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Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with ␤(2-6) and ␤(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, 13 C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by 13 C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55°C, with a drastic decrease at 60°C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.Levansucrase and inulosucrase enzymes, collectively called fructansucrases (FSs) or fructosyltransferases (FTFs), polymerize the fructose moiety of their substrate sucrose into fructans which possess either levan or inulin structures with ␤(2-6) and ␤(2-1) linkages, respectively. Possible applications of inulin and its oligosaccharides have been reviewed earlier (19). Inulin-type fructans of a higher degree of polymerization are of particular interest due to their demonstrated pronounced in vitro prebiotic effects (45). In the food industry, inulin is used as a fat substitute and to provide texture and stability in several products, such as desserts, baked goods, and fermented dairy products, as well as infant formula (36). Inulin polymers also have a potential application as surfactants. Carbamoylated inulin has the ability to reduce interfacial tension, thus providing a biodegradable surface-active agent (40).Inulosucrase enzymes (EC 2.4.1.9) are classified in glycoside hydrolase family GH68, along with other bacterial FS enzymes (http://www.cazy.org). Recently, a few mutagenesis studies focused on determining the structure-function relationship among FS enzymes have been published. For instance, modification of residues located at the Ϫ1 sugar-binding subsite of inulosucrase from Lactobacillus reuteri 121 strongly affected the size of the ...

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with b(2-6) and b(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS.

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