Abstract
Akkermansia muciniphila (AKK) is a mucin-degrading gut symbiont with emerging probiotic potential. Among its carbohydrate-active enzymes, Amuc_0517, a glycoside hydrolase family 36 (GH36) protein, has been identified as a highly specific α-galactosidase. In this study, the Amuc_0517 gene was cloned into pET-28a(+), expressed in Escherichia coli BL21, and purified via Ni(2+)-NTA affinity chromatography. Bioinformatic analysis indicated the presence of a signal peptide and α-galactosidase domain. Enzyme assays confirmed its ability to cleave α-1,6-glycosidic bonds in pNPGal, with no detectable activity toward pNPGlu, and molecular dynamics simulations revealed stronger binding affinity and lower free energy with pNPGal, supporting its substrate specificity. Given that α-galactosidases are widely applied in the dairy industry to hydrolyze galactose-containing oligosaccharides in milk and whey, the biochemical features of Amuc_0517 suggest its potential as a novel biocatalyst for functional dairy processing and probiotic-enriched dairy product development.