5-01: Activity improvement and understanding of cellobiohydrolase function through enzyme engineering

We have recently engineered new Family 7 processive cellobiohydrolases to understand the thermodynamic and kinetic contributions to enzyme activity. New, engineered cellobiohydrolase I enzymes were created by gene synthesis, expressed in an in-house developed expression strain of Trichoderma reesei (Hypocrea jecorina), and evaluated for activity on dilute-acid pretreated corn stover. Concurrently, wild type and mutant enzymes were analyzed in silico using molecular dynamics simulations of interactions with crystalline cellulose to gain understanding of the molecular mechanisms responsible for observed biochemical differences between the different enzymes. One of these engineered enzymes demonstrated a nearly threefold improvement on dilute-acid pretreated corn stover in a ternary enzyme digestion assay at industrially-relevant enzyme loadings. This work demonstrated that significant improvement of cellobiohydrolase I activity is possible using protein engineering techniques and contributes to a fundamental understanding of this well-studied, yet poorly understood class of enzymes that is crucial to current cellulosic biofuels production processes.