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

Abstract

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.

Enzymatic Synthesis and Characterization of Fructooligosaccharides and Novel Maltosylfructosides by Inulosucrase from Lactobacillus gasseri DSM 20604

“…Briefly, a 1.7-kb inuGB DNA fragment was PCR amplified using HS Primer Start DNA polymerase (TaKaRa) and primers 806 (5=-GAACTCTAGAGGGTATTA ATAatggatgcggtaaaacaagatgaaaaag) and 779 (5=-GCTATTAATGATGAT GATGATGATGttgatgtggctttaagttatatcc) (the nucleotides pairing the inuGB gene sequence are written in lowercase letters; the start and stop codons are indicated in bold). The inuGB gene was amplified lacking the 137 N-terminal and the 59 C-terminal amino acid residues without affecting the catalytic domain as described previously (14). The 1.7-kb purified PCR product was inserted into pURI2-Cter vector using a restriction enzyme-free and ligation-free cloning strategy (23).…”

“…A fragment of the recombinant IS protein lacking the cell-anchoring motif from L. gasseri DSM 20604 (14) was overproduced in Escherichia coli and purified. Briefly, a 1.7-kb inuGB DNA fragment was PCR amplified using HS Primer Start DNA polymerase (TaKaRa) and primers 806 (5=-GAACTCTAGAGGGTATTA ATAatggatgcggtaaaacaagatgaaaaag) and 779 (5=-GCTATTAATGATGAT GATGATGATGttgatgtggctttaagttatatcc) (the nucleotides pairing the inuGB gene sequence are written in lowercase letters; the start and stop codons are indicated in bold).…”

“…It was previously shown that L. gasseri strain DSM 20604 synthesized both inulin poly-and oligosaccharides, products of its inuGB IS enzyme (14). A fragment of the recombinant IS protein lacking the cell-anchoring motif also converted sucrose or raffinose into a range of FOS (14).…”

“…A fragment of the recombinant IS protein lacking the cell-anchoring motif also converted sucrose or raffinose into a range of FOS (14). In the present investigation, the synthesis of FOS and novel oligosaccharides, termed maltosylfructosides (MFOS), by the L. gasseri inuGB fragment was studied in the presence of sucrose and maltose upon transfructosylation reaction by optimization of synthesis conditions such as IS concentration, temperature, pH, sucrose/maltose molar ratio, and reaction time.…”

“…While numerous levansucrases (EC 2.4.1.10) are known, only a few inulosucrase (IS) genes/enzymes (EC 2.4.1.9) with the ability to synthesize potential bioac-tive oligosaccharides have recently been characterized from lactic acid bacteria, including some strains of L. gasseri (14).…”