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

Catalysts

Daniela Barile¹  JoshuaL. Cohen¹  JulianaM. L. N. de Moura Bell¹  Sercan Karav² 

[1] Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA;Department of Molecular Biology and Genetics, Canakkale Onsekiz Mart University, 17100 Canakkale, Turkey;

关键词: N-glycans;    mass spectrometry;    immobilization;    prebiotic;    glycosidase;    recombinant;    kinetic;    nano-LC Chip Q-ToF MS;   

DOI  :  10.3390/catal8070278

来源: DOAJ

【 摘 要 】

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-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.

【 授权许可】

Unknown