Immobilization of an Endo-?-N-acetylglucosaminidase for the Release of Bioactive N-glycans

Basit Öğe Kaydı
dc.authoridbarile, daniela/0000-0002-3889-1596
dc.authoridKARAV, SERCAN/0000-0003-4056-1673
dc.contributor.authorCohen, Joshua L.
dc.contributor.authorKarav, Sercan
dc.contributor.authorBarile, Daniela
dc.contributor.authorBell, Juliana M. L. N. de Moura
dc.date.accessioned2025-01-27T21:01:38Z
dc.date.available2025-01-27T21:01:38Z
dc.date.issued2018
dc.departmentÇanakkale Onsekiz Mart Üniversitesi
dc.description.abstractAs more is learned about glycoproteins' roles in human health and disease, the biological functionalities of N-linked glycans are becoming more relevant. Protein deglycosylation allows for the selective release of N-glycans and facilitates glycoproteomic investigation into their roles as prebiotics or anti-pathogenic factors. To increase throughput and enzyme reusability, this work evaluated several immobilization methods for an endo-beta-N-acetylglucosaminidase recently discovered from the commensal Bifidobacterium infantis. Ribonuclease B was used as a model glycoprotein to compare N-glycans released by the free and immobilized enzyme. Amino-based covalent method showed the highest enzyme immobilization. Relative abundance of N-glycans and enzyme activity were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Kinetic evaluation demonstrated that upon immobilization, both V-max and the K-m decreased. Optimal pH values of 5 and 7 were identified for the free and immobilized enzyme, respectively. Although a higher temperature (65 vs. 45 degrees C) favored rapid glycan release, the immobilized enzyme retained over 50% of its original activity after seven use cycles at 45 degrees C. In view of future applications in the dairy industry, we investigated the ability of this enzyme to deglycosylate whey proteins. The immobilized enzyme released a higher abundance of neutral glycans from whey proteins, while the free enzyme released more sialylated glycans, determined by nano-LC Chip Q-ToF MS.
dc.description.sponsorshipCenter for Advanced Processing and Packaging Studies (CAPPS); National Institutes of Health [R01AT007079, R01AT008759]; USDA: NIFA Hatch project [232719]; Training Program in Biomolecular Technology at the University of California, Davis [T32-GM008799]
dc.description.sponsorshipThis research was supported by the Center for Advanced Processing and Packaging Studies (CAPPS), the National Institutes of Health awards R01AT007079 and R01AT008759, the USDA: NIFA Hatch project 232719, and was partially supported by an industry/campus supported fellowship under the Training Program in Biomolecular Technology (T32-GM008799) at the University of California, Davis. The authors would like to thank David Mills for providing the endo-beta-N-acetylglucosaminidase enzyme and Nannan Tao from Bruker Daltonics for assistance with MALDI-ToF MS applications.
dc.identifier.doi10.3390/catal8070278
dc.identifier.issn2073-4344
dc.identifier.issue7
dc.identifier.pmid30881698
dc.identifier.scopus2-s2.0-85050009391
dc.identifier.scopusqualityQ1
dc.identifier.urihttps://doi.org/10.3390/catal8070278
dc.identifier.urihttps://hdl.handle.net/20.500.12428/27130
dc.identifier.volume8
dc.identifier.wosWOS:000440016400025
dc.identifier.wosqualityQ2
dc.indekslendigikaynakWeb of Science
dc.indekslendigikaynakScopus
dc.indekslendigikaynakPubMed
dc.language.isoen
dc.publisherMdpi
dc.relation.ispartofCatalysts
dc.relation.publicationcategoryinfo:eu-repo/semantics/openAccess
dc.rightsinfo:eu-repo/semantics/openAccess
dc.snmzKA_WoS_20250125
dc.subjectN-glycans
dc.subjectmass spectrometry
dc.subjectimmobilization
dc.subjectprebiotic
dc.subjectglycosidase
dc.subjectrecombinant
dc.subjectkinetic
dc.subjectnano-LC Chip Q-ToF MS
dc.titleImmobilization of an Endo-?-N-acetylglucosaminidase for the Release of Bioactive N-glycans
dc.typeArticle

Dosyalar

Koleksiyon

WoS İndeksli Yayınlar Koleksiyonu
PubMed İndeksli Yayınlar Koleksiyonu
Scopus İndeksli Yayınlar Koleksiyonu