Interrelationship of Substrate Crystallinity, Enzyme Binding Strength, and Cellulase Activity

Abstract

AbstractStructural polysaccharides are difficult to degrade due to their crystalline structure. Hence, industrial conversion of biomass has focused on both substrate pretreatment and enzyme engineering to improve the biochemical conversion of biomass into fuels and chemicals. However, few studies have explored the interrelationship between substrate crystallinity and cellulase activity. Here, we systematically investigated the kinetics of structurally diverse cellulases on five cellulosic substrates with varying crystallinity. Regardless of enzyme structure and catalytic mechanism, we observed a linear scaling of the kinetic parameters (KMandkcat) in a log-log plot, indicating a linear free energy relationship (LFER) between binding and activation energy. LFERs were observed for all investigated substrates, but their slopes varied distinctly and appeared linked to the substrate crystallinity. Substrates with low crystallinity exhibited LFERs with a slope near 1, while highly crystalline substrates had a slope of approximately 0.25, providing insights into the transition state (TS) for the rate-limiting step. We propose that maximal turnover was limited by slow dissociation, with the TS structurally close to the enzyme-ligand complex on crystalline substrate, while on amorphous substrate, the TS structure was closer to the dissociated system. We suggest that these observations reflect competing interactions of the ligand with respectively the enzyme binding cleft and the substrate matrix. This study emphasizes the interconnected nature of substrate pretreatment and enzyme engineering, urging a holistic approach to propel the biochemical conversion of lignocellulosic biomass, crucial for advancing sustainable production of fuels and chemicals.