Recombinant bifidobacterial endo-β-N-acetylglucosaminidases enable efficient release of bioactive N-glycans from glycoproteins
Abstract
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-Nacetylglucosaminidases (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), K m values, expressed on a total protein mass basis, were 3.44, 7.18, and 2.85 mg/mL and V max 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 Nglycan 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.
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BibTeX
@article{duman2026recombinant,
title = {{Recombinant bifidobacterial endo-β-N-acetylglucosaminidases enable efficient release of bioactive N-glycans from glycoproteins}},
author = {Hatice Duman and Merve Kaplan and Berfin Sucu and Melda Karyelioğlu and Ayşenur Arslan and Furkan Eker and Yalçın Mert Yalçıntaş and Emir Akdaşçi and İzzet Avcı and Hacı Mehmet Kayılı and Bekir Salih and Mikhael Bechelany and Sercan Karav},
journal = {Enzyme and Microbial Technology},
year = {2026},
month = sep,
doi = {10.1016/j.enzmictec.2026.110962},
url = {https://hal.umontpellier.fr/hal-05769496v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Recombinant bifidobacterial endo-β-N-acetylglucosaminidases enable efficient release of bioactive N-glycans from glycoproteins
AU - Hatice Duman
AU - Merve Kaplan
AU - Berfin Sucu
AU - Melda Karyelioğlu
AU - Ayşenur Arslan
AU - Furkan Eker
AU - Yalçın Mert Yalçıntaş
AU - Emir Akdaşçi
AU - İzzet Avcı
AU - Hacı Mehmet Kayılı
AU - Bekir Salih
AU - Mikhael Bechelany
AU - Sercan Karav
PY - 2026
DA - 2026/09/29
JO - Enzyme and Microbial Technology
DO - 10.1016/j.enzmictec.2026.110962
UR - https://hal.umontpellier.fr/hal-05769496v1
AB - 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-Nacetylglucosaminidases (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), K m values, expressed on a total protein mass basis, were 3.44, 7.18, and 2.85 mg/mL and V max 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 Nglycan 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.
SN - 0141-0229
ER -