In-Depth Characterization of Prebiotic Galacto-oligosaccharides by a Combination of Analytical Techniques (original) (raw)

Recently, a novel glucansucrase-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121, and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized, and turned out to be inactive with sucrose, the natural glucansucrase substrate. Instead, GTFB acted on malto-oligosaccharides, thereby yielding elongated gluco-oligomers/polymers containing besides (14) also (16) glycosidic linkages, and it was classified as a 4,6--glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of maltooligosaccharides and their corresponding alditols [degree of polymerization, DP2(-ol)– DP7(-ol)] were carried out, and (purified) products were structurally analyzed with matrixassisted laser-desorption ionization time-of-flight mass spectrometry and onedimensional/ two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto/malto-oligomers, in case of DP7 up to DP >35.

Abstract Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1→ 4) also (α1→ 6) glycosidic linkages, and it was classified as a 4, 6-α-glucanotransferase.

The application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) as a tool for the characterization of complex carbohydrate structures was investigated for konjac glucomannan (KGM) oligosaccharide mixtures and the monitoring of their structural changes during 72 h of in vitro fermentation with human gut flora. Different types of KGM oligosaccharide mixtures were produced from a KGM polysaccharide using endo-beta-(1,4)-mannanase and endo-beta-(1,4)-glucanase. Distinction of structures emerging from different enzymatic KGM digests and detection of acetylated oligosaccharides were possible by both CE-LIF and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Using CE-LIF it could be shown that the endo-beta-(1,4)-glucanase digest exhibited a large degradability of the DP2, DP3, DP5, and DP6 components during in vitro fermentation, whereas the endo-beta-(1,4)-mannanase digest was digested only slightly, thereby highlighting the influence of structural characteristics on the fermentability by human gut flora.

The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 → 4, 1 → 6)-α-D-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D (1)H/(13)C NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (α1 → 4) and (α1 → 6) linkages, yielding linear gluco-oligosaccharides up to at least DP ~ 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP ~ 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 × 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (α1 → 4)/(α1 → 6) alternating structure.