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Enzyme Microb Technol. 2015 Sep;77:46-53. doi: 10.1016/j.enzmictec.2015.05.007. Epub 2015 Jun 3.

Kinetic characterization of a novel endo-β-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.

Author information

1
Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA.
2
Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA; Foods for Health Institute, University of California, One Shields Avenue, Davis, CA 95616, USA; Department of Viticulture and Enology, University of California, Davis, CA, USA.
3
Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA; Foods for Health Institute, University of California, One Shields Avenue, Davis, CA 95616, USA.
4
Department of Viticulture and Enology, University of California, Davis, CA, USA; Department of Chemical Engineering and Materials Science, University of California, Davis, CA, USA. Electronic address: deblock@ucdavis.edu.

Abstract

EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans.

KEYWORDS:

Deglycosylation; Endo-β-N-acetylglucosaminidase; N-glycans

PMID:
26138399
PMCID:
PMC4733529
DOI:
10.1016/j.enzmictec.2015.05.007
[Indexed for MEDLINE]
Free PMC Article

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