Abstract
Exopolysaccharides (EPS) can affect the rheological properties of foods, act as stabilizers or stimulate preferential growth of bifidobacteria in the gut and therefore function as prebiotics. The latter is referred to fructans, which are synthesized from sucrose by fructosyl transferases (FTFs). In this work, the FTF enzyme of Lactobacillus panis TMW1.648 isolated from sourdough was characterized. The coding gene was identified, sequenced and expressed heterologously in E. coli. Enzyme activity was maximal at pH 4.0–4.6, 45°C and a substrate concentration of 300 mmol l−1. It produced free fructose, a high molecular fructan and the oligosaccharide kestose from sucrose. Calcium ions proved to be essential for the enzymatic activity. In comparison to published data of other FTF enzymes of lactobacilli the described enzyme showed significant differences.
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Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Dal Bello FD, Walter J, Hertel C, Hammes WP (2001) In vitro study of prebiotic properties of levan-type exopolysaccharides from Lactobacilli and non-digestible carbohydrates using denaturing gradient gel electrophoresis. Syst Appl Microbiol 24:232–237
Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. In: Gilbert HJ, Davies GJ, Henrissat B, Svensson B (eds) Recent advances in carbohydrate bioengineering. The Royal Society of Chemistry, Cambridge, pp 3–12
Davies GJ, Wilson KS, Henrissat B (1997) Nomenclature for sugar-binding subsites in glycosyl hydrolases. Biochem J 321(Pt 2):557–559
Delzenne NM, Kok N (2001) Effects of fructans-type prebiotics on lipid metabolism. Am J Clin Nutr 73:456S–458S
Hanahan D (1985) Techniques for transformation of E. coli. In: Glover DM (ed) DNA cloning: a practical approach. IRL Press, Oxford, p 109
Hernandez L et al (1995) Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane. Biochem J 309(Pt 1):113–118
Korakli M, Gänzle MG, Vogel RF (2002) Metabolism by bifidobacteria and lactic acid bacteria of polysaccharides from wheat and rye, and exopolysaccharides produced by Lactobacillus sanfranciscensis. J Appl Microbiol 92:958–965
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lewington J, Greenaway SD, Spillane BJ (1987) Rapid small scale preparations of bacterial genomic DNA, suitable for cloning and hybridization analysis. Lett Appl Microbiol 5:51–53
Miller JH (1972) A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria.
Morales-Arrieta S, Rodriguez ME, Segovia L, Lopez-Munguia A, Olvera-Carranza C (2006) Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B–512 F. Gene 376:59–67
Ozimek LK, Euverink GJ, van der Maarel MJ, Dijkhuizen L (2005) Mutational analysis of the role of calcium ions in the Lactobacillus reuteri strain 121 fructosyltransferase (levansucrase and inulosucrase) enzymes. FEBS Lett 579:1124–1128
Ozimek LK, Kralj S, Kaper T, van der Maarel MJ, Dijkhuizen L (2006a) Single amino acid residue changes in subsite-1 of inulosucrase from Lactobacillus reuteri 121 strongly influence the size of products synthesized. FEBS J 273(17):4104–4113
Ozimek LK, Kralj S, van der Maarel MJ, Dijkhuizen L (2006b) The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions. Microbiology 152:1187–1196
Ozimek LK, van Hijum SA, van Koningsveld GA, van der Maarel MJ, van Geel-Schutten GH, Dijkhuizen L (2004) Site-directed mutagenesis study of the three catalytic residues of the fructosyltransferases of Lactobacillus reuteri 121. FEBS Lett 560:131–133
Salminen S, Isolauri E, Salminen E (1996) Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges. Antonie Van Leeuwenhoek 70:347–358
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbour Laboratory Press, Woodburry
Stolz P, Böcker G, Hammes WP, Vogel RF (1995) Utilization of electron acceptors by lactobacilli isolated from sourdough. I. Lactobacillus sanfrancisco. Z Lebensm Unters Forsch 201:91–96
Tieking M, Ehrmann MA, Vogel RF, Gänzle MG (2005a) Molecular and functional characterization of a levansucrase from the sourdough isolate Lactobacillus sanfranciscensis TMW 1.392. Appl Microbiol Biotechnol 66:655–663
Tieking M, Kühnl W, Gänzle MG (2005b) Evidence for formation of heterooligosaccharides by Lactobacillus sanfranciscensis during growth in wheat sourdough. J Agric Food Chem 53:2456–2461
van Hijum SA, Bonting K, van der Maarel MJ, Dijkhuizen L (2001) Purification of a novel fructosyltransferase from Lactobacillus reuteri strain 121 and characterization of the levan produced. FEMS Microbiol Lett 205:323–328
van Hijum SA, Kralj S, Ozimek LK, Dijkhuizen L, van Geel-Schutten IG (2006) Structure-function relationships of glucansucrase and fructansucrase enzymes from lactic acid bacteria. Microbiol Mol Biol Rev 70:157–176
van Hijum SA, Szalowska E, van der Maarel MJ, Dijkhuizen L (2004) Biochemical and molecular characterization of a levansucrase from Lactobacillus reuteri. Microbiology 150:621–630
van Hijum SA, van Geel-Schutten GH, Rahaoui H, van der Maarel MJ, Dijkhuizen L (2002) Characterization of a novel fructosyltransferase from Lactobacillus reuteri that synthesizes high-molecular-weight inulin and inulin oligosaccharides. Appl Environ Microbiol 68:4390–4398
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Communicated by Erko Stackebrandt.
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Waldherr, F.W., Meissner, D. & Vogel, R.F. Genetic and functional characterization of Lactobacillus panis levansucrase. Arch Microbiol 190, 497–505 (2008). https://doi.org/10.1007/s00203-008-0404-4
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DOI: https://doi.org/10.1007/s00203-008-0404-4