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.
Sercan Karava, Annabelle Le Parca, Juliana Maria Leite Nobrega de Moura Bella, Camille Rouquiéa, David A. Millsa,b,c , Daniela Barilea,b, and David E. Blockc,d,* aDepartment of Food Science and Technology, University of California, One Shields Avenue, Davis, CA 95616, USA bFoods for Health Institute, University of California, One Shields Avenue, Davis, CA 95616, USA cDepartment of Viticulture and Enology, University of California, Davis, CA, USA dDepartment of Chemical Engineering and Materials Science, University of California, Davis, CA