# 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.

## Key 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.