Substrate Versatility of a Recombinant Bifidobacterial Endoglycosidase: N-Glycan Release and Profiling from Milk and Plant Glycoproteins

N-linked glycans (N-glycans) are complex carbohydrate structures covalently attached to the asparagine residues of glycoproteins and play a substantial role in protein folding, stability and biological activity. One of the major challenges in glycomics is the enzymatic release of intact N-glycans from native glycoproteins, as conventional deglycosylation approaches often require prior protein denaturation and exhibit limited activity toward structurally diverse substrates. In this study, we recombinantly produced EndoBI-1, an endo-β-N-acetylglucosaminidase derived from Bifidobacterium longum subsp. infantis ATCC 15697, and investigated its N-glycan release activity on two structurally distinct glycoprotein substrates: bovine lactoferrin (bLF), an animal-derived glycoprotein, and soy protein, a plant-derived glycoprotein. EndoBI-1 was produced using an in vivo cloning approach and purified to homogeneity, as validated by SDS-PAGE analysis. The released N-glycan profiles of each substrate were characterized by HILIC-FLD-QTOF-MS/MS analysis, which showed different glycan compositions depending on the origin: complex-type and sialylated N-glycans were the major structures in the bLF derived fractions, while the fractions derived from soy protein mainly contained high-mannose-type structures. Together, these results demonstrate the capacity of EndoBI-1 to release structurally intact N-glycans from both animal- and plant-derived glycoproteins under native, non-denaturing conditions, establishing this enzyme as a biocatalytic tool for the preparative-scale production of bioactive N-glycans from diverse protein sources.