Site-specific Glycosylation via Metal-Targeted Conjugation Enhances Biochemical Functions of Bovine Lactoferrin

Bovine lactoferrin (BLF) has attracted increasing attention due to its multiple nutritional benefits. However, the precise modification of BLF remains a significant challenge. Here, a site-specific glycosylation strategy, based on a copper assisted sequence-specific binding tag and prediction of glycosylation sites, was designed to enable the precise and controlled glycosylation of BLF in vitro. An optimal site-specific glycosylation conjugate was further fabricated to promote the biochemical functions, which prevented the original structural disruption caused by the random introduction of chitosan oligosaccharide, thereby maintaining the structure of native BLF. Site-specific glycosylation significantly improved the antioxidant capacity of the protein, achieving 68.34% ABTS radical scavenging activity. Moreover, the antibacterial effect of the glycoprotein was obviously enhanced, especially against Escherichia coli, with an antibacterial rate as high as 61.05% after 6 h. This work boosted the poor stability in native BLF and broadened its biochemical properties, as well as application prospects.