Cloning, expression, purification and characterization of thermophilic enzymes catalyzing plant biomass degradation

Abstract

With the ever-increasing demand for food and crops, which are the primary source of production of 1st generation biofuels, the world is moving towards 2nd generation biofuels. The most abundant constituent of biomass, cellulose, is a regenerating source of biomass energy, however, it is present in a meshwork of lignin and hemicellulose, which are complex polymers and hence rigid and recalcitrant. Thus, the most essential step in the generation of 2nd generation biofuels from biomass is efficient enzymatic depolymerization. Thermophilic and hyperthermophilic cellulases which are very advantageous, owing to their ability to survive high temperatures, could be the best candidates for cellulose depolymerization. Here, we discuss our efforts to clone, express, purify and characterize some of the most potent and effective thermostable cellulases from the genome of Clostridium thermocellum and Pyrococcus furiosus. Through structural characterization, most of the enzymes were observed to be thermostable as well as resistant to chemical denaturation by urea and guanidine chloride. We also describe the attempts to study their activity on the substrate carboxymethyl cellulose with varying temperatures.