Cataloging natural sialic acids and other nonulosonic acids (NulOs), and their representation using the Symbol Nomenclature for Glycans.

Nonulosonic acids or non-2-ulosonic acids (NulOs) are an ancient family of 2-ketoaldonic acids (α-ketoaldonic acids) with a 9-carbon backbone. In nature, these monosaccharides occur either in a 3-deoxy form (referred to as "sialic acids") or in a 3,9-dideoxy "sialic-acid-like" form. The former sialic acids are most common in the deuterostome lineage, including vertebrates, and mimicked by some of their pathogens. The latter sialic-acid-like molecules are found in bacteria and archaea. NulOs are often prominently positioned at the outermost tips of cell surface glycans, and have many key roles in evolution, biology and disease. The diversity of stereochemistry and structural modifications among the NulOs contributes to more than 90 sialic acid forms and 50 sialic-acid-like variants described thus far in nature. This paper reports the curation of these diverse naturally occurring NulOs at the NCBI sialic acid page (https://www.ncbi.nlm.nih.gov/glycans/sialic.html) as part of the NCBI-Glycans initiative. This includes external links to relevant Carbohydrate Structure Databases. As the amino and hydroxyl groups of these monosaccharides are extensively derivatized by various substituents in nature, the Symbol Nomenclature For Glycans (SNFG) rules have been expanded to represent this natural diversity. These developments help illustrate the natural diversity of sialic acids and related NulOs, and enable their systematic representation in publications and online resources.

Enzymatic depolymerization of alginate by two novel thermostable alginate lyases from Rhodothermus marinus.

Alginate (alginic acid) is a linear polysaccharide, wherein (1→4)-linked ß-D-mannuronic acid and its C5 epimer, α-L-guluronic acid, are arranged in varying sequences. Alginate lyases catalyze the depolymerization of alginate, thereby cleaving the (1→4) glycosidic linkages between the monomers by a ß-elimination mechanism, to yield unsaturated 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid (Δ) at the non-reducing end of resulting oligosaccharides (α-L-erythro configuration) or, depending on the enzyme, the unsaturated monosaccharide itself. In solution, the released free unsaturated monomer product is further hydrated in a spontaneous (keto-enol tautomerization) process to form two cyclic stereoisomers. In this study, two alginate lyase genes, designated alyRm3 and alyRm4, from the marine thermophilic bacterium Rhodothermus marinus (strain MAT378), were cloned and expressed in Escherichia coli. The recombinant enzymes were characterized, and their substrate specificity and product structures determined. AlyRm3 (PL39) and AlyRm4 (PL17) are among the most thermophilic and thermostable alginate lyases described to date with temperature optimum of activity at ∼75 and 81°C, respectively. The pH optimum of activity of AlyRm3 is ∼5.5 and AlyRm4 at pH 6.5. Detailed NMR analysis of the incubation products demonstrated that AlyRm3 is an endolytic lyase, while AlyRm4 is an exolytic lyase, cleaving monomers from the non-reducing end of oligo/poly-alginates.

Trans-α-glucosylation of stevioside by the mutant glucansucrase enzyme Gtf180-ΔN-Q1140E improves its taste profile.

The adverse health effects of sucrose overconsumption, typical for diets in developed countries, necessitate use of low-calorie sweeteners. Following approval by the European Commission (2011), steviol glycosides are increasingly used as high-intensity sweeteners in food. Stevioside is the most prevalent steviol glycoside in Stevia rebaudiana plant leaves, but it has found limited applications in food products due to its lingering bitterness. Enzymatic glucosylation is a strategy to reduce stevioside bitterness, but reported glucosylation reactions suffer from low productivities. Here we present the optimized and efficient α-glucosylation of stevioside using the mutant glucansucrase Gtf180-ΔN-Q1140E and sucrose as donor substrate. Structures of novel products were elucidated by NMR spectroscopy, mass spectrometry and methylation analysis; stevioside was mainly glucosylated at the steviol C-19 glucosyl moiety. Sensory analysis of the α-glucosylated stevioside products by a trained panel revealed a significant reduction in bitterness compared to stevioside, resulting in significant improvement of edulcorant/organoleptic properties.

Exploration of the Sialic Acid World.

Sialic acids are cytoprotectors, mainly localized on the surface of cell membranes with multiple and outstanding cell biological functions. The history of their structural analysis, occurrence, and functions is fascinating and described in this review. Reports from different researchers on apparently similar substances from a variety of biological materials led to the identification of a 9-carbon monosaccharide, which in 1957 was designated "sialic acid." The most frequently occurring member of the sialic acid family is N-acetylneuraminic acid, followed by N-glycolylneuraminic acid and O-acetylated derivatives, and up to now over about 80 neuraminic acid derivatives have been described. They appeared first in the animal kingdom, ranging from echinoderms up to higher animals, in many microorganisms, and are also expressed in insects, but are absent in higher plants. Sialic acids are masks and ligands and play as such dual roles in biology. Their involvement in immunology and tumor biology, as well as in hereditary diseases, cannot be underestimated. N-Glycolylneuraminic acid is very special, as this sugar cannot be expressed by humans, but is a xenoantigen with pathogenetic potential. Sialidases (neuraminidases), which liberate sialic acids from cellular compounds, had been known from very early on from studies with influenza viruses. Sialyltransferases, which are responsible for the sialylation of glycans and elongation of polysialic acids, are studied because of their significance in development and, for instance, in cancer. As more information about the functions in health and disease is acquired, the use of sialic acids in the treatment of diseases is also envisaged.

Glucansucrase (mutant) enzymes from Lactobacillus reuteri 180 efficiently transglucosylate Stevia component rebaudioside A, resulting in a superior taste.

Steviol glycosides from the leaves of the plant Stevia rebaudiana are high-potency natural sweeteners but suffer from a lingering bitterness. The Lactobacillus reuteri 180 wild-type glucansucrase Gtf180-ΔN, and in particular its Q1140E-mutant, efficiently α-glucosylated rebaudioside A (RebA), using sucrose as donor substrate. Structural analysis of the products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that both enzymes exclusively glucosylate the Glc(ß1→C-19 residue of RebA, with the initial formation of an (α1→6) linkage. Docking of RebA in the active site of the enzyme revealed that only the steviol C-19 ß-D-glucosyl moiety is available for glucosylation. Response surface methodology was applied to optimize the Gtf180-ΔN-Q1140E-catalyzed α-glucosylation of RebA, resulting in a highly productive process with a RebA conversion of 95% and a production of 115 g/L α-glucosylated products within 3 h. Development of a fed-batch reaction allowed further suppression of α-glucan synthesis which improved the product yield to 270 g/L. Sensory analysis by a trained panel revealed that glucosylated RebA products show a significant reduction in bitterness, resulting in a superior taste profile compared to RebA. The Gtf180-ΔN-Q1140E glucansucrase mutant enzyme thus is an efficient biocatalyst for generating α-glucosylated RebA variants with improved edulcorant/organoleptic properties.

Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation.

The wild-type Gtf180-ΔN glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the α-glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a transglucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is specifically α-d-glucosylated at the steviol C-19 ß-d-glucosyl moiety (55% conversion). The main product is a mono-(α1 â†’ 6)-glucosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating (α1 â†’ 3)- and (α1 â†’ 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modification of their naturally occurring carbohydrate moieties.

Stevia Glycosides: Chemical and Enzymatic Modifications of Their Carbohydrate Moieties to Improve the Sweet-Tasting Quality.

Stevia glycosides, extracted from the leaves of the plant Stevia rebaudiana Bertoni, display an amazing high degree of sweetness. As processed plant products, they are considered as excellent bio-alternatives for sucrose and artificial sweeteners. Being noncaloric and having beneficial properties for human health, they are the subject of an increasing number of studies for applications in food and pharmacy. However, one of the main obstacles for the successful commercialization of Stevia sweeteners, especially in food, is their slight bitter aftertaste and astringency. These undesirable properties may be reduced or eliminated by modifying the carbohydrate moieties of the steviol glycosides. A promising procedure is to subject steviol glycosides to enzymatic glycosylation, thereby introducing additional monosaccharide residues into the molecules. Depending on the number and positions of the monosaccharide units, the taste quality and sweetness potency of the compounds will vary. Many studies have been performed already, and this review summarizes the structures of native steviol glycosides and the recent data of modifications of the carbohydrate moieties that have been published to provide an overview of the current progress.

Modification of linear (ß1→3)-linked gluco-oligosaccharides with a novel recombinant ß-glucosyltransferase (trans-ß-glucosidase) enzyme from Bradyrhizobium diazoefficiens.

Recently, we have shown that glycoside hydrolases enzymes of family GH17 from proteobacteria (genera Pseudomonas, Azotobacter) catalyze elongation transfer reactions with laminari-oligosaccharides generating (ß1→3) linkages preferably and to a lesser extent (ß1→6) or (ß1→4) linkages. In the present study, the cloning and characterization of the gene encoding the structurally very similar GH17 domain of the NdvB enzyme from Bradyrhizobium diazoefficiens, designated Glt20, as well as its catalytic properties are described. The Glt20 enzyme was strikingly different from the previously investigated bacterial GH17 enzymes, both regarding substrate specificity and product formation. The Azotobacter and Pseudomonas enzymes cleaved the donor laminari-oligosaccharide substrates three or four moieties from the non-reducing end, generating linear oligosaccharides. In contrast, the Glt20 enzyme cleaved donor laminari-oligosaccharide substrates two glucose moieties from the reducing end, releasing laminaribiose and transferring the remainder to laminari-oligosaccharide acceptor substrates creating only (ß1→3)(ß1→6) branching points. This enables Glt20 to transfer larger oligosaccharide chains than the other type of bacterial enzymes previously described, and helps explain the biologically significant formation of cyclic ß-glucans in B. diazoefficiens.

Sialylated galacto-oligosaccharides and 2'-fucosyllactose reduce necrotising enterocolitis in neonatal rats.

Necrotising enterocolitis (NEC) is one of the most frequent and fatal intestinal disorders in preterm infants and has very limited treatment options. Breast-fed infants are at a 6-10-fold lower NEC risk than formula-fed infants, and we have previously shown that human milk oligosaccharides (HMO) improved survival and reduced pathology in a rat NEC model. The HMO disialyllacto-N-tetraose (DSLNT) was most effective, and sialylation was shown to be essential for its protective effect. Galacto-oligosaccharides (GOS), currently added to some infant formula, but not containing sialic acid, had no effect. In addition to DSLNT, our previous work also showed that the neutral HMO fraction, which contains high concentrations of 2'-fucosyllactose (2'FL), slightly improved pathology scores. Here, we assessed the in vivo efficacy of 2'FL, as well as of GOS that we enzymatically sialylated (Sia-GOS). Neonatal rats were randomised into the following study groups - dam-fed (DF), formula-fed (FF), FF containing pooled HMO (10 mg/ml), GOS (8 mg/ml), Sia-GOS (500 µm) or 2'FL (2 mg/ml) - and subjected to the established NEC protocol. The DF and HMO groups had the lowest pathology scores with mean values of 0·67 (sd 0·34) and 0·90 (sd 0·47), respectively. The FF group had significantly elevated pathology scores of 2·02 (sd 0·63). Although the addition of GOS to the formula had no protective effect and generated scores of 2·00 (sd 0·63), the addition of Sia-GOS or 2'FL significantly lowered pathology scores to 1·32 (sd 0·56) (P<0·0034) and 1·43 (sd 0·51) (P<0·0040), respectively. The results warrant further studies to investigate the underlying mechanisms and to assess safety and efficacy in human neonates.

Comparative structural characterization of 7 commercial galacto-oligosaccharide (GOS) products.

Many ß-galactosidase enzymes convert lactose into a mixture of galacto-oligosaccharides (GOS) when incubated under the right conditions. Recently, the composition of commercial Vivinal GOS produced by Bacillus circulans ß-galactosidase was studied in much detail in another study by van Leeuwen et al. As a spin-off of this study, we used the developed analytical strategy for the evaluation of 6 anonymous commercial GOS products, in comparison with Vivinal GOS. These GOS products were first subjected to HPLC-SEC, calibrated HPAEC-PAD profiling (glucose units in relation to a malto-oligosaccharide ladder), and 1D (1)H NMR spectroscopy. For a more detailed analysis and support of the conclusions based on the initial analysis, the GOS products were separated into DP-pure subpools on Bio-Gel P-2 (MALDI-TOF-MS analysis), which were subjected to calibrated HPAEC-PAD profiling and (1)H NMR analysis. Unidentified peaks from different GOS products, not present in Vivinal GOS, were isolated for detailed structural characterization. In this way, the differences between the various GOS products in terms of DP distribution and type of glycosidic linkages were established. A total of 13 new GOS structures were characterized, adding structural-reporter-group signals and HPAEC-PAD based glucose unit G.U. values to the analytical toolbox. The newly characterized products enhance the quality of the database with GOS structures up to DP4. The combined data provide a firm basis for the rapid profiling of the GOS products of microbial ß-galactosidase enzymes.

Synthesis of oligo- and polysaccharides by Lactobacillus reuteri 121 reuteransucrase at high concentrations of sucrose.

GTFA, a glucansucrase enzyme of the probiotic bacterium Lactobacillus reuteri 121, is capable of synthesizing an α-glucan polysaccharide with (1 → 4) and (1 → 6) linkages from sucrose. With respect to its biosynthesis, the present study has shown that the ratio of oligosaccharide versus polysaccharide synthesized was directly proportional to the concentration of sucrose. It appears that the size distribution of products is kinetically controlled, but the linkage distribution in the polysaccharide material is not changed. At high sucrose concentrations the sucrose isomers leucrose and trehalulose were synthesized, using the accumulated fructose as acceptor, together with 4'- and 6'-α-D-glucosyl-leucrose and 6'-α-D-glucosyl-trehalulose. The finding of an additional branched hexasaccharide demonstrates that the enzyme is able to introduce branch-points already in relatively short oligosaccharides.

Complexity and Diversity of the Mammalian Sialome Revealed by Nidovirus Virolectins.

Sialic acids (Sias), 9-carbon-backbone sugars, are among the most complex and versatile molecules of life. As terminal residues of glycans on proteins and lipids, Sias are key elements of glycotopes of both cellular and microbial lectins and thus act as important molecular tags in cell recognition and signaling events. Their functions in such interactions can be regulated by post-synthetic modifications, the most common of which is differential Sia-O-acetylation (O-Ac-Sias). The biology of O-Ac-Sias remains mostly unexplored, largely because of limitations associated with their specific in situ detection. Here, we show that dual-function hemagglutinin-esterase envelope proteins of nidoviruses distinguish between a variety of closely related O-Ac-Sias. By using soluble forms of hemagglutinin-esterases as lectins and sialate-O-acetylesterases, we demonstrate differential expression of distinct O-Ac-sialoglycan populations in an organ-, tissue- and cell-specific fashion. Our findings indicate that programmed Sia-O-acetylation/de-O-acetylation may be critical to key aspects of cell development, homeostasis, and/or function.

Enzymatic Decoration of Prebiotic Galacto-oligosaccharides (Vivinal GOS) with Sialic Acid Using Trypanosoma cruzi trans-Sialidase and Two Bovine Sialoglycoconjugates as Donor Substrates.

Decoration of prebiotic galacto-oligosaccharides (GOS) with sialic acid yields mixtures of GOS and sialylated GOS (Sia-GOS), novel products that are expected to have both prebiotic and antiadhesive functionalities. The recombinantly produced trans-sialidase enzyme from Trypanosoma cruzi (TcTS), an enzyme with the ability to transfer (α2-3)-linked sialic acid from sialogalactoglycans to asialogalactoglycans, was employed to catalyze this sialylation. As sialic acid acceptor substrates, Vivinal GOS and derived fractions of specific degree of polymerization were taken. As sialic acid donor substrates, bovine κ-casein-derived glycomacropeptide [>99% N-acetylneuraminic acid (Neu5Ac); <1% N-glycolylneuraminic acid (Neu5Gc)] and bovine blood plasma glycoprotein mixture (45% Neu5Ac; 55% Neu5Gc) were selected, yielding potential food and feed products, respectively. High-pH anion-exchange chromatography, matrix-assisted laser-desorption ionization time-of-flight mass spectrometry, and nuclear magnetic resonance spectroscopy were used for product analysis.

Structural analysis of novel trehalose-based oligosaccharides from extremely stress-tolerant ascospores of Neosartorya fischeri (Aspergillus fischeri).

Different fungi, including the genera Neosartorya, Byssochlamys and Talaromyces, produce (asco)spores that survive pasteurization treatments and are regarded as the most stress-resistant eukaryotic cells. Here, the NMR analysis of a series of trehalose-based oligosaccharides, being compatible solutes that are accumulated to high levels in ascospores of the fungus Neosartorya fischeri, is presented. These oligosaccharides consist of an α,α-trehalose backbone, extended with one [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp; isobemisiose], two [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp] or three [α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 → 6)-α-D-Glcp-(1 ↔ 1)-α-D-Glcp] glucose units. The tetra- and pentasaccharide, dubbed neosartose and fischerose, respectively, have not been reported before to occur in nature.

Truncation of domain V of the multidomain glucansucrase GTF180 of Lactobacillus reuteri 180 heavily impairs its polysaccharide-synthesizing ability.

Glucansucrases are exclusively found in lactic acid bacteria and synthesize a variety of α-glucans from sucrose. They are large multidomain enzymes belonging to the CAZy family 70 of glycoside hydrolase enzymes (GH70). The crystal structure of the N-terminal truncated GTF180 of Lactobacillus reuteri 180 (GTF180-ΔN) revealed that the polypeptide chain follows a U shape course to form five domains, including domains A, B, and C, which resemble those of family GH13 enzymes, and two extra and novel domains (domains IV and V), which are attached to the catalytic core. To elucidate the functional roles of domain V, we have deleted the domain V fragments from both the N- and C-terminal ends (GTF180-ΔNΔV). Truncation of domain V of GTF180-ΔN yielded a catalytically fully active enzyme but with heavily impaired polysaccharide synthesis ability. Instead, GTF180-ΔNΔV produced a large amount of oligosaccharides. Domain V is not involved in determining the linkage specificity, and the size of polysaccharide produced as the polysaccharide produced by GTF180-ΔNΔV was identical in size and structure with that of GTF180-ΔN. The data indicates that GTF180-ΔNΔV acts nonprocessively, frequently initiating synthesis of a new oligosaccharide from sucrose, instead of continuing the synthesis of a full size polysaccharide. Mutations L940E and L940F in GTF180-ΔNΔV, which are involved in the acceptor substrate binding, restored polysaccharide synthesis almost to the level of GTF180-ΔN. These results demonstrated that interactions of growing glucan chains with both domain V and acceptor substrate binding sites are important for polysaccharide synthesis.

(1)H NMR analysis of the lactose/ß-galactosidase-derived galacto-oligosaccharide components of Vivinal® GOS up to DP5.

Vivinal® GOS is a galacto-oligosaccharide (GOS) product, prepared from lactose by incubation with Bacillus circulans ß-galactosidase (EC 3.2.1.23). This complex mixture of saccharides with degree of polymerization (DP) between 1 and 8 is generally applied in infant nutrition. Here, a detailed structural description of the commercial product up to the DP5 level is given. First, Vivinal® GOS was subjected to DP analysis using HPLC-SEC (Rezex RSO-01 oligosaccharide Ag(+) column) and (1)H NMR analysis. Then, the product was fractionated on Bio-Gel P-2, and the obtained fractions were pooled according to DP, as indicated by MALDI-TOF-MS analysis. Finally, fractions of single DP, as well as their subfractions obtained by HPAEC-PAD on CarboPac PA-1, were analyzed by 1D/2D (1)H/(13)C NMR spectroscopy and linkage analysis. In total, over 40 structures, providing a structural coverage of over 99% of the product, have been characterized. Detailed (1)H and (13)C NMR data, as well as G.U. values (glucose units; malto-oligosaccharide ladder) on CarboPac PA-1 of all oligosaccharides are included.

Isomalto/malto-polysaccharide, a novel soluble dietary fiber made via enzymatic conversion of starch.

Dietary fibers are at the forefront of nutritional research because they positively contribute to human health. Much of our processed foods contain, however, only small quantities of dietary fiber, because their addition often negatively affects the taste, texture, and mouth feel. There is thus an urge for novel types of dietary fibers that do not cause unwanted sensory effects when applied as ingredient, while still positively contributing to the health of consumers. Here, we report the generation and characterization of a novel type of soluble dietary fiber with prebiotic properties, derived from starch via enzymatic modification, yielding isomalto/malto-polysaccharides (IMMPs), which consist of linear (α1 → 6)-glucan chains attached to the nonreducing ends of starch fragments. The applied Lactobacillus reuteri 121 GTFB 4,6-α-glucanotransferase enzyme synthesizes these molecules by transferring the nonreducing glucose moiety of an (α1 → 4)-glucan chain to the nonreducing end of another (α1 → 4)-α-glucan chain, forming an (α1 → 6)-glycosidic linkage. Once elongated in this way, the molecule becomes a better acceptor substrate and is then further elongated with (α1 → 6)-linked glucose residues in a linear way. Comparison of 30 starches, maltodextrins, and α-glucans of various botanical sources, demonstrated that substrates with long and linear (α1 → 4)-glucan chains deliver products with the highest percentage of (α1 → 6) linkages, up to 92%. In vitro experiments, serving as model of the digestive power of the gastrointestinal tract, revealed that the IMMPs, or more precisely the IMMP fraction rich in (α1 → 6) linkages, will largely pass the small intestine undigested and therefore end up in the large intestine. IMMPs are a novel type of dietary fiber that may have health promoting activity.

Residue Leu940 has a crucial role in the linkage and reaction specificity of the glucansucrase GTF180 of the probiotic bacterium Lactobacillus reuteri 180.

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu(940) in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu(940) of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67-100% of (α1→6) linkages) were produced by GTF180 and its Leu(940) mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu(940) in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.

Development of a (1)H NMR structural-reporter-group concept for the analysis of prebiotic galacto-oligosaccharides of the [ß-d-Galp-(1→x)]n-d-Glcp type.

Some ß-galactosidases (EC 3.2.1.23) are capable of producing mixtures of linear and branched galacto-oligosaccharides (GOS) with various types of glycosidic linkages [degree of polymerization (DP) 2-8; mainly GalnGlc] when incubated under specific conditions with lactose. These products are generally applied in infant formula. However, for most galacto-oligosaccharide products only major components (low DP) or linkage patterns have been described. To build up a library of (1)H and (13)C NMR data, a detailed NMR study on commercially available GOS di- and trisaccharides, and some larger GOS oligosaccharides was carried out. Based on the fully assigned (1)H and (13)C chemical shifts of these model compounds, a (1)H NMR structural-reporter-group concept was formulated to function as a tool in the structural analysis of single GOS components and GOS mixtures.

Galactosyl-lactose sialylation using Trypanosoma cruzi trans-sialidase as the biocatalyst and bovine κ-casein-derived glycomacropeptide as the donor substrate.

trans-Sialidase (TS) enzymes catalyze the transfer of sialyl (Sia) residues from Sia(α2-3)Gal(ß1-x)-glycans (sialo-glycans) to Gal(ß1-x)-glycans (asialo-glycans). Aiming to apply this concept for the sialylation of linear and branched (Gal)nGlc oligosaccharide mixtures (GOS) using bovine κ-casein-derived glycomacropeptide (GMP) as the sialic acid donor, a kinetic study has been carried out with three components of GOS, i.e., 3'-galactosyl-lactose (ß3'-GL), 4'-galactosyl-lactose (ß4'-GL), and 6'-galactosyl-lactose (ß6'-GL). This prebiotic GOS is prepared from lactose by incubation with suitable ß-galactosidases, whereas GMP is a side-stream product of the dairy industry. The trans-sialidase from Trypanosoma cruzi (TcTS) was expressed in Escherichia coli and purified. Its temperature and pH optima were determined to be 25°C and pH 5.0, respectively. GMP [sialic acid content, 3.6% (wt/wt); N-acetylneuraminic acid (Neu5Ac), >99%; (α2-3)-linked Neu5Ac, 59%] was found to be an efficient sialyl donor, and up to 95% of the (α2-3)-linked Neu5Ac could be transferred to lactose when a 10-fold excess of this acceptor substrate was used. The products of the TcTS-catalyzed sialylation of ß3'-GL, ß4'-GL, and ß6'-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated ß3'-GL and ß4'-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of ß6'-GL, TcTS was shown to sialylate the 3 position of both the internal and terminal Gal moieties, yielding two different monosialylated products and a disialylated structure. Kinetic analyses showed that TcTS had higher affinity for the GL substrates than lactose, while the Vmax and kcat values were higher in the case of lactose.