α-L-Fucosidases from an Alpaca Faeces Metagenome: Characterisation of Hydrolytic and Transfucosylation Potential

In various life forms, fucose-containing glycans play vital roles in immune recognition, developmental processes, plant immunity, and host-microbe interactions. Together with glucose, galactose, N-acetylglucosamine, and sialic acid, fucose is a significant component of human milk oligosaccharides (HMOs). Fucosylated HMOs benefit infants by acting as prebiotics, preventing pathogen attachment, and potentially protecting against infections, including HIV. Although the need for fucosylated derivatives is clear, their availability is limited. Therefore, synthesis methods for various fucosylated oligosaccharides are explored, employing enzymatic approaches and alpha-L-fucosidases. This work aimed to characterise alpha-L-fucosidases identified in an alpaca faeces metagenome. Based on bioinformatic analyses, they were confirmed as members of the GH29A subfamily. The recombinant alpha-L-fucosidases were expressed in Escherichia coli and showed hydrolytic activity towards p-nitrophenyl-alpha-L-fucopyranoside and 2 '-fucosyllactose. Furthermore, the enzymes' biochemical properties and kinetic characteristics were also determined. All four alpha-L-fucosidases could catalyse transfucosylation using a broad diversity of fucosyl acceptor substrates, including lactose, maltotriose, L-serine, and L-threonine. The results contribute insights into the potential use of alpha-L-fucosidases for synthesising fucosylated amino acids.