Characterization of a Novel α-l-Fucosidase from Truepera sp. for Efficient Transfucosylation and 2′-Fucosyllactose Biosynthesis

Fucose is a key deoxyhexose found in polysaccharides, glycolipids, and glycoproteins. 2′-Fucosyllactose (2′FL), a major human milk oligosaccharide with health benefits for infants, faces production challenges due to the limited availability of efficient α-l-fucosidases. Here, we present a novel α-l-fucosidase, True-Fuc, from Truepera sp. heterologously expressed for 2′FL biosynthesis. True-Fuc, a GH29A family enzyme (50 kDa), showed optimal activity at 50 °C and pH 8.0, hydrolyzing para-nitrophenyl-α-l-fucopyranoside (pNP-α-Fuc), Lewisa, and Lewisx substrates. It catalyzed 2′FL synthesis via transfucosylation using pNP-α-Fuc and lactose with minimal degradation of 2′FL and 3-fucosyllactose (3FL). Molecular dynamics simulations revealed that loops 1–4 surrounding the substrate pocket mediate substrate recognition, while the flexible C-terminal loop 5 plays a minor role. These results establish True-Fuc as a promising tool for cost-effective 2′FL production and novel glycoside synthesis.