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.

Structure and Dynamics of Actin-Like Cytomotive Filaments in Plasmid Segregation.

One of the well-known functions of the bacterial cytoskeleton is plasmid segregation. Type II plasmid segregation systems, among the best characterized with respect to the mechanism of action, possess an actin-like cytomotive filament as the motor component. This chapter describes the essential components of the plasmid segregation machinery and their mechanism of action, concentrating on the actin-like protein family of the bacterial cytoskeleton. The structures of the actin-like filaments depend on their nucleotide state and these in turn contribute to the dynamics of the filaments. The components that link the filaments to the plasmid DNA also regulate filament dynamics. The modulation of the dynamics facilitates the cytomotive filament to function as a mitotic spindle with a minimal number of components.