Direct Determination of the Interaction between Carbohydrate-Binding Modules and Lignin with Different Phenol Hydroxyl Contents

The nonproductive adsorption between lignin and cellulase hinders bioethanol production, with phenolic hydroxyl (Ph-OH) groups believed to play a crucial role in the lignin-cellulase interaction. However, the adsorption mechanism remains unclear and unverified. In this work, lignins with different Ph-OH contents were systematically prepared by acetylation or phenolation of softwood kraft lignin (KL). Single-molecule dynamic force spectroscopy with immobilized carbohydrate-binding modules was employed to directly quantify these interactions. A linear increase in both CBM adsorption on lignin and interaction force (2.31-8.33 nN) was observed with the Ph-OH content, providing direct evidence that the Ph-OH content is an important contributing factor affecting CBM-lignin interactions. Physicochemical characterization showed increased surface negative charge and decreased hydrophobicity. Thermodynamic analyses suggested that electrostatic attraction, hydrogen bonding, and hydrophobic interactions all contribute to CBM-lignin association, while increasing the Ph-OH content enhanced the relative contribution of electrostatic attraction and hydrogen bonding. This study emphasizes the contribution of Ph-OH to cellulase adsorption, offering a basis for lignin modification strategies in lignocellulosic biorefineries.