Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity

Casein glycomacropeptide (cGMP) and lactose, which are purified (or semi-purified) components obtained from side streams from dairy industry operations, were used as substrates for enzyme catalyzed production of 3′-sialyllactose, a model case compound for human milk oligosaccharides (HMOs). The enzyme employed was a mutated sialidase, Tr6, derived from Trypanosoma rangeli, and expressed in Pichia pastoris after codon-optimization. The Tr6 contained 6 point mutations and exhibited trans-sialidase activity. The Tr6 trans-sialidase reaction conditions were tuned for maximizing Tr6 catalyzed 3′-sialyllactose production by optimizing pH, temperature, acceptor, and donor concentrations using response surface designs. At the optimum reaction conditions, the Tr6 catalyzed the transfer of sialic acid from cGMP to lactose at high efficiency without substantial hydrolysis of the 3′-sialyllactose product. The robustness of the Tr6 catalyzed reaction was verified at 5 L-scale providing a yield of 3.6 g 3′-sialyllactose at an estimated molar trans-sialylation yield of 50% on the 3′-sialyl in cGMP. Lacto-N-tetraose and lacto-N-fucopentaoses also functioned as acceptor molecules demonstrating the versatility of the Tr6 trans-sialidase for catalyzing sialyl-transfer for generating different HMOs. The data signify the applicability of enzymatic trans-sialylation on dairy side-stream components for production of human milk oligosaccharides.     

Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans

This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been used to investigate the structural requirements for trans-sialidase activity. We observed that the T. cruzi trans-sialidase has a seven-amino-acid motif (197–203) at the border of the substrate binding cleft. The motif differs substantially in chemical properties and substitution probability from the homologous sialidase, and we hypothesised that this motif is important for trans-sialidase activity. The 197–203 motif is strongly positively charged with a marked change in hydrogen bond donor capacity as compared to the sialidase. To investigate the role of this motif, we expressed and characterised a T. rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13''s acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose. Sialic acid is important in association with human milk oligosaccharides, and Tr13 was shown to sialylate a number of established and potential prebiotics. Initial evaluation of prebiotic potential using pure cultures demonstrated, albeit not selectively, growth of Bifidobacteria. Since the 197–203 motif stands out in the native trans-sialidase, is markedly different from the wild-type sialidase compared to previous mutants, and is shown here to confer efficient and broad trans-sialidase activity, we suggest that this motif can serve as a framework for future optimization of trans-sialylation towards prebiotic production.     

唾液酸转移酶对肿瘤中唾液酸化结构的影响

随着侵袭能力的增强,肿瘤细胞表面经常会出现唾液酸化糖链结构的增加。现在已经证实许多特殊的唾液酸化结构,如TF类抗原、sLew类抗原、α2-6唾液酸化的乳糖胺结构、PSA结构、神经节苷脂结构都参与细胞间的相互作用。本文综述了与肿瘤相关的特殊的唾液酸化结构的改变与相应的唾液酸转移酶在其中发挥的作用。     

NKG2D and CD94 bind to multimeric α2,3-linked N-acetylneuraminic acid

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to α2,3-sialylated human α₁-acid glycoprotein (AGP) but not to α2,6-sialylated AGP. Mutagenesis revealed that ¹⁵²Y of NKG2D and ¹⁴⁴F and ¹⁶⁰N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to α2,3-sialylated but not to α2,6-sialylated multi-antennary N-glycans.     

Polyoxometalates as effective inhibitors for sialyl- and sulfotransferases

Sialylated and/or sulfated carbohydrate chains in glycoproteins and glycolipids play important roles in infection by microorganisms and diseases including cancer. Inhibitors of sialyl/sulfotransferases, responsible for the biosynthesis of these carbohydrate chains, could be medical agents against such infections and diseases. Polyoxometalates (PMs) are inorganic polyanionic molecules that have been shown to exhibit activity against tumors and infectious microorganisms; however, the effects of PMs on carbohydrate biosynthesis have never been investigated. Here, we found that some types of PMs can inhibit the enzymatic activities of specific sialyl/sulfotransferases. Several tungstate-type PMs inhibited Gal: α2,3-sialyltransferase-I (ST3Gal-I) activity at sub-nanomolar levels. The half-inhibitory concentration of the best inhibitors was 0.2 nM and the inhibition was non-competitive for both donor and acceptor substrates (Ki values ∼0.5 nM). By certain vanadate-type PMs, ST3Gal-I and Gal 3-O-sulfotransferase-2 (Gal3ST-2) were specifically inhibited at nanomolar levels. The inhibitory effect of a tungstate-type PM on ST3Gal-I was reversible and electrostatic. A ST3Gal-I mutant protein which was converted ³³⁵Arg residue in the C-terminal region to Glu, was rather insensitive to the PM, suggesting that specific C-terminal basic amino acid of ST3Gal-I is involved in the binding to PMs. Collectively, PMs are novel inhibitors of specific sialyl/sulfotransferases.     

Immobilization of Bacillus circulans β-galactosidase and its application in the synthesis of galacto-oligosaccharides under repeated-batch operation

A highly active and stable derivate of immobilized Bacillus circulans β-galactosidase was prepared for the synthesis of galacto-oligosaccharides (GOS) under repeated-batch operation. B. circulans β-galactosidase was immobilized on monofunctional glyoxyl agarose and three heterofunctional supports: amino-, carboxy-, and chelate-glyoxyl agarose. Glyoxyl agarose was the support with highest immobilization yield and stability being selected for the optimization of immobilization conditions and application in GOS synthesis. A central composite rotatable design was conducted to optimize contacted protein and immobilization time, using maximum catalytic potential as the objective function. Optimal conditions of immobilization were 28.9 mg/g and 36.4 h of contact, resulting in a biocatalyst with 595 IU/g and a half-life 89-fold higher than soluble enzyme. Immobilization process did not alter the synthetic capacity of β-galactosidase, obtaining the same GOS yield and product profile than the free enzyme. GOS yield and productivity remained unchanged along 10 repeated batches, with values of 39% (w/w) and 5.7 g GOS/g of biocatalyst·batch. Total product obtained after 10 batches of reaction was 56.5 g GOS/g of biocatalyst (1956 g GOS/g protein). Cumulative productivity in terms of mass of contacted protein was higher for the immobilized enzyme than for its soluble counterpart from the second batch of synthesis onwards.     

Characterization of cancer associated mucin type O-glycans using the exchange sialylation properties of mammalian sialyltransferase ST3Gal-II

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galβ1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.     

Glucooligosaccharide production by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> fermentation with efficient pH control,using a calcium hydroxide-sucrose solution

95.3% of the sucrose in a feed batch fermentation (300 g/L) was hydrolyzed by Leuconostoc mesenteroides subp. mesenteroides NRRL B-23188 glucansucrase. Further, the glucose of sucrose formed glucooligosaccharides (GOS) of degree of polymerization (DP) over 2, together with 91.6% of the maltose (200 g/L). Lime saccharate (lime sucrate) was used to control the pH during fermentation. The GOS products had DP between 2 and 7. When Streptococcus mutans mutansucrase (0.1 U/mL) reacted with 0.1% sucrose, addition of 0.1 ~ 10% GOS to the mutansucrase reaction digest resulted in a 56 ~ 90% reduction of mutan formation. GOS also reduced E. coli (72.2%) and Salmonella sp. (over 40.0%) growth, when 2.5% GOS was used as a single carbon source, compared to growth using glucose. The calculated glycemic index and glycemic load of GOS was 8 and 1, respectively, based on a 10 g carbohydrate serving. GOS was calculated to have 2.43 kcal/g. After a glucose tolerance test was performed using C57BL/6 mice, we found that mice treated with GOS showed a 59.4% lower increase in plasma glucose than those treated with maltose.     

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.     

Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.     

Production of glucooligosaccharides by an acceptor reaction using two types of glucansucrase from Streptococcus sobrinus

Glucooligosaccharides (GOS) were produced by using an acceptor reaction with two types of glucansucrase (GTF-S and GTF-I) from Streptococcus sobrinus. Acceptor reactions of GTF-S with maltose acceptor, gave a great number of GOS ranging from DP(degree of polymerization) 2 to DP15. At the both acceptor reactions with GTF-S or GTF-I, as the sucrose/maltose ratio was decreased, the amount of dextran and DP of oligosaccharides was decreased. A maximum GOS yield of 69% was achieved at the acceptor reaction with GTF-I and when the molar ratio of sucrose/maltose is 2:1, in which GOS of DP6~DP9 were major oligosaccharides and 17% of dextran. The polymeric size of GOS could be controlled by varying the ratio of sucrose to the acceptor (maltose in this work).     

Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis

Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His²⁸⁴ in the active site replaced by Asn, Asp or Tyr showed up to 10⁴-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His²⁸⁴ in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.     

An exo-alpha-sialidase from bifidobacteria involved in the degradation of sialyloligosaccharides in human milk and intestinal glycoconjugates

Bifidobacteria are health-promoting enteric commensals that are assumed to proliferate predominantly in the intestines of breast-fed infants by assimilating human milk oligosaccharides (HMOs) that are frequently fucosylated and/or sialylated. We previously identified two different α-l-fucosidases in Bifidobacterium bifidum and showed that the strain furnishes an extracellular degradation pathway for fucosylated HMOs. However, the catabolism of sialylated HMOs by bifidobacteria has remained unresolved. Here we describe the identification and characterization of an exo-α-sialidase in bifidobacteria. By expression cloning, we isolated a novel exo-α-sialidase gene (siabb2) from B. bifidum JCM1254, which encodes a protein (SiaBb2) consisting of 835-amino-acid residues with a predicted molecular mass of 87 kDa. SiaBb2 possesses an N-terminal signal sequence, a sialidase catalytic domain classified into the glycoside hydrolase family 33 (GH33) and a C-terminal transmembrane region, indicating that the mature SiaBb2 is an extracellular membrane-anchored enzyme. The recombinant enzyme expressed in Escherichia coli showed the highest activity in an acidic pH range from 4.0 to 5.0, and at 50 °C. Notably, 80% activity remained after 30 min incubation at 80 °C, indicating that the enzyme is highly thermostable. SiaBb2 liberated sialic acids from sialyloligosaccharides, gangliosides, glycoproteins and colominic acid; however, the linkage preference of the enzyme was remarkably biased toward the α2,3-linkage rather than α2,6- and α2,8-linkages. Expression of siabb2 in B. longum 105-A, which has no endogenous exo-α-sialidase, enabled this strain to degrade sialyloligosaccharides present in human milk. Our results suggest that SiaBb2 plays a crucial role in bifidobacterial catabolism of sialylated HMOs.     

Biosynthesis of L-selectin ligands: sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases

The leukocyte adhesion molecule L-selectin mediates lymphocyte homing to secondary lymphoid organs and to certain sites of inflammation. The cognate ligands for L-selectin possess the unusual sulfated tetrasaccharide epitope 6-sulfo sialyl Lewis x (Siaalpha2-->3Galbeta1-->4[Fucalpha1-->3][SO(3)-->6]GlcNAc). Sulfation of GlcNAc within sialyl Lewis x is a crucial modification for L-selectin binding, and thus, the underlying sulfotransferase may be a key modulator of lymphocyte trafficking. Four recently discovered GlcNAc-6-sulfotransferases are the first candidate contributors to the biosynthesis of 6-sulfo sLex in the context of L-selectin ligands. Here we report the in vitro activity of the four GlcNAc-6-sulfotransferases on a panel of synthetic oligosaccharide substrates that comprise structural motifs derived from sialyl Lewis x. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). Surprisingly, for three of the enzymes, significant activity was observed with sialylated LacNAc, and two of the enzymes were capable of detectable sulfation of GlcNAc in the context of sialyl Lewis x. On the basis of these results, we propose possible pathways for 6-sulfo sialyl Lewis x biosynthesis and suggest that sulfation may be an early committed step.     

Characteristics of Korean-Saanen goat milk caseins and somatic cell counts in comparison with Holstein cow milk counterparts

Characteristics of α- and β-casein fractions in the milk of Korean-Saanen goats were compared with those of Holstein cow milk using capillary electrophoresis (CE) analysis. The αs₁-CN content of the Saanen goat milk samples varied from 2.4% to 9.3% of total proteins. Total αs-CN content of the goat milk varied from 10.1% to 17.0%. Total β-CN content containing β₁-CN and the β₂-CN varied from 49.6% to 61.0% of total proteins. Average αs-CN to β-CN ratio of the Saanen goat milk from different farms was 0.24 ± 0.04, ranging from 0.17 to 0.33. The αs-CN (αs₁-CN + αs₀-CN) to β-CN (β_A1-CN + β_A2-CN) ratio of Holstein cow milk was 0.81, which was much higher than that of Korean-Saanen goat milk. The goat milk samples having more than 1.5 million cells/ml somatic cell counts (SCC) contained higher αs-CNs (P < 0.01) and lower β-CNs (P < 0.05) contents than milks with <1.5 million SCC. This resulted in a higher αs-CN to β-CN ratio (P < 0.01) in the milk with >1.5 million SCC.     

Bioconversion of inulin to 2,3-butanediol by a newly isolated Klebsiella pneumoniae producing inulinase

Inulin could be converted to bio-based chemicals by an inulinase producer without external inulinase, and the production of 2,3-butanediol was less than 50 g/L. In this work, a novel inulinase producer of Klebsiella pneumoniae H3 was isolated, and inulinase catalytic properties as well as 2,3-butanediol fermentation were investigated. The enzyme was an intracellular inulinase with an optimal pH of 6 ∼ 7 and a temperature of 30 °C. The use of inulin by H3 was dependent on the degree of polymerization (DP), and the average DP of inulin in fermentation broth increased from 2.82 to 8.08 in 24-h culture of batch fermentation. Acidic pretreatment was developed to increase inulin utilization by adjusting medium pH to 3.0 prior to sterilization. In batch fermentation with optimized medium and fermentation conditions, the concentration of target product (2,3-butanediol and acetoin) was 80.4 g/L with a productivity of 2.23 g/(L⋅h), and a yield of 0.426 g/g inulin.     

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.     

Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing

2,3-Butanediol is a promising valuable chemical that can be used in various areas as a liquid fuel and a platform chemical. Here, 2,3-butanediol production in Saccharomyces cerevisiae was improved stepwise by eliminating byproduct formation and redox rebalancing. By introducing heterologous 2,3-butanediol biosynthetic pathway and deleting competing pathways producing ethanol and glycerol, metabolic flux was successfully redirected to 2,3-butanediol. In addition, the resulting redox cofactor imbalance was restored by overexpressing water-forming NADH oxidase (NoxE) from Lactococcus lactis. In a flask fed-batch fermentation with optimized conditions, the engineered adh1Δadh2Δadh3Δadh4Δadh5Δgpd1Δgpd2Δ strain overexpressing Bacillus subtilis α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD), S. cerevisiae 2,3-butanediol dehydrogenase (Bdh1), and L. lactis NoxE from a single multigene-expression vector produced 72.9 g/L 2,3-butanediol with the highest yield (0.41 g/g glucose) and productivity (1.43 g/(L·h)) ever reported in S. cerevisiae.     

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(β1-x)-glycans (sialo-glycans) to Gal(β1-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 (β3′-GL), 4′-galactosyl-lactose (β4′-GL), and 6′-galactosyl-lactose (β6′-GL). This prebiotic GOS is prepared from lactose by incubation with suitable β-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 β3′-GL, β4′-GL, and β6′-GL, using GMP as the sialic acid donor, were purified, and their structures were elucidated by nuclear magnetic resonance spectroscopy. Monosialylated β3′-GL and β4′-GL contained Neu5Ac connected to the terminal Gal residue; however, in the case of β6′-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 V_max and k_cat values were higher in the case of lactose.     

Polysaccharide of nectarine gum exudate: Comparison with that of peach gum

The gum exudate polysaccharide from the trunk of nectarine (PPNEC) was compared with that of peach, being composed of Ara, Xyl, Man, Gal, and uronic acids in 37:13:2:42:6 molar ratio and had M_w 3.93 × 10⁶ g mol^?1, compared with 5.61 × 10⁶ g mol^?1 for peach gum polysaccharide. Methylation analysis of PPNEC indicated a highly branched structure with relatively high amounts of di- (16%) and tri-O-substituted (9%) Galp units and nonreducing end-units of Araf (26%) and Xylp (17%). Combination with ¹³C NMR data, showed the presence of α-l-Araf (nonreducing end, 3-O-, 5-O-, and 2,5-di-O-subst.), β-l-Arap (4-O- and 2,4-di-O-subst.), β-d-Galp (3-O-, 2,3-di-O-, 3,6-di-O-, and 3,4,6-tri-O-subst.), and α- and/or β-d-Xylp nonreducing end-units. A signal appeared from 4-O-Me-α-d-GlcpA units. PPNEC had structures similar to those of polysaccharide from peach tree gum, although in different proportions and with a lower M_w.