Abstract:Beta-glucosidase is one of the rate-limiting enzymes in the cellulase system. In order to study the connection mode of the substrate，as well as the optimum substrate of β-glucosidase in the process of enzymatic hydrolysis，the β-glucosidase from Aspergillus niger 3.316 was used as the research object.Molecular docking of three substrates was performed and the kinetic parameters were tested.The results showed that all of the three substrates were connected to the β-glucosidase complex via hydrogen bonding.Among the three transition state complexes，the complex between cellobiose and the enzyme was the most stable；this was probably due to the formation of H-π conjugation.Additionally，the kinetic parameters showed that the catalytic rate and maximum reaction rate were the highest when cellobiose was the substrate；on the other hand，4-nitrophenyl-β-D-glucopyranoside（pNPG）had the strongest affinity to β-glucosidase.This study revealed a suitable substrate for βglucosidase，and found that the effect on catalytic efficiency and catalytic rate might due to H-π conjugation，providing a valuable reference for the understanding of the molecular transformation of β-glucosidase.