Purification and Characterization of a Thermostable Cellobiohydrolase from Thermotoga petrophila

Background: Cellulose, being the most abundant biopolymer found in nature, can be utilized for bioethanol production to cater the future energy needs. Due to increased usage of fossil fuel it has been predicted that fossil fuel reserves may be depleted by year 2050. These concerns need serious attention and focus should be diverted to renewable fuels that are based on natural resources. Cellulases including exoglucanase (cellobiohydrolases) are the key enzymes that are produced by cellulolytic micro-organisms for the biodegradation of natural resource (cellulose) into fermentable reducing sugars. Many members of genus Clostridium possess supramolecular structures known as cellulosomes which contain various cellulases. Cellulase are composed of catalytic subunits that include endoglucanase, β-glucosidase and cellobiohydrolases which concurrently can catalyse and subsequently convert cellulose into glucose and other sugars. After the action of cellulases, the sugars can be conveniently converted into bioethanol. Objective: In the present study, characterization of a thermostable cellobiohydrolase enzyme from Thermotoga petrophila was carried out. The main purpose of this study is the utilization of thermostable cellobiohydrolase along with other cellulases in the process of saccharification of the cellulosic biomass to produce fermentable sugars that could in turn be converted into bioethanol which is the fuel of the future. Method: In this article, we propose a framework for achieving our a forementioned object. We started with the cloning of thermophilic cellobiohydrolase gene in mesophilic hosts to ease enzyme production. After cloning of cellobiohydrolase gene, submerged fermentation was performed for intracellular enzyme production. Microbial pellet obtained after centrifugation was sonicated and subjected to ammonium sulphate precipitation. The fraction obtained was purified to isoelectric homogeneity through ion exchange chromatography. Finally SDS analysis of purified cellobiohydrolase was carried out alongwith its characterization, kinetics and thermodynamics studies. Results: Purification fold of 4.05 was obtained along with enzyme activity and specific activity of 11.5 U ml-1 min-1 and 66.5 U mg-1, respectively. The molecular mass of purified recombinant enzyme was 37 kDa as calculated by means of SDS-PAGE analysis. The enzyme showed 50% residual activity at 90°C and also at a wide pH range of 4-10. The enzyme retained its activity in the presence of most of the metal ions except Fe+2, Hg+2 and Pb+2. EDTA has an inhibitory effect on the function of the enzyme. The catalytic activity of the enzyme was maintained in the presence of the organic solvents. The enzyme had a Km and Vmax of 4.6 mM and 25.64±1.87 µM min-1 for PNP-β- D-cellobioside under optimal conditions. Conclusion: The present study demonstrated that cellobiohydrolase produced from Thermotoga petrophila can be employed in many industries like paper and pulp and food processing. Most recent application of the cellobiohydrolases is their utilization in the production of bioethanol. Keywords: Thermotoga petrophila, cellobiohydrolase, thermostable, characterization, purification, biofuels.