EndoBI-1 is a recently isolated endo-beta-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. infands. 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. K-m values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and V-max values ranging from 3.5 x 10(-3) to 5.09 x 10(-3), 4.5 x 10(-3) to 7.75 x 10(-3) and 1.9 x 10(-2)to 5.2 x 10(-2) mg/mL x 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. (C) 2015 Published by Elsevier Inc.