Abstract:Prebiotics are in high demand due to their health benefits，yet the varieties of commercially available products are limited.Low-molecular-weight pectin-derived oligosaccharides（POS）serve as a new type of prebiotic candidates，and the correlations between their structures and activities have become a key focus of research. The interactions between Gal4Ara（Gal1→4Gal1→4Gal1→4Gal1→3Ara），a neutral oligosaccharide prepared via enzymatic hydrolysis of commercial apple pectin，and 27 glycosidases derived from the gut microbiota were analyzed. The structure-activity relationship of Gal4Ara was then clarified based on relevant parameters of the colonic fermentation model. The results showed that with other glycosidases except A7KGA5 had the binding free energy lower than-7.00 kcal/mol（1 kcal=4.184 kJ）with Gal4Ara，indicating successful docking. The binding free energy between Gal4Ara and the glycosidases from beneficial bacteria in healthy intestines was lower than -9.00 kcal/mol，and the affinity was significantly higher than that with the glycosidases from pathogenic bacteria（binding free energy higher than -8.50 kcal/mol）. Glutamic acid（Glu）residues were the key residues in the interactions between Gal4Ara and glycosidases. They acted as not only the catalytic residues of glycosidases but also the main residues for binding to Gal4Ara. Meanwhile，differences in the interaction forces between different glycosidases and Gal4Ara led to variations in dissociation constant（Kd）and half-saturation constant（K1POS），which in turn affected hydrolysis kinetics. In conclusion，Gal4Ara exhibited high affinity for glycosidases from the beneficial bacteria of gut microbiota and showed significant prebiotic potential.Moreover，Glu residues played a core role in the interactions between Gal4Ara and glycosidases.