The Probiotic Lactobacillus johnsonii NCC 533 Produces High-Molecular-Mass Inulin from Sucrose by Using an Inulosucrase Enzyme

Abstract

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 ...

Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products

“…Instead, a locus tag, LGAS_1266, annotated as a pseudogene, shows high similarity to the inulosucrase (inuJ, orf LJ0913; GenBank accession no. AE017198) of L. johnsonii NCC 533 (Anwar et al, 2008;Pridmore et al, 2004). The complement of this gene spans the region 1262127-1264493 bp of the L. gasseri DSM 20243 genome.…”

“…Michaelis' barbital sodium acetate buffer (Chipperfield & Taylor, 1970), pH 3.5-8.5 was used to study the effect of pH on the activity of recombinant FTF enzymes at 37 uC, as described previously (Anwar et al, 2008). The effect of temperature on the enzyme activity was studied in 25 mM sodium acetate buffer supplemented with 500 mM sucrose and 1 mM CaCl 2 .…”

“…The effect of temperature on the enzyme activity was studied in 25 mM sodium acetate buffer supplemented with 500 mM sucrose and 1 mM CaCl 2 . The effect on enzyme activity of scavenging calcium ions from the reaction buffer by EDTA (0-1000 mM) was studied as described previously (Anwar et al, 2008).…”

“…Culture supernatants were dialysed for 24 h against this sodium acetate buffer, which was replaced four times during dialysis. Total, hydrolysis and transglycosylation activities in the washed cells and supernatants were measured as described previously (Anwar et al, 2008) at 37 uC in 25 mM sodium acetate buffer, pH 5.5, containing 1 mM CaCl 2 , using 62 ml cell suspension or supernatant in the reaction mixture as the source of enzyme. Sucrose was used as substrate at 500 mM final concentration.…”

“…(Wada et al, 2003), L. reuteri TMW 1.106 (Schwab et al, 2007) and Lactobacillus johnsonii NCC 533 (Anwar et al, 2008), while numerous levansucrases are known (http://www.cazy.org/fam/GH68.html) from both Grampositive and Gram-negative bacteria. This limited knowledge of inulosucrase enzymes and lack of 3D structures hamper our efforts to understand the differences between inulo-and levansucrases.…”