Immobilization of an Endo-β-N-acetylglucosaminidase for the Release of Bioactive N-glycans
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-β-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 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. View Full-Text
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Cohen, J.L.; Karav, S.; Barile, D.; De Moura Bell, J.M.L.N. Immobilization of an Endo-β-N-acetylglucosaminidase for the Release of Bioactive N-glycans. Catalysts 2018, 8, 278.
Cohen JL, Karav S, Barile D, De Moura Bell JMLN. Immobilization of an Endo-β-N-acetylglucosaminidase for the Release of Bioactive N-glycans. Catalysts. 2018; 8(7):278.Chicago/Turabian Style
Cohen, Joshua L.; Karav, Sercan; Barile, Daniela; De Moura Bell, Juliana M.L.N. 2018. "Immobilization of an Endo-β-N-acetylglucosaminidase for the Release of Bioactive N-glycans." Catalysts 8, no. 7: 278.
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