Expression, Purification, and Functional Exploration of an α-Galactosidase from Akkermansia muciniphila

TL;DR

Scientists cloned and purified an α-galactosidase from Akkermansia muciniphila and found it could break down specific sugar bonds, suggesting potential use in dairy processing.

Contribution

The study reports the cloning, purification, and functional characterization of a novel α-galactosidase from Akkermansia muciniphila.

Findings

Amuc_0517 was successfully cloned, expressed in E. coli, and purified using Ni2+-NTA affinity chromatography.

The enzyme specifically cleaves α-1,6-glycosidic bonds in pNPGal with no activity toward pNPGlu.

Molecular dynamics simulations confirmed stronger binding affinity and lower free energy with pNPGal, supporting its substrate specificity.

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 Ni2+-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.

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