title: Kinetic characterization of a novel endo-β-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.
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.
authors.affiliation: Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Sercan
authors.initials: S
authors.lastname: Karav
authors.affiliation: Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Annabelle Le
authors.initials: AL
authors.lastname: Parc
authors.affiliation: Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Juliana Maria Leite Nobrega
authors.initials: JM
authors.lastname: de Moura Bell
authors.affiliation: Department of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Camille
authors.initials: C
authors.lastname: Rouquié
authors.affiliation: 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.
authors.forename: David A
authors.initials: DA
authors.lastname: Mills
authors.affiliation: 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.
authors.forename: Daniela
authors.initials: D
authors.lastname: Barile
authors.affiliation: 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.
authors.forename: David E
authors.initials: DE
authors.lastname: Block
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001644:Bifidobacterium / Q000201:enzymology / Q000235:genetics; D002417:Cattle; D003126:Colostrum / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005798:Genes, Bacterial; D006031:Glycosylation; D007700:Kinetics; D007781:Lactoferrin / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D056692:Prebiotics; D011247:Pregnancy; D012260:Ribonucleases / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D000067796:Whey / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011134:Polysaccharides; D056692:Prebiotics; D012260:Ribonucleases; C029600:ribonuclease B; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D007781:Lactoferrin
keywords: Deglycosylation; Endo-β-N-acetylglucosaminidase; N-glycans