Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri

1 Centre for Carbohydrate Bioengineering, TNO-RUG, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands 2 Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands 3 Innovative Ingredients and Products Department, TNO Nutrition and Food Research, Rouaanstraat 27, 9723 CC Groningen, The Netherlands Correspondence L. Dijkhuizen L.Dijkhuizen{at}biol.rug.nl Lactobacillus reuteri strain 121 employs a fructosyltransferase (FTF) to synthesize a fructose polymer [a fructan of the levan type, with (2 6) linkages] from sucrose or raffinose. Purification of this FTF (a levansucrase), and identification of peptide amino acid sequences, allowed isolation of the first Lactobacillus levansucrase gene ( lev ), encoding a protein (Lev) consisting of 804 amino acids. Lev showed highest similarity with an inulosucrase of L. reuteri 121 [Inu; producing an inulin polymer with (2 1)-linked fructosyl units] and with FTFs from streptococci. Expression of lev in Escherichia coli resulted in an active FTF (Lev 773His) that produced the same levan polymer [with only 2–3 % (2 1 6) branching points] as L. reuteri 121 cells grown on raffinose. The low degree of branching of the L. reuteri levan is very different from bacterial levans known up to now, such as that of Streptococcus salivarius , having up to 30 % branches. Although Lev is unusual in showing a higher hydrolysis than transferase activity, significant amounts of levan polymer are produced both in vivo and in vitro . Lev is strongly dependent on Ca 2+ ions for activity. Unique properties of L. reuteri Lev together with Inu are: (i) the presence of a C-terminal cell-wall-anchoring motif causing similar expression problems in Escherichia coli , (ii) a relatively high optimum temperature for activity for FTF enzymes, and (iii) at 50 °C, kinetics that are best described by the Hill equation. Abbreviations: FTF, fructosyltransferase; HPSEC-MALLS, high-performance size-exclusion chromatography multi-angle laser light scattering The GenBank accession number for the L. reuteri 121 gene encoding levansucrase ( lev ) and its flanking regions is AF465251.