Combined Computational and Experimental Investigations of Processive Cellulases for Engineering Activity Improvements

The Family 7 cellobiohydrolase (Cel7A) from Hypocrea jecorina (Trichoderma reesei) is one of the most well-characterized, active cellulases known for deconstruction of cellulose to soluble sugars. With the aim to engineer enhanced cellulase specific activity, we have investigated the Cel7A mechanism on cellulose with molecular dynamics simulations and biochemical experiments. We have elucidated new functions for the carbohydrate-binding module, determined the effect of glycosylation on the stability of the linker and carbohydrate-binding module, examined the role of aromatic and polar residues in the catalytic domain tunnel, and examined the structure of the catalytically-active complex of Cel7A on crystalline cellulose up to the microsecond time scale. These new insights combined with experimental activity data have led to a large-scale experimental screening of new enzymes to examine both natural diversity and engineered enzymes for activity and stability improvements. Our combined computational and experimental approach has demonstrated that unprecedented improvements in specific activities of processive cellulases are possible, which when combined in industrial cocktails, will result in lower enzyme loadings for cell wall deconstruction and lower overall production costs for renewable biofuels.