Boosting Enzyme Activity in Biomass Conversion by Modulating the Hydrolysis Process of Cellobiohydrolases

Cellobiohydrolases (CBHs) are the most significant cellulose-degrading enzymes, the performance of which determines the cost-effective utilization of renewable lignocellulosic resources. Most engineering strategies for improving CBH hydrolysis are currently focused on accelerating the noncatalytic enzyme–substrate dissociation by increasing the flexibility of eight substrate-enclosing loops (SELs), which does not take the catalysis into account or even deteriorates it. Here, in the model Trichoderma reesei CBHI, we identified a key SEL that affects the dissociation by examining enzyme–enzyme/substrate interactions. Furthermore, through analyzing the hydrogen-bonding network for the catalytic region, we detected a crucial residue D262. Root-mean-square-fluctuation analysis indicates that its replacement with valine (D262V) markedly improves the stability of the catalytic triad. Through QM/MM simulations, we determined that this mutation diminished the free-energy barrier against catalysis by 2.3 kcal/mol and increased kcat by 53.1%, as determined in kinetic experiments. Additionally, the substitution caused a significant enhancement of SEL flexibility and led to a lowered dissociation barrier by 2.1 kcal/mol. The cellobiose yield was increased by 49.8%, owing to the impact of the single valine replacement on the enzyme hydrolysis. This work unlocks a brand-new engineering direction for industrially important CBHs, contributing to more efficient depolymerization of renewable lignocellulose.