Boosting Enzyme\nActivity in Biomass Conversion by\nModulating the Hydrolysis Process of Cellobiohydrolases

Cellobiohydrolases (CBHs) are the most significant cellulose-degrading\nenzymes, the performance of which determines the cost-effective utilization\nof renewable lignocellulosic resources. Most engineering strategies\nfor improving CBH hydrolysis are currently focused on accelerating\nthe noncatalytic enzyme–substrate dissociation by increasing\nthe flexibility of eight substrate-enclosing loops (SELs), which does\nnot take the catalysis into account or even deteriorates it. Here,\nin the model Trichoderma reesei CBHI,\nwe identified a key SEL that affects the dissociation by examining\nenzyme–enzyme/substrate interactions. Furthermore, through\nanalyzing the hydrogen-bonding network for the catalytic region, we\ndetected a crucial residue D262. Root-mean-square-fluctuation analysis\nindicates that its replacement with valine (D262V) markedly improves\nthe stability of the catalytic triad. Through QM/MM simulations, we\ndetermined that this mutation diminished the free-energy barrier against\ncatalysis by 2.3 kcal/mol and increased <i>k</i><sub>cat</sub> by 53.1%, as determined in kinetic experiments. Additionally, the\nsubstitution caused a significant enhancement of SEL flexibility and\nled to a lowered dissociation barrier by 2.1 kcal/mol. The cellobiose\nyield was increased by 49.8%, owing to the impact of the single valine\nreplacement on the enzyme hydrolysis. This work unlocks a brand-new\nengineering direction for industrially important CBHs, contributing\nto more efficient depolymerization of renewable lignocellulose.