Production of recombinant glycosidases fused with Usp45 and SpaX to avoid the purification and immobilization stages.

The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ±â€¯0.36, 5.81 ±â€¯0.29 and 9.90 ±â€¯0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ±â€¯0.09 mg/mL, 4.11 ±â€¯0.21 mg/mL, and 4.08 ±â€¯0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.

Characterization and stabilization of the α-L-fucosidase set from Lacticaseibacillus rhamnosus INIA P603.

This study describes the molecular identification, biochemical characterization, and stabilization of three recombinant AlfA, AlfB, and AlfC fucosidases from Lacticaseibacillus rhamnosus INIA P603. Even though previous studies revealed the presence of fucosidase activity in L. rhamnosus extracts, the identification of the fucosidases, their physicochemical properties, and the substrate spectrum remained unknown. Although the presence of alfB is not common in strains of L. rhamnosus, fucosidases from L. rhamnosus INIA P603 were selected because this strain exhibited higher fucosidase activity in culture and the complete set of fucosidases. A high yield of purified recombinant AlfA, AlfB, and AlfC fucosidases was obtained (8, 12, and 18 mg, respectively). AlfA, AlfB, and AlfC showed their optimal activities at pH 5.0 and 4.0 at 60 °C, 40 °C, and 50 °C, respectively. Unlike 3-fucosyllactose, all three recombinant fucosidases were able to hydrolyze 2'-fucosyllactose (2'-FL), and their activities were improved through their immobilization on agarose supports. Nevertheless, immobilized AlfB exhibited the highest hydrolysis, releasing 39.6 µmol of fucose mg enzyme-1 min-1. Only the immobilized AlfB was able to synthetize 2'-FL. In conclusion, the enzymatic properties elucidated in this study support the potential ability of fucosidases from L. rhamnosus INIA P603 to hydrolyze fucosylated substrates as well as justifying interest for further research into AlfB for its application to catalyze the synthesis of fucosylated prebiotics. KEY POINTS: • Few strains of L. rhamnosus exhibited alfB on their chromosomes. • Fucosidases from L. rhamnosus INIA P603 were characterized and stabilized. • Although all the fucosidases hydrolyzed 2'-FL, only AlfB transfucosylated lactose.