affinity chromatography. The enzyme activities were assayed using the model substrate 4-Nitrophenyl β-D-glucopyranoside (pNPGlu) and the natural substrate cellobiose. The optimum temperature and pH are 70 °C and 7.1 for B8CYA8, 80°C and 6.5 for O08324 respectively. The steady state enzyme kinetics was determined along with the effect of different concentrations of glucose on the kinetics. Each of these enzymes showed glucose tolerance with subtle differences and enhanced thermo stability in the presence of glucose. These results indicate that B8CYA8 and O08324 are ideal candidates for further research and industrial applications as an important constituent of a cellulase cocktail.

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