(672f) Understanding the Interactions between T. Reesei Cel7a and the Plant Cell Wall Cellulose Substrate

Understanding the complex interaction between the substrate chemistry and enzyme function is critical to improving enzymatic biomass conversion. Crystalline cellulose forms the core of carbohydrate microfibrils that provide structure and strength to plant cell walls. It has long been recognized that crystalline cellulose substrate is the most recalcitrant carbohydrate to enzymatic hydrolysis, making its conversion rate limiting in many processes. Carbohydrate binding modules (CBMs), which are a component of the Trichoderma reesei Cel7A cellobiohydrolase, may play an important role in the initial decrystallzation of cellulose. We are evaluating the effect of cellulose crystallinity on T. reesei Cel7A action using pure cellulose samples of varying crystallinities (as measured by solid state NMR) and by site-directed mutagenesis of residues on the CBM. We have evaluated a set of 'disordered' or 'amorphous' celluloses generated from pure cellulose substrates (filter paper, Avicel, cotton, Sigma alpha cellulose and Sigmacell 50). Digestion studies with T. reesei Cel7A have confirmed that the new material is dramatically more digestible, both in initial digestion rates and extent of digestibility. All four of the amorphous cellulose substrates appear, however, to have an upper limit of digestion between 70 to 80%. Whether the reason for this digestion limit is due to substrate or enzyme effects will be evaluated using cellobiohydrolase mutants with altered cellulose binding modules.