Characterization of a Novel α-<scp>l</scp>-Fucosidase from <i>Truepera sp.</i> 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 <i>Truepera</i> 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 (<i>p</i>NP-α-Fuc), Lewis^a, and Lewis^x substrates. It catalyzed 2'FL synthesis via transfucosylation using <i>p</i>NP-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.