Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates

TL;DR

This study explores new levansucrase enzymes from various bacteria, analyzing their ability to synthesize fructooligosaccharides and their structural features influencing substrate specificity and product diversity.

Contribution

It uncovers novel levansucrases with enhanced transfructosylating activity and broader acceptor promiscuity through genome mining and structural analysis.

Findings

Gluconobacter oxydans and Novosphingobium aromaticivorans LS produce fructooligosaccharides up to 13 units.

LS enzymes show high acceptor promiscuity with monosaccharides, disaccharides, and alcohols.

Raffinose is a preferred donor over sucrose for certain LS enzymes.

Abstract

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via $\beta$-(2$\to$6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.