Unveiling the role of long-range and short-range forces in the non-productive adsorption between lignin and cellulases at different temperatures.

Quantitatively understanding of interaction mechanism between lignin and cellulases is essential for the efficient improvement of lignocellulose enzymatic hydrolysis. However, the individual contribution of multiple forces between lignin and cellulases to the non-productive adsorption of enzymes still remains deeply ambiguous, especially in situations of near enzymatic hydrolysis temperatures. Herein, atomic force microscopy (AFM) and computational simulations were utilized to quantitatively analyze the intermolecular forces between lignin and enzyme at 25 °C and 40 °C. Our results unveiled that an increase in temperature obviously improved adsorption capacity and total intermolecular forces between lignin and cellulases. This positive relationship mainly comes from the increase in the decay length of hydrophobic forces for lignin-cellulases when temperature increases. Different from the hydrophobic interaction which provides long-range part of attractions, van der Waals forces dominate the intermolecular force only at approaches < 2 nm. On the other hand, electrostatic forces exhibited repulsive effects, and its intensity and distance were limited due to the low surface potential of cellulases. Short-range forces including hydrogen bonding (main) and π-π stacking (minor) stabilize the non-specific binding of enzymes to lignin, but increasing temperature reduces hydrogen bond number. Therefore, the relative contribution of long-range forces increased markedly at higher temperatures, which benefits protein capture and brings lignin and cellulase close together. Finally, the structure-activity relationships between lignin physicochemical properties and its inhibitory effect to enzymes indicated that hydrophobic interactions, hydrogen bonding, and steric effects drive the final adsorption capacity and glucose yields. This work provides quantitative and basic insights into the mechanism of lignin-cellulase interfacial interactions and guides design of saccharification enhancement approaches.