Mining for new levansucrase enzymes with high levan production, transfructosylating activity, and thermal stability and studying their kinetics and acceptor specificity.

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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 β-(2→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.

BACKGROUND: Levansucrase (LS)-catalyzed-transfructosylation reaction is a potential approach for the synthesis of fructooligosaccharides (FOSs) and levan as health promoting compounds. This biocatalytic approach is hindered by low thermal stability of LS and its high rate of hydrolysis. In the present study, LS from Bacillus amyloliquefaciens was immobilized onto modified and unmodified epoxy-activated supports (Eupergit ® C; Sepabeads ® ) and on modified cross-linked-agarose beads, to increase its thermal stability and modulate its reaction selectivity (hydrolysis/transfructosylation).

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