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- Title
Immobilization of an Endo-β-N-acetylglucosaminidase for the Release of Bioactive N-glycans.
- Authors
Cohen, Joshua L.; Karav, Sercan; Barile, Daniela; De Moura Bell, Juliana M. L. N.
- Abstract
As more is learned about glycoproteins’ roles in human health and disease, the biological functionalities of <italic>N</italic>-linked glycans are becoming more relevant. Protein deglycosylation allows for the selective release of <italic>N</italic>-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-β-<italic>N</italic>-acetylglucosaminidase recently discovered from the commensal <italic>Bifidobacterium infantis</italic>. Ribonuclease B was used as a model glycoprotein to compare <italic>N</italic>-glycans released by the free and immobilized enzyme. Amino-based covalent method showed the highest enzyme immobilization. Relative abundance of <italic>N</italic>-glycans and enzyme activity were determined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Kinetic evaluation demonstrated that upon immobilization, both Vmax and the Km decreased. Optimal pH values of 5 and 7 were identified for the free and immobilized enzyme, respectively. Although a higher temperature (65 vs. 45 °C) favored rapid glycan release, the immobilized enzyme retained over 50% of its original activity after seven use cycles at 45 °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.
- Subjects
BIOACTIVE compounds; GLYCAN analysis; GLYCOPROTEINS; DEGLYCOSYLATION; PREBIOTICS; ENZYME analysis
- Publication
Catalysts (2073-4344), 2018, Vol 8, Issue 7, p278
- ISSN
2073-4344
- Publication type
Article
- DOI
10.3390/catal8070278