# Sequence‐Based and Functional Analysis for the Discovery of N‐Glycan Degrading Glycosidases From the Microbial Metagenome of the Infant Gut

## TL;DR

This study identifies and characterizes glycosidases in the infant gut microbiome that break down N-glycans, revealing their potential role in shaping the microbiota.

## Contribution

The paper discovers and functionally characterizes novel glycosidases from the infant gut metagenome that act on N-glycans.

## Key findings

- Nine β-galactosidases were identified, with two (Gal1b and Gal99) capable of removing galactose from glycoconjugates.
- Exo360 uniquely hydrolyzes GlcNAc at both α1,3 and α1,6 branches of N-glycans, while Endo38 and Endo358 release full N-glycan structures.
- The infant gut microbiota shows a diverse range of glycosidases with distinct substrate preferences for N-glycan degradation.

## Abstract

The role of bacterial glycosyl hydrolases (GHs) in degrading free human milk oligosaccharides is well documented. However, their activity on glycoconjugates is less well known. Here, an in silico analysis of the metagenome of the fecal microbiome of breastfed infants was employed to identify GH2 β‐galactosidases, GH20 exo‐N‐acetylglucosaminidases and GH18 endo‐N‐acetylglucosaminidases active on N‐glycans. A total of nine β‐galactosidases were recombinantly expressed and two of them, Gal1b and Gal99, were able to remove galactose from the G2 peptide and asialofetuin. Gal1b, Gal25, Gal37c, Gal99 and Gal296 hydrolyzed lactose and N‐acetyllactosamine, indicating specificity for galactose β1,4‐linked to glucose or GlcNAc. All of the exo‐β‐N‐acetylglucosaminidases studied here (Exo10a, Exo18, Exo38, Exo39b, Exo360 and Exo399) hydrolyzed the disaccharide N‐acetylglucosaminyl‐β1,2‐mannose, which forms part of the N‐glycan structures. Exo10a, Exo38 and Exo360 hydrolyzed N‐acetylglucosamine (GlcNAc) from the G2 peptide pretreated with Gal1b. Notably, Exo360 hydrolyzed GlcNAc at both the α1,3 and α1,6 branches of the G2 peptide core mannose simultaneously, whereas Exo10a showed a preference for GlcNAc at one branch. Exo38 and Exo360 also release GlcNAc from asialofetuin once galactose has been removed. The whole structures of N‐glycans were liberated from glycoproteins by the action of the endo‐N‐acetylglucosaminidases Endo38 and Endo358. These enzymes hydrolyze the N,N’‐diacetylchitobiose core of N‐linked glycans of the high‐mannose and non‐sialylated complex types, respectively. Overall, these results provide insight into the range of glycosyl hydrolases present in the infant gut microbiota that act on glycoconjugates, which may play a role in the establishment and composition of the newborn microbiota.

This research offers valuable understanding of how N‐glycans are broken down by the gut microbiota of infants, focusing on glycoside hydrolase families GH2, GH20, and GH18. It reveals that the gut microbiota of breastfed infants has a diverse array of genes coding for these enzymes. The functional analysis of GH2 β‐galactosidases, GH20 exo‐N‐acetylglucosaminidases, and GH18 endo‐N‐acetylglucosaminidases shows distinct substrate preferences, highlighting their involvement in the degradation of dietary and host‐derived glycans.