Phenolic compound-mediated covalent crosslinking as a universal mechanism for cellulase deactivation during lignocellulose saccharification.

Efficient enzymatic saccharification is critical for lignocellulose bioconversion, yet it is often challenged by various inhibitors in lignocellulosic biomass that suppress cellulase activity. This study identifies the phenolic compounds (PCs), particularly phenolic aldehydes, as key mediators of cellulase crosslinking, significantly impacting lignocellulosic bio-saccharification. We demonstrate that PCs, originating not only from lignin derivatives but also from raw extractives, covalently bond with protein side chains primarily via an oxygen-dependent mechanism, leading to cellulase deactivation. While PC-mediated crosslinking broadly affects various cellulases, its impact can be mitigated by removing oxygen or introducing amino group-containing competitors. This study provides insights into the origins and inhibitory effects of PCs on cellulase activity, highlights the importance of optimizing pretreatment processes to reduce residual PCs in extractives and minimize PC generation from lignin, and offers practical strategies to alleviate their adverse impacts, thereby enhancing saccharification efficiency.