Immobilization of an Endo-?-N-acetylglucosaminidase for the Release of Bioactive N-glycans
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Tarih
2018
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Mdpi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
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-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.
Açıklama
Anahtar Kelimeler
N-glycans, mass spectrometry, immobilization, prebiotic, glycosidase, recombinant, kinetic, nano-LC Chip Q-ToF MS
Kaynak
Catalysts
WoS Q Değeri
Q2
Scopus Q Değeri
Q1
Cilt
8
Sayı
7
