To enhance the yield of l-fucosylated molecules synthesized via transfucosylation, we employed α-l-transfucosidases, enzymes engineered from α-l-fucosidases to favour transglycosylation over hydrolysis. This study investigated the transglycosylation potential of mutated variants of α-l-fucosidase iso1 from Paenibacillus thiaminolyticus designed by structural comparison with Thermotoga maritima α-l-transfucosidase. Using site-directed mutagenesis, point mutations (S237A, S237G, S237P, S237V, Y189F) were introduced, and the corresponding recombinant proteins were successfully expressed and purified. Among them, the S237V variant achieved the highest overall yield of fucosylated lactose and increased the transglycosylation/hydrolysis ratio more than 20-fold compared to the wild-type enzyme. This variant enabled regioselective synthesis of 3'-and 6'-fucosyllactose, as well as a functionalized 3'-fucosyllactose bearing a Boc-protected ethylthioureidyl linker (3'-FucLac-tBoc), with all structures confirmed by NMR spectroscopy. The engineered α-l-transfucosidase iso1 from Paenibacillus thiaminolyticus catalysed the regioselective synthesis of structurally defined fucosyllactose derivatives, including C-1 functionalized analogs suitable for immobilization on biosensor surfaces or macromolecular scaffolds, thus expanding the biocatalytic toolbox and demonstrating the potential of semi-rational enzyme design for targeted HMO-based glycoengineering applications.
Vodičková et al. (Wed,) studied this question.