Unveiling the Inhibition of Enzymatic Hydrolysis of Cellulose by Lignin-Derived Phenolics: Interfacial Kinetics and Molecular Simulations

Enzymatic hydrolysis of cellulose is a crucial step in lignocellulose sugar-platform biorefineries, and this process can be significantly inhibited by lignin-derived phenolics. This study investigated the inhibition of the enzymatic hydrolysis of cellulose by phenolics, focusing on its impact on Cel7A, a key enzyme involved in cellulose hydrolysis. Phenolics primarily inhibited the productive binding of Cel7A to cellulose, likely due to interference with catalytic domain (CD) binding. Surprisingly, phenolics increased Cel7A processivity. Kinetic modeling revealed that while phenolics increase Cel7A processivity, they decrease their association (kon) and dissociation (koff), and the dissociation step is considered as the limiting factor in hydrolysis in the presence of phenolics. Molecular dynamics simulations provided further insights, showing that phenolics form stable complexes with Cel7A through hydrogen bonds, hydrophobic interactions, and π-stacking. This binding altered Cel7A's structure, resulting in a more enclosed catalytic tunnel and less flexible loops, likely accounting for the observed changes in processivity and koff. Our study bridges the gap between interfacial kinetics and molecular simulations, thereby shedding light on the molecular mechanisms of phenolic inhibition. This knowledge paves the way for enzyme engineering strategies to enhance cellulase efficiency for biorefinery applications.