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Biochemical and molecular characerization of a levansucrase from Lactobacillus reuteri

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Author: Hijum, S.A.F.T. van · Szalowska, E. · Maarel, M.J.E.C. van der · Dijkhuizen, L.
Type:article
Date:2004
Institution: TNO Voeding Centraal Instituut voor Voedingsonderzoek TNO
Source:Microbiology, 150, 621-630
Identifier: 39281
Keywords: Nutrition · Food technology · Amino acid · Bacterial enzyme · Calcium ion · Fructose · Inulin · Levan · Levansucrase · Polymer · Raffinose · Sucrose · Transferase · Bacterial cell · Bacterial gene · Bacterial strain · Bacterium isolation · Carboxy terminal sequence · Cell anchorage · Cell growth · Chemical analysis · Controlled study · Enzyme activity · Enzyme kinetics · Enzyme purification · Enzyme synthesis · Escherichia coli · Genetic code · High temperature · Hydrolysis · In vitro study · In vivo study · Lactobacillus reuteri · Molecular biology · Nonhuman · Nucleotide sequence · Priority journal · Protein expression · Protein motif · Sequence analysis · Streptococcus salivarius · Amino Acid Sequence · Base Sequence · Carbohydrate Conformation · DNA, Bacterial · Escherichia coli · Fructans · Genes, Bacterial · Hexosyltransferases · Kinetics · Lactobacillus · Molecular Sequence Data · Recombinant Proteins · Sequence Deletion · Sequence Homology, Amino Acid · Bacteria (microorganisms) · Escherichia coli · Lactobacillus · Lactobacillus reuteri · Streptococcus · Streptococcus salivarius

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 2.4.1.10; Recombinant Proteins. Molecular Sequence Numbers: GENBANK: AF459437, AL162757, CAA05973, L08445, M18954, P11701, Q06447, Q55242, X02730, AF465251;