Recombinant expression of α-galactosidases and screening of targeted enzymes for guar gum side chains

β-Mannan oligosaccharides (β-MOS) are a class of high-performance prebiotics with significant application value in food, feed, and related fields. Guar gum, rich in a β-mannan backbone, is an ideal raw material for β-MOS production, but the α-galactose residues on its side chains severely hinder the efficient preparation and application promotion of β-MOS. To overcome this critical technical bottleneck, four novel α-galactosidase genes (BfGal27A, BfGal27B, BfGal36A, BfGal36B) were cloned from Bacteroides fragilis NCTC9343. Soluble expression and purification of recombinant proteins were achieved using an Escherichia coli heterologous expression system, and their enzymatic properties were systematically characterized. The results showed that all recombinant enzymes exhibit weakly alkaline catalytic preference and mesophilic adaptability, with different metal ions regulating their catalytic activities. Substrate-specific screening based on guar gum demonstrated that BfGal36B displays highly efficient targeted catalytic activity toward α-galactose residues in guar gum side chains. A strong binding affinity between BfGal36B and guar gum was verified via molecular docking, and the stable hydrogen bond network formed between its active center and the substrate provides a key structural basis for efficient degradation. BfGal36B was successfully screened as a specific degrading enzyme for guar gum side chains in this study for guar gum side chains, enriching the glycoside hydrolase resources library and providing important theoretical and technical support for the large-scale production of functional β-MOS, high-value processing of agricultural products, and industrial application of glycosidases.