Thiol Modification of β-Glucosidase via N-Methoxycarbonylmaleimide for Enhancement of Catalytic Efficiency and Environmental Tolerance

β-Glucosidase (BGL) is limited by the catalytic efficiency and environmental tolerance in the biomass biorefinery process. In this study, maleimide and its derivatives were used to modify BGL. FT-IR, circular dichroism, ζ-potential, ultraviolet, and fluorescence were used to characterize the conformational alteration of the thiol-modified enzyme and induced the enhancement of rigidity and substrate affinity. Molecular docking revealed that the modified BGL, especially Moc-Mal-BGL, had lower binding energy (−6.8 kcal mol–1) and shorter average hydrogen bond length (2.018 Å) with the substrate. The activity of Moc-Mal-BGL increased by 110.6% compared with native BGL and realized a 2.6-fold increase in catalytic efficiency kcat/Km (13.92 s–1/mM). The corresponding cellobiose conversion rate increased by 19.0%. Thermal stability, storage stability, solvent (GVL and DMSO) tolerance, and glucose tolerance of Moc-Mal-BGL were significantly improved. This work provides a rapid strategy to improve the catalytic efficiency and environmental tolerance of BGL and investigates the mechanism in depth.