Kinetics and products of Thermotoga maritima ß-glucosidase with lactose and cellobiose.

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by ß-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many ß-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable ß-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the ß-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly ß(1 → 3) and ß(1 → 6) elongating activity, but also some ß(1 → 4) elongation was observed. The ß-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: • ß-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima ß-glucosidase has high activity and high thermostability. • Thermotoga maritima ß-glucosidase GOS contains mainly (ß1-3) and (ß1-6) linkages.

Synthesis and Characterization of Sialylated Lactose- and Lactulose-Derived Oligosaccharides by Trypanosoma cruzi Trans-sialidase.

Sialylated oligosaccharides contribute 12.6-21.9% of total free oligosaccharides in human milk ( hMOS). These acidic hMOS possess prebiotic properties and display antiadhesive effects against pathogenic bacteria. Only limited amounts of sialylated hMOS are currently available. The aim of our work is to enzymatically synthesize sialylated oligosaccharides mimicking hMOS functionality. In this study, we tested mixtures of glucosylated-lactose (GL34), galactosylated-lactulose (LGOS), and galacto-oligosaccharide (Vivinal GOS) molecules, as trans-sialylation acceptor substrates. The recombinant trans-sialidase enzyme from Trypanosoma cruzi (TcTS) was used for enzymatic decoration, transferring (α2→3)-linked sialic acid from donor substrates to nonreducing terminal ß-galactopyranosyl units of these acceptor substrates. The GL34 F2 2-Glc-Lac compound with an accessible terminal galactosyl residue was sialylated efficiently (conversion degree of 47.6%). TcTS sialylated at least 5 LGOS structures and 11 Vivinal GOS DP3-4 compounds. The newly synthesized sialylated oligosaccharides are interesting as potential hMOS mimics for applications in biomedical and functional-food products.

Regional variations in human milk oligosaccharides in Vietnam suggest FucTx activity besides FucT2 and FucT3.

Breastfeeding is the normal way of providing young infants with the nutrients they need for healthy growth and development (WHO). Human milk oligosaccharides (hMOS) constitute a highly important class of nutrients that are attracting strong attention in recent years. Several studies have indicated that hMOS have prebiotic properties, but also are effective in anti-adhesion of pathogens, modulating the immune system and providing nutrients for brain growth and development. Most of the latter functions seem to be linked to the presence of fucose-containing immunodeterminant epitopes, and Neu5Ac-bearing oligosaccharides. Analysis of hMOS isolated from 101 mothers' milk showed regional variation in Lewis- and Secretor based immunodeterminants. Lewis-negative milk groups could be sub-divided into two sub-groups, based on the activity of a third and hitherto unidentified fucosyltransferase enzyme. Analysis of hMOS remaining in faeces showed three sub-groups based on hMOS surviving passage through the gut, full consumption, specific partial consumption and non-specific partial consumption, fitting previous findings.

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.

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.

Capillary electrophoresis with lamp-based wavelength-resolved fluorescence detection for the probing of protein conformational changes.

Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation.