Characterization of a Novel Fructosyltransferase from Lactobacillus crispatus, InuCA, That Attaches to the Cell Surface by Electrostatic Interaction.

ABSTRACT

Fructosyltransferases (FTases), a group of carbohydrate-active enzymes, synthesize fructooligosaccharides (FOS) and fructans, which are promising prebiotics for human health. Here, we identified a novel FTase, InuCA, from Lactobacillus crispatus, a dominant species in the vaginal microbiota of human. InuCA was characterized by the shortest C terminus and the highest isoelectric point among the reported Lactobacillus FTases. InuCA was an inulosucrase and produced a series of FOS using sucrose as the substrate at a moderate temperature. Surprisingly, the C-terminal deletion mutant synthesized oligosaccharides with the fructosyl chain longer than that of the wild type, suggesting that the C-terminal part blocked the binding of long-chain receptor. Moreover, InuCA bound to the cell surface by electrostatic interaction, which was dependent on the environmental pH and represented a distinctive binding mode in FTases. The catalytic and structural properties of InuCA will contribute to FTase engineering and the knowledge of the adaptation of L. crispatus in the vaginal environment. IMPORTANCE L. crispatus is one of the most important species in human vaginal microbiotas, and its persistence is strongly negatively correlated with vaginal diseases. Our research reveals that a novel inulosucrase, InuCA, is present in L. crispatus. InuCA keeps the ability to synthesize prebiotic fructo-oligosaccharides, although it lacks a large part of the C-terminal region compared to other FTases. Remarkably, the short C terminus of InuCA blocks the transfructosylation activity for producing oligosaccharides with longer chains, which is meaningful for the directional modification of FTases and the oligosaccharide products. Besides the catalytic activity, InuCA is anchored on the cell surface, depending on the environmental pH, and also may be involved in the adhesion of L. crispatus to the vaginal epithelial cells. Since L. crispatus plays an essential role in the normal vaginal micro-ecosystem, the described work will be helpful to elucidate the functional genes and colonization mechanism of the dominant species.