Structural characterization of glucosylated GOS derivatives synthesized by the Lactobacillus reuteri GtfA and Gtf180 glucansucrase enzymes

β-Galacto-oligosaccharides (GOS) are used commercially in infant nutrition, aiming to functionally replace human milk oligosaccharides (hMOS). Glucansucrases Gtf180-ΔN and GtfA-ΔN of Lactobacillus reuteri strains convert sucrose into α-glucans with (α1→6)/(α1→3) and (α1→4)/(α1→6) glucosidic linkages, respectively. Previously we reported that both glucansucrases glucosylate lactose, producing a minimum of 5 compounds (degree of polymerization 3–4) (GL34 mixture) with (α1→2/3/4) linkages. This GL34 mixture exhibited growth stimulatory effects on various probiotic bacteria. Aiming to obtain additional compounds mimicking hMOS in structure and function, we here studied glucosylation of 3 commercially available galactosyl-lactose GOS compounds. Both Gtf180-ΔN and GtfA-ΔN were unable to use 3′-galactosyl-lactose (β3′-GL), but used sucrose to add a single glucose moiety to 4′-galactosyl-lactose (β4′-GL) and 6′-galactosyl-lactose (β6′-GL). β6′-GL was elongated at its reducing glucosyl unit with an (α1→2)-linked moiety and at its non-reducing end with an (α1→ 4) linked moiety; β4′-GL was only elongated at its reducing end with an (α1→2) linked moiety. Glucansucrases Gtf180-ΔN and GtfA-ΔN thus can be used to produce galactosyl-lactose-derived oligosaccharides containing (α1→2) and (α→4) glucosidic linkages, potentially with valuable bioactive (prebiotic) properties.


Trans-glucosylation of GOS
β3′-GL, β4′-GL and β6′-GL (0.02 M) were incubated with sucrose (0.05 M) plus the Gtf180-ΔN or GtfA-ΔN enzymes, at 37°C and pH 4.7. Blank reactions minus β-GL compounds used sucrose as both acceptor and donor substrate, resulting in α-glucan synthesis. Incubation mixtures were sampled after 0 h, 5 h and 24 h, and subjected to HPAEC-PAD analysis (Fig. 1). No trans-glucosylation products were observed with β3′-GL (data not shown). Gtf180-ΔN and GtfA-ΔN incubated with sucrose plus β6′-GL yielded similar HPAEC-PAD profiles of oligosaccharides synthesized ( Fig. 1a and c), with three major peaks at retention times between 22 and 29 min. In case of Gtf180-ΔN, there are several minor peaks eluting later in time, which most likely are higher DP oligosaccharides (DP5 -DP9). The areas of the Gtf180-ΔN peaks at 23.0 min and 25.5 min, called GL1 and GL2 respectively, are much more significant than those from GtfA-ΔN. Regarding the glucosylation of β4′-GL, one significant peak at retention time of 31.9 min, called GL3, was observed in HPAEC-PAD profiles of the reaction mixtures of both Gtf180-ΔN and GtfA-ΔN ( Fig. 1b and d). Especially with Gtf180-ΔN, there are several minor peaks that elute later than GL3. Similarly, the intensity of peak GL3 in the profile of Gtf180-ΔN is much more significant than that of GtfA-ΔN, especially after 5 h of incubation. The areas of the peaks GL1, GL2 and GL3, decreased upon prolonged incubation. This may be due to further glucansucrase catalyzed elongation reactions using these transglycosylation products as intermediate acceptor substrates. Further studies involving structural elucidation of the higher DP trans-glycosylation products are required to fully understand this. In case of Gtf180-ΔN, β6′-GL and β4′-GL were converted for 26 and 32% ( Fig. 1a and b; 24 h incubation time), estimated from their peak areas. Only limited amounts of β6′-GL and β4′-GL were available, and further optimizations of reaction conditions and product yields remain to be done.

Structural analysis of trans-glycosylation products
The three major glucosylation products corresponding to the peaks GL1, GL2 and GL3 of Gtf180-ΔN decorating β6′-GL and β4′-GL ( Fig. 1) were isolated from the incubation mixture for structural analysis by MALDI-TOF-MS and 1D/2D 1 H and 13 C NMR spectroscopy. The purity and retention time of each fraction was confirmed by reinjection on an analytical CarboPac PA-1 (4 × 250 mm) column. The fragment size distribution of each fraction was determined by MALDI-TOF MS. The data showed that all three major products corresponded to tetrasaccharides, as evidenced by a pseudo-molecular sodium adduct ion at 689 m/z.

Fraction GL1
Tetrasaccharide GL1 includes 4 hexose residues, namely A, B and C (one glucosyl and two galactosyl residues from the β6′-GL substrate), and D (transferred glucosyl residue from sucrose) (Scheme 1). The  (Table 1 and Fig. 2). Residue A showed the 1 H and 13 C pattern fitting with a reducing residue. Moreover, the set of chemical shifts of this residue matches very well with the corresponding residue from the structure β6′-GL [31]. These data show that there is no other substitution at residue A except for the 4-substitution with the galactosyl residue B. There are  Table 1.
in preparation]. However, the linear structure β4′-GL also was readily digested by commensal bacteria like Bacteroides thetaiotaomicron and other bacteria encoding endo-β-galactanase GH53 enzymes [37]. Elongation of β4′-GL with an (α1→2) linked glucose moiety may improve its resistance towards consumption by commensal bacteria, and may promote its non-digestible and prebiotic properties. The β6′-GL compound was not digested by commensal bacteria and predicted to stimulate growth of beneficial gut bacteria in a similar manner as hMOS [37]. Glucosylation of β6′-GL may even further enhance its selectivity and thus provides another hMOS mimicking compound. The potential stimulatory effects of these new GL1-GL3 DP4 compounds on growth of probiotic bacteria, and other functional properties, remain to be studied. Optimization of the reaction conditions, to enhance galactosyllactose conversion and product yields, are required to obtain sufficient amounts of the GL1-GL3 compounds for such functional studies. Transglucosylation of galactosyl-lactose compounds with glucansucrase enzymes is likely to further expand the already well-known prebiotic GOS status.

Trans-glucosylation reactions
The total activity of purified Gtf180-ΔN or GtfA-ΔN was measured as initial rates with sucrose by methods described previously by Van Geel-Schutten et al. [39]. The products of the trans-glucosylation reaction were prepared by incubating a mixture of 0.05 M sucrose (donor substrate) and 0.02 M GOS (acceptor substrate) with 3 U mL −1 glucansucrase at 37°C in 50 mM sodium acetate buffer with 0.1 mM CaCl 2 at pH 4.7. A volume of 10 μL of the reaction mixtures was taken at 0 h, 5 h and 24 h and then mixed with 190 μL DMSO. The diluted samples were analyzed by High-pH anion-exchange chromatography (HPAEC-PAD).

Isolation and purification of oligosaccharide products
The reactions were carried out in a volume of 20 mL under the conditions described in the previous section of Trans-glucosylation reactions. Afterwards the reaction mixtures were diluted with two volumes of cold ethanol 20% and stored at 4°C overnight to precipitate any polysaccharide material present. After centrifugation at 10,000 g for 10 min, the supernatants were applied to a rotatory vacuum evaporator to remove ethanol. The aqueous fractions were then absorbed onto a CarboGraph SPE column (GRACE, USA) using acetonitrile:water = 1:3 as eluent, followed by evaporation of acetonitrile under an N 2 stream before being freeze-dried. This was followed by fractionation HPAEC on a Dionex ICS-5000 workstation (Dionex, Amsterdam, the Netherlands), equipped with a CarboPac PA-1 column (250 × 9 mm; Dionex) and an ED40 pulsed amperometric detector (PAD). The collected fractions were neutralized by acetic acid 20% and then desalted using a CarboGraph SPE column as described earlier.

HPAEC-PAD analysis
The profiles of the oligosaccharides products were analyzed by HPAEC-PAD on a Dionex ICS-3000 work station (Dionex, Amsterdam, the Netherlands) equipped with an ICS-3000 pulse amperometric detection (PAD) system and a CarboPac PA-1 column (250 × 4 mm; Dionex). The analytical separation was performed at a flow rate of 1.0 mL min −1 using a complex gradient of effluents A (100 mM NaOH); B (600 mM NaOAc in 100 mM NaOH); C (Milli-Q water); and D (50 mM NaOAc). The gradient started with 10% A, 85% C, and 5% D in 25 min-40% A, 10% C, and 50% D, followed by a 35-min gradient to 75% A, 25% B, directly followed by 5 min washing with 100% B and reconditioning for 7 min with 10% A, 85% B, and 5% D.

MALDI-TOF mass spectrometry
Molecular masses of the compounds in the reaction mixtures were determined by MALDI-TOF mass spectrometry on an Axima™ Performance mass spectrometer (Shimadzu Kratos Inc., Manchester, UK), equipped with a nitrogen laser (337 nm, 3 ns pulse width). Iongate cut-off was set to m/z 200 and sampling resolution was softwareoptimized for m/z 1500. Samples were prepared by mixing 1 μL with 1 μL aqueous 10% 2,5-dihydroxybenzoic as matrix solution.

NMR spectroscopy
The structures of oligosaccharides of interest were elucidated by 1D and 2D 1 H NMR, and 2D 13 C NMR. A Varian Inova 500 Spectrometer and 600 Spectrometer (NMR center, University of Groningen) were used at probe temperatures of 25°C with acetone as internal standard (chemical shift of δ 2.225). The aliquot samples were exchanged twice with 600 μL of 99.9 %atom D 2 O (Cambridge Isotope Laboratories, Inc., Andover, MA) by freeze-drying, and then dissolved in 0.65 mL D 2 O, containing internal acetone. In the 1D 1 H NMR experiments, the data were recorded at 8 k complex data points, and the HOD signal was suppressed using a WET1D pulse. In the 2D 1 H-1 H NMR COSY experiments, data were recorded at 4000 Hz for both directions at 4 k complex data points in 256 increments. 2D 1 H-1 H NMR TOCSY data were recorded with 4000 Hz at 30, 60, 100 spinlock times in 200 increments. In the 2D 1 H-1 H NMR ROESY, spectra were recorded with 4800 Hz at a mixing time of 300 ms in 256 increments of 4000 complex data points. MestReNova 9.1.0 (Mestrelabs Research SL, Santiago de Compostela, Spain) was used to process NMR spectra, using Whittaker Smoother baseline correction.