Print Email Facebook Twitter Biochemical and molecular characerization of a levansucrase from Lactobacillus reuteri Title Biochemical and molecular characerization of a levansucrase from Lactobacillus reuteri Author van Hijum, S.A.F.T. Szalowska, E. van der Maarel, M.J.E.C. Dijkhuizen, L. TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO Publication year 2004 Abstract Lactobacillus reuteri strain 121 employs a fructosyltransferase (FTF) to synthesize a fructose polymer [a fructan of the levan type, with β(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 β(2→1)-linked fructosyl units] and with FTFs from streptococci. Expression of lev in Escherichia coli resulted in an active FTF (LevΔ773His) that produced the same levan polymer [with only 2-3 % β(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 Ca2+ 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. © 2004 SGM. Chemicals / CAS: amino acid, 65072-01-7; calcium ion, 14127-61-8; fructose, 30237-26-4, 57-48-7, 7660-25-5, 77907-44-9; inulin, 9005-80-5; levan, 50815-13-9, 9013-95-0; levansucrase, 9030-17-5; raffinose, 512-69-6; sucrose, 122880-25-5, 57-50-1; transferase, 9047-61-4; DNA, Bacterial; Fructans; Hexosyltransferases, EC 2.4.1.-; levansucrase, EC 220.127.116.11; Recombinant Proteins. Molecular Sequence Numbers: GENBANK: AF459437, AL162757, CAA05973, L08445, M18954, P11701, Q06447, Q55242, X02730, AF465251; Subject NutritionFood technologyAmino acidBacterial enzymeCalcium ionFructoseInulinLevanLevansucrasePolymerRaffinoseSucroseTransferaseBacterial cellBacterial geneBacterial strainBacterium isolationCarboxy terminal sequenceCell anchorageCell growthChemical analysisControlled studyEnzyme activityEnzyme kineticsEnzyme purificationEnzyme synthesisEscherichia coliGenetic codeHigh temperatureHydrolysisIn vitro studyIn vivo studyLactobacillus reuteriMolecular biologyNonhumanNucleotide sequencePriority journalProtein expressionProtein motifSequence analysisStreptococcus salivariusAmino Acid SequenceBase SequenceCarbohydrate ConformationDNA, BacterialEscherichia coliFructansGenes, BacterialHexosyltransferasesKineticsLactobacillusMolecular Sequence DataRecombinant ProteinsSequence DeletionSequence Homology, Amino AcidBacteria (microorganisms)Escherichia coliLactobacillusLactobacillus reuteriStreptococcusStreptococcus salivarius To reference this document use: http://resolver.tudelft.nl/uuid:456154a8-63c9-48ac-a9aa-0df10e51c707 TNO identifier 39281 ISSN 1350-0872 Source Microbiology, 150, 621-630 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.