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


ABSTRACT: As 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-?-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.

SUBMITTER: Cohen JL 

PROVIDER: S-EPMC6419974 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Immobilization of an Endo-β-<i>N</i>-acetylglucosaminidase for the Release of Bioactive <i>N-</i>glycans.

Cohen Joshua L JL   Karav Sercan S   Barile Daniela D   de Moura Bell Juliana M L N JMLN  

Catalysts (Basel, Switzerland) 20180710 7


As more is learned about glycoproteins' roles in human health and disease, the biological functionalities of <i>N</i>-linked glycans are becoming more relevant. Protein deglycosylation allows for the selective release of <i>N</i>-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-<i>N</i>-acetylglucosaminidase recently discov  ...[more]

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