TOWARDS ASSEMBLY OF EFFICIENT CELLULOLYTIC COCKTAIL BASED ON CLOSTRIDIUM THERMOCELLUM ENZYMES

Second generation bioethanol production processes involve an enzymatic hydrolysis step, in which the lignocellulosic biomass is degraded into fermentable sugars. This step is one of the rate-limiting obstacles, due to the relatively high costs of enzyme production. Thus, assembly of a reinforced enzyme mixture, capable of hydrolyzing biomass in an efficient manner, is critical. A general scheme of cellulose hydrolytic enzymes consists of three main groups: endoglucanases, exoglucanases and β-glucosidases. Abstract In order to promote the assembly of a Clostridium thermocellum-based cellulosic mixture, two different approaches were implemented: A) Screening: We have developed an easy-to-use scalable method, designed to screen and classify cellulosic enzymes. Using this method, over thirty six C. thermocellum enzymes were analyzed and classified, thus establishing a database of C. thermocellum enzymatic activities. Such databases enable us to compare the activities, and thus can provide insight on key enzymes for use in future cellulolytic mixtures. B) Improving activity: The role of β-glucosidase is critical to prevent feedback inhibition of the cellulases. Thus, we applied directed-evolution techniques for mutagenesis and consequent improvement of the C. thermocellum enzyme, β-glucosidase A. A mutant clone with increased thermal stability properties was thus achieved. Such a mutant will enable decreased enzyme dosage in the cocktail and will enable reactions at higher temperatures.