Recombinant bifidobacterial endo-β-N-acetylglucosaminidases enable efficient release of bioactiveN-glycans from glycoproteins.

Glycosylation, one of the most significant and prevalent post-translational modifications, engenders a diverse array of protein structures, with over half of eukaryotic proteins assuming glycosylated forms. Given the limitations of alternative deglycosylation methods, identifying and characterizing novel glycosidases is important for understanding the diverse roles of glycans. In this study, the enzymatic activities of three recombinant endo-β-N-acetylglucosaminidases (ENGases) from Bifidobacterium pullorum subsp. pullorum (EndoBP), Bifidobacterium kashiwanohense (EndoBK), and Bifidobacterium bohemicum (EndoBbo) were assessed using ribonuclease B as a model glycoprotein. Furthermore, the optimal pH and temperature conditions for each enzyme were elucidated. Released N-glycans were quantified by a phenol-sulphuric acid assay, and the resulting N-glycan compositions were profiled by MALDI-MS. Moreover, comparative structural models were generated to provide structural context for the conserved GH18 catalytic regions of the enzymes. Based on the apparent kinetic analysis of the three enzymes on a glycoprotein mixture (whey, bovine IgG, bovine lactoferrin, and soy protein), Km values, expressed on a total protein mass basis, were 3.44, 7.18, and 2.85 mg/mL and Vmax values were 2.29 x 10-2, 2.52 x 10-2, and 1.35 x 10-2 mg/mL×min for EndoBP, EndoBK, and EndoBbo, respectively. MALDI-TOF/TOF-MS profiling of whey-derived N-glycans revealed distinct enzyme-dependent patterns, with 31, 21, and 10 N-glycan compositions detected after treatment with EndoBP, EndoBK, and EndoBbo, respectively. EndoBP exhibited the broadest observed glycan-release profile, whereas EndoBbo produced a comparatively narrower profile under the conditions examined. These findings highlight distinct glycan-release behaviors among the three ENGases and support the enzymatic recovery of structurally diverse, value-added N-glycan fractions from whey as a potential whey-valorization strategy.