Release of bifidogenic N-glycans from native bovine colostrum proteins by an endo-ß-N-acetylglucosaminidase.

Milk glycoproteins play various biological roles including antibacterial, antiviral activities, modulating immune responses in living organisms. Released N-glycans from milk glycoproteins act as growth substrates for infant-associated bifidobacteria, which are key members of the breastfed infant's gut. To date, the mechanisms, and contributions of glycans to the biological activities of glycoproteins remain to be elucidated. Only by testing both the released glycans and the deglycosylated protein in their native (i.e., non-denatured) form, can the individual contribution to the biological activity of glycoproteins be elucidated. However, for conventional enzymatic and chemical deglycosylation strategies to work efficiently, glycoprotein denaturation is required, which alters the protein native shape, hindering further investigations of its biological roles. An endo-ß-N-acetylglucosaminidase (EndoBI-1) from Bifidobacterium longum subsp. infantis ATCC 15697 (B. infantis) was characterized as having the ability to release N-glycans from bovine milk glycoproteins efficiently, without the denaturation. In this study, the activity of EndoBI-1 was compared to a commercial enzyme to release N-glycans, the peptide-N-glycosidase F (PNGase F), using dairy glycoproteins as the substrate. The kinetic evaluation showed that EndoBI-1 displayed higher activity on native glycoproteins than PNGase F, with 0.036 mg/mL×min and 0.012 mg/mL×min glycan release, respectively. EndoBI-1 released a broader array of glycan structures compared to PNGase F from native glycoproteins. Thirty-two and fifteen distinct compositions were released from the native glycoproteins by EndoBI-1 and PNGase F, respectively, as characterized by advanced mass spectrometry. EndoBI-1 can be considered a promising enzyme for the release of N-glycans and their protein backbone in the native form, which will enable effective glycan release and will facilitate subsequent investigations to reveal their contribution to glycoproteins' biological roles.

Effects of Industrial Thermal Treatments on the Release of Bovine Colostrum Glycoprotein N-Glycans by Endo-ß-N-acetylglucosaminidase.

N-Glycans are structurally similar to human milk oligosaccharides, the gold standard prebiotics for infants. Bovine milk N-glycans released by endo-ß-N-acetylglucosaminidase (EndoBI-1) were shown to have similar prebiotic selectivity as human milk oligosaccharides, explaining the interest for N-glycan recovery for use as prebiotics. Industrial thermal treatments such as high-temperature short-time (HTST) and ultra-high-temperature (UHT) might favor the enzymatic deglycosylation of N-glycans through promoting protein denaturation. We investigated the effects of HTST (72 °C for 15 s) and UHT (135 °C for 3 s) on N-glycan release from bovine colostrum glycoproteins by nonimmobilized and amino-immobilized EndoBI-1. A total of 104 N-glycans including isomers/anomers were identified by high-resolution mass spectrometry. In both EndoBI-1 forms, HTST increased the release of N-glycans; however, the impact of UHT on releasing N-glycans was comparable to the nonthermal treatment. Although the amino-immobilized enzyme similarly released neutral N-glycans as the free form, it released fewer sialylated and fucosylated N-glycans.