Mutations at the glucose-stimulation site in the gatekeeper region of β-glucosidase CbiCba3 from Cellulomonas biazote reveal an activity-stability trade-off.

β-Glucosidase (BGL) catalyzes the hydrolysis of cellooligosaccharides into glucose during lignocellulosic biomass saccharification and is widely used in industrial applications such as bioethanol and biorefinery processes, food and beverage production, and platform chemical manufacturing. The BGL CbiCba3 exhibits glucose-product stimulation and glucose tolerance, making it an attractive enzyme for industrial applications. Although the glucose-stimulation site of CbiCba3 has been identified within the gatekeeper region, its molecular function has not been fully elucidated. In this study, structural and functional analyses were performed to investigate the role of the gatekeeper region in glucose stimulation. The single mutants N293A and Q295A, which are involved in glucose-binding residues in the gatekeeper region, showed reduced catalytic efficiency and weaker glucose-stimulated activity compared with the wild-type enzyme. However, the N293A and N293A/Q295A mutants exhibited improved thermal stability. Structural analysis indicated that alanine substitutions at the glucose-binding residues increased the volume of the substrate-entry region but resulted in a less favorable environment for substrate binding owing to increased hydrophobicity. Molecular dynamics simulations revealed that mutations in the gatekeeper region increased the flexibility of loop regions near the substrate entrance and perturbed the structural environment of the glucose-binding site. These results provide mechanistic insight into the role of the gatekeeper region in regulating glucose stimulation and reveal an activity-stability trade-off associated with mutations at the glucose-stimulation site. These findings offer guidance for engineering BGLs for enhanced industrial applications.