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In an attempt to develop a process of enzymatic peeling of mandarin segments suitable for use on an industrial scale, the cell wall fraction of the segment membrane of Satsuma mandarin fruits was extracted to obtain a chelating agent-soluble pectin fraction (ChSS), a dilute sodium hydroxide-soluble pectin fraction (DASS), a 1M sodium hydroxide-soluble hemicellulose fraction (1MASS), a 4M sodium hydroxide-soluble hemicellulose fraction (4MASS) and a cellulose-rich residue (3.1, 0.9, 0.4, 0.7 and 1.6%w/w of fresh membrane, respectively). The ChSS pectin consisted mainly of galacturonic acid followed by arabinose and galactose. The DASS fraction contained less galacturonic acid and more neutral sugars than ChSS. Eighty-nine percent of the galacturonic acid present in the segment membranes was recovered in the above two pectin fractions. The two hemicellulosic fractions consisted of two different molecular weight populations, which also differed in their sugar composition. Arabinose, xylose, mannose, galactose and glucose were the main sugar constituents of these hemicellulose fractions. In addition to an (arabino)xylan and a xyloglucan, the presence of an arabinogalactan is suggested by the sugar composition of both hemicelluloses. The pectin fractions were also characterised by their degradability by the pectic enzymes polygalacturonase, pectinmethylesterase and rhamnogalacturonan hydrolase. However the degree of degradation of the pectin fractions by enzymes differed, and the amount of the polymeric materials resistant to further degradation and the oligomeric products also differed. Using pectic enzymes it is possible to obtain peeled mandarin segments ready to eat or for canning., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Enzymes from a balanced human gut flora are promising tools to design prebiotic oligosaccharides. In this study, we investigated the action of enzymes from fecal bacteria on the complex polysaccharide konjac glucomannan (KGM). The oligosaccharides produced were compared to oligosaccharides from KGM digests with fungal endo-β-(1,4)-glucanase (EG) or endo-β-(1,4)-mannanase (EM). For this purpose, the oligosaccharides from the different digests were first studied for their structural characteristics like monosugar composition and exo-enzymatic degradability, as monitored by capillary electrophoresis with laser-induced fluorescence detection. Whereas the oligosaccharides produced by EG and EM were characteristic for the selectivity of the respective enzyme in cleaving the mannose-/glucose-sugar linkages of KGM, oligosaccharides produced by the fecal enzymes did not point to a sugar-selective degradation. The oligosaccharide fragments from the different digests indicated the KGM polysaccharide to be composed of a backbone composed of short mannose and glucose sequences, to which branches rich in mannose are attached.

The characterization of oligosaccharides in the feces of breast-fed babies is a valuable tool for monitoring the gastrointestinal fate of human milk oligosaccharides (HMOs). In the present study we monitored fecal oligosaccharide profiles together with the HMO-profiles of the respective breast milks up to six months postpartum, by means of capillary electrophoresis-laser induced fluorescence detection and mass spectrometry. Eleven mother/child pairs were included. Mother's secretor- and Lewis-type included all combinations [Le(a-b+), Le(a+b-), Le(a-b-)]. The fecal HMO-profiles in the first few months of life are either predominantly composed of neutral or acidic HMOs and are possibly effected by the HMO-fingerprint in the respective breast milk. Independent of the initial presence of acidic or neutral fecal HMOs, a gradual change to blood-group specific oligosaccharides was observed. Their presence pointed to a gastrointestinal degradation of the feeding-related HMOs, followed by conjugation with blood group specific antigenic determinants present in the gastrointestinal mucus layer. Eleven of these 'hybrid'-oligosaccharides were annotated in this study. When solid food was introduced, no HMOs and their degradation- and metabolization products were recovered in the fecal samples., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

So far, little is known on the fate of oligosaccharides in the colon of breast- and formula-fed babies. Using capillary electrophoresis with laser induced fluorescence detector coupled to a mass spectrometer (CE-LIF-MS(n)), we studied the fecal oligosaccharide profiles of 27 two-month-old breast-, formula- and mixed-fed preterm babies. The interpretation of the complex oligosaccharide profiles was facilitated by beforehand clustering the CE-LIF data points by agglomerative hierarchical clustering (AHC). In the feces of breast-fed babies, characteristic human milk oligosaccharide (HMO) profiles, showing genetic fingerprints known for human milk of secretors and non-secretors, were recognized. Alternatively, advanced degradation and bioconversion of HMOs, resulting in an accumulation of acidic HMOs or HMO bioconversion products was observed. Independent of the prebiotic supplementation of the formula with galactooligosaccharides (GOS) at the level used, similar oligosaccharide profiles of low peak abundance were obtained for formula-fed babies. Feeding influences the presence of diet-related oligosaccharides in baby feces and gastrointestinal adaptation plays an important role herein. Four fecal oligosaccharides, characterized as HexNAc-Hex-Hex, Hex-[Fuc]-HexNAc-Hex, HexNAc-[Fuc]-Hex-Hex and HexNAc-[Fuc]-Hex-HexNAc-Hex-Hex, highlighted an active gastrointestinal metabolization of the feeding-related oligosaccharides. Their presence was linked to the gastrointestinal mucus layer and the blood-group determinant oligosaccharides therein, which are characteristic for the host's genotype., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The complex formation between β-lactoglobulin and pectins of varying overall charge and local charge density were investigated. Isothermal titration calorimetry experiments were carried out to determine the enthalpic contribution to the complex formation at pH 4.25 and various ionic strengths. Complex formation was found to be an exothermic process for all conditions. Combination with previously published binding constants by Sperber et al. (Sperber, B. L. H. M.; Cohen Stuart, M. A.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246-3252) allows for the determination of the changes in the Gibbs energy and the change in entropy of the system upon complex formation between β-lactoglobulin and pectin. The local charge density of pectin is found to determine the balance between enthalpic and entropic contributions. For a high local charge density pectin, the main contribution to the Gibbs energy is of an enthalpic nature, supported by a favorable entropy effect due to the release of small counterions. A pectin with a low local charge density has a more even distribution of the enthalpic and entropic part to the change of the Gibbs energy. The enthalpic part is reduced due to the lower charge density, while the relative increase of the entropic contribution is thought to be caused by a change in the location of the binding place for pectin on the β-lactoglobulin molecule. The association of the hydrophobic methyl esters on pectin with an exposed hydrophobic region on β-lg results in the release of water molecules from the hydrophobic region and surrounding the methyl esters of the pectin molecule. An increase in the ionic strength decreases the enthalpic contribution due to the shielding of electrostatic attraction in favor of the entropic contribution, supporting the idea that the release of water molecules from hydrophobic areas plays a part in the complex formation.

Recently, various branched arabino-oligosaccharides as present in a sugar beet arabinan digest were characterized using NMR. Although HPAEC often has been the method of choice to monitor the enzymatic degradation reactions of polysaccharides, it was shown that HPAEC was incapable to separate all known linear and branched arabino-oligosaccharides present. As this lack of resolution might result in an incorrect interpretation of the results, other separation techniques were explored for the separation of linear and branched arabino-oligosaccharides. The use of porous-graphitized carbon liquid chromatography with evaporative light scattering and mass detection as well as capillary electrophoresis with laser-induced fluorescence and mass detection demonstrated the superiority of both the techniques toward HPAEC by enabling the separation and unambiguous identification of almost all the linear and branched arabino-oligosaccharides available. The elution behavior of all arabino-oligosaccharides for the three tested separation techniques was correlated with their chemical structures and conclusions were drawn for the retention mechanisms of the arabino-oligosaccharides on the different chromatographic and electrophoretic systems. The combination of the elution/migration behavior on LC/CE and the MS fragmentation patterns of the arabino-oligosaccharides led to the prediction of structures for new DP6 arabino-oligosaccharides in complex enzyme digests., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The proliferation and apoptosis of metastatic melanoma cells are often abnormal. We have evaluated the action of a pectic rhamnogalacturonan obtained by hot buffer extraction of okra pods (okra RG-I) on melanoma cell growth and survival in vitro. We added okra RG-I containing an almost pure RG-I carrying very short galactan side chains to 2D (on tissue culture polystyrene, tPS) and 3D (on poly(2-hydroxyethylmethacrylate), polyHEMA) cultures of highly metastatic B16F10 mouse melanoma cells. We then analyzed cell morphology, proliferation index, apoptosis, cell cycle progression and the expression of adhesion molecules. Immunostaining and western blotting were used to assay galectin-3 (Gal-3) protein.Incubation with okra RG-I altered the morphology of B16F10 cells and significantly reduced their proliferation on both tPS and polyHEMA. The cell cycle was arrested in G2/M, and apoptosis was induced, particularly in cells on polyHEMA. The expression of N-cadherin and alpha5 integrin subunit was reduced and that of the multifunctional carbohydrate-binding protein, Gal-3, at the cell membrane increased.These findings suggest that okra RG-I induces apoptosis in melanoma cells by interacting with Gal-3. As these interactions might open the way to new melanoma therapies, the next step will be to determine just how they occur.

Sugar beet arabinan consists of an alpha-(1,5)-linked backbone of L-arabinosyl residues, which can be either single or double substituted with alpha-(1,2)- and/or alpha-(1,3)-linked L-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an alpha-(1,5)-linked backbone of L-arabinosyl residues. One series carries single substituted alpha-(1,3)-linked L-arabinosyl residues at the backbone, whereas the other series consists of a double substituted alpha-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Cell wall materials derived from leaves and hypocotyls of Arabidopsis mutant and wild type plants have been incubated with a mixture of pure and well-defined pectinases, hemicellulases, and cellulases. The resulting oligosaccharides have been subjected to MALDI-TOF MS and CE-LIF analysis. MALDI-TOF MS analysis provided a fast overview of all oligosaccharides released, whereas CE-LIF-measurements enabled separation and characterization of many oligosaccharides under investigation. Both methods have been validated with leaf material of known mutant Arabidopsis plants and were shown to be able to discriminate mutant from wild type plants. Downscaling of the MALDI-TOF MS and CE-LIF approaches toward the hypocotyl level was established, and the performance of MALDI-TOF MS and CE-LIF was shown in the successful recognition of the Arabidopsis mutant gaut13 as an interesting candidate for further analysis.

The analysis and quantification of (galacto)oligosaccharides from food matrices demands both a reproducible extraction method as well as a sensitive and accurate analytical method. Three typical matrices, namely, infant formula, fruit juice, and a maltodextrin-rich preparation, to which a commercial galactooligosaccharide mixture was added in a product concentration range from 1.25 to 30%, served as model substrates. Solid-phase extraction on graphitized carbon material upon enzymatic amyloglucosidase pretreatment enabled a good recovery and a selective purification of the different galactooligosaccharide structures from the exceeding amounts of particularly lactose and maltodextrins. With the implementation of capillary electrophoresis in combination with laser-induced fluorescence (CE-LIF) detection, a new possibility facilitating a sensitive qualitative and quantitative determination of the galactooligosaccharide contents in the different food matrices is outlined. Simultaneous monitoring and quantifying prebiotic oligosaccharides embedded in food matrices presents a promising and important step toward an efficient monitoring of individual oligosaccharides and is of interest for research areas dealing with small quantities of oligosaccharides embedded in complex matrices, e.g., body liquids.

Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MS(n)-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar beta-(1,4)-xylo- and beta-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of beta-(1,4)-galacto-, beta-(1,4)-manno- and alpha-(1,4)-gluco-oligosaccharides. Charged oligomers such as alpha-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

The formation of complexes between proteins and polysaccharides is of great importance for many food systems like foams, emulsions, acidified milk drinks, and so on. The complex formation between beta-lactoglobulin (beta-lg) and pectins with a well-defined physicochemical fine structure has been studied to elucidate the influence of overall charge and local charge density of pectin on the complex formation. Binding isotherms of beta-lg to pectin are constructed using fluorescence anisotropy, which is shown to be an excellent technique for this purpose, as it is fast and requires low sample volumes. From the binding isotherms the maximal adsorbed amount, binding constant (k(obs)) and the cooperativity of binding are obtained at different ionic strengths. The Hill model is used to fit the binding isotherms and is shown to be preferable over a Langmuir fit. At pH 4.25, k(obs) shows a maximum at an ionic strength of 10 mM when using a low methyl esterified pectin (LMP) due to the balance of attractive and repulsive electrostatic forces between beta-lg and pectin and beta-lg neighbors. For two high methyl esterified pectins, one with a blockwise distribution of methyl esters (HMP(B)) and one with a random distribution (HMP(R)), this ionic strength maximum is absent and k(obs) decreases with increasing ionic strength. k(obs) is found to be largest for LMP and HMP(B) and considerably lower for HMP(R). A positive cooperativity is observed for both LMP (above an ionic strength of 45 mM) and HMP(R) (above an ionic strength of 15 mM) but not for HMP(B). Positive cooperativity is thought to be caused by a rearrangement of the pectin helix structure caused by binding of beta-lg, thus creating new or binding sites with a higher affinity. To attain strong binding of beta-lg to pectin it is preferable to use a pectin with a blockwise distribution of methyl esters. When complex formation takes place in high ionic strength media an LMP gives the best results, while at low ionic strength a high methyl esterified pectin with blockwise distribution may give better results, due to reduced electrostatic repulsion between both pectin and beta-lg and beta-lg neighbors.

Interactions of proteins with phenolic compounds occur in food products containing vegetable sources, such as cocoa, cereals, or yogurts containing fruit. Such interactions can modify protein digestion and protein industrial properties. Noncovalent interactions between globular proteins (proteins important in industry) and procyanidins (phenolic compounds present in large quantity in fruits) were studied. The affinity constants between procyanidins of various average degrees of polymerization (DP) and lysozyme or alpha-lactalbumin were measured by isothermal titration calorimetry. The effects of these interactions on protein solubility and foam properties were examined using alpha-lactalbumin and BSA. Weak interactions were found with epicatechin and procyanidin dimers. Procyanidins of n = 5.5 and n = 7.4 showed medium (1.5 x 10(5) M(-1)) and high (8.69 x 10(9) M(-1)) affinities, respectively, for alpha-lactalbumin at pH 5.5, with n the average number of subunits per oligomer. A positive cooperativity of binding at low procyanidin:protein molar ratios was observed. The affinities of alpha-lactalbumin and lysozyme for procyanidins increased when the pH was close to the isoelectric pH. Solubility of lysozyme was strongly decreased by procyanidins of n = 5.5, whereas alpha-lactalbumin and BSA were less affected. Protein solubility in the presence of procyanidins was not affected by increased ionic strength but increased slightly with temperature. Procyanidins of n = 5.5 and n = 7.4 stabilized the average bubble diameter of foam formed with alpha-lactalbumin but had no effect on foam made from BSA. These results indicate that procyanidins of medium can lead to an undesirable decrease of protein solubility, but may play a positive role in foam stability.

Okra pods are commonly used in Asia as a vegetable, food ingredient, as well as a traditional medicine for many different purposes; for example, as diuretic agent, for treatment of dental diseases and to reduce/prevent gastric irritations. The healthy properties are suggested to originate from the high polysaccharide content of okra pods, resulting in a highly viscous solution with a slimy appearance when okra is extracted with water. In this study, we present a structural characterisation of all major cell wall polysaccharides originating from okra pods. The sequential extraction of okra cell wall material yielded fractions of soluble solids extractable using hot buffer (HBSS), chelating agent (CHSS), dilute alkaline (DASS) and concentrated alkaline (CASS). The HBSS fraction was shown to be rich in galactose, rhamnose and galacturonic acid in the ratio 1.3:1:1.3. The degree of acetylation is relatively high (DA=58) while the degree of methyl esterification is relatively low (DM=24). The CHSS fraction contained much higher levels of methyl esterified galacturonic acid residues (63% galacturonic acid; DM=48) in addition to minor amounts of rhamnose and galactose. The ratio of galactose to rhamnose to galacturonic acid was 1.3:1.0:1.3 and 4.5:1.0:1.2 for HBSS and CHSS, respectively. These results indicated that the HBSS and CHSS fractions contain rhamnogalacturonan type I next to homogalacturonan, while the latter is more prevailing in CHSS. Also the DASS fraction is characterised by high amounts of rhamnose, galactose, galacturonic acid and some arabinose, indicating that rhamnogalacturonan I elements with longer arabinose- and galactose-rich side chains were part of this fraction. Partial digestion of HBSS and CHSS by pectin methyl esterase and polygalacturonase resulted in a fraction with a lower Mw and lower viscosity in solution. These samples were subjected to NMR analysis, which indicated that, in contrast to known RG I structure, the acetyl groups in HBSS are not located on the galacturonic acid residues, while for CHSS only part of the acetyl groups are located on the RG I galacturonic acid residues. The CASS fraction consisted of XXXG-type xyloglucan and 4-methylglucuronoxylan as shown by their sugar (linkage) composition and enzymatic digestion.

The okra plant, Abelmoschus esculentus (L.) Moench, a native plant from Africa, is now cultivated in many other areas such as Asia, Africa, Middle East, and the southern states of the USA. Okra pods are used as vegetables and as traditional medicines. Sequential extraction showed that the Hot Buffer Soluble Solids (HBSS) extract of okra consists of highly branched rhamnogalacturonan (RG) I containing high levels of acetyl groups and short galactose side chains. In contrast, the CHelating agent Soluble Solids (CHSS) extract contained pectin with less RG I regions and slightly longer galactose side chains. Both pectic populations were incubated with homogeneous and well characterized rhamnogalacturonan hydrolase (RGH), endo-polygalacturonase (PG), and endo-galactanase (endo-Gal), monitoring both high and low molecular weight fragments. RGH is able to degrade saponified HBSS and, to some extent, also non-saponified HBSS, while PG and endo-Gal are hardly able to degrade either HBSS or saponified HBSS. In contrast, PG is successful in degrading CHSS, while RGH and endo-Gal are hardly able to degrade the CHSS structure. These results point to a much higher homogalacturonan (HG) ratio for CHSS when compared to HBSS. In addition, the CHSS contained slightly longer galactan side chains within its RG I region than HBSS. Matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated the presence of acetylated RG oligomers in the HBSS and CHSS enzyme digests and electron spray ionization-ion trap-mass spectrum showed that not only galacturonosyl residues but also rhamnosyl residues in RG I oligomers were O-acetylated. NMR spectroscopy showed that all rhamnose residues in a 20kDa HBSS population were O-acetylated at position O-3. Surprisingly, the NMR data also showed that terminal alpha-linked galactosyl groups were present as neutral side chain substituents. Taken together, these results demonstrate that okra contained RG I structures which have not been reported before for pectic RG I.

Enzyme-resistant pectin or modified hairy regions were subjected to size exclusion (HPSEC) and weak anion exchange (WAX) chromatography. Fractions collected after separation were tested for the presence of different pectic epitopes using the monoclonal antibodies LM2, LM5, LM6, and JIM7. Separation by HPSEC showed that based on molecular weight the different epitopes were restricted to distinct molecular weight populations. WAX chromatography resulted in an even better separation of the different pectic epitopes present. A clear separation between arabino galactan type II epitopes and the RG I side chains, (1,5)-alpha-l-arabinan and (1,4)-beta-d-galactan, could be established. Arabinogalactan type II was found in the first populations eluting off the WAX column. The observations made within the ELISA assays of the collected fractions could be confirmed by determination of the sugar composition of the individual populations obtained. The sugar composition of the AGII positive populations eluting off the WAX column shows the presence of significant amounts of rhamnose and galacturonic acid. Together with the delay on an anion exchanger, this observation indicates a possible linkage between RGI and AGII. The volume of the individual fractions collected provides enough material for a maximum of 20 different antibodies to be tested from one analytical separation.

A mixture of single side chains from white cabbage pectin were obtained by anion exchange chromatography after applying mild chemical conditions promoting beta-elimination. These pectin fragments were characterized by their molecular weight distribution, sugar composition, 13C-NMR, and MALDI-TOF-MS analysis. These analyses revealed that the large oligosaccharides released by beta-eliminative treatment were composed of alpha-1,5 linked arabinosyl residues with 2- and 3-linked alpha-arabinosyl side chains, and, or beta-1,4 linked galactosyl side chains. Fractions were tested for complement-fixing activity in order to determine their interaction with the complement system. These results strongly indicated that there was a minimal unit size responsible for the complement-fixing activity. Neutral pectin fragments (8 kDa) obtained from beta-elimination were inactive in the complement system, although they contained a sugar composition previously shown to be highly active. Larger pectin fragments (17 kDa) retained some activity, but much lower than polymers containing rhamnogalacturonan type 1 (RGI) structures isolated from the same source. This implied that structural elements containing multiple side chains is necessary for efficient complement-fixing activity.

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.

Lignan macromolecule from flaxseed hulls is composed of secoisolariciresinol diglucoside (SDG) and herbacetin diglucoside (HDG) moieties ester-linked by 3-hydroxy-3-methylglutaric acid (HMGA), and of p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) moieties ester-linked directly to SDG. The linker molecule HMGA was found to account for 11% (w/w) of the lignan macromolecule. Based on the extinction coefficients and RP-HPLC data, it was determined that SDG contributes for 62.0% (w/w) to the lignan macromolecule, while CouAG, FeAG, and HDG contribute for 12.2, 9.0, and 5.7% (w/w), respectively. Analysis of fractions of lignan macromolecule showed that the higher the molecular mass, the higher the proportion of SDG was. An inverse relation between the molecular mass and the proportion (%) CouAG+FeAG was found. Together with the structural information of oligomers of lignan macromolecule obtained after partial saponification, it is hypothesized that the amount of CouAG+FeAG present during biosynthesis determines the chain length of lignan macromolecule. Furthermore, the chain length was estimated from a model describing lignan macromolecule based on structural and compositional data. The average chain length of the lignan macromolceule was calculated to be three SDG moieties with CouAG or FeAG at each of the terminal positions, with a variation between one and seven SDG moieties.

As pectin molecules are too large and heterogeneous to analyze as a whole, the polymer is usually degraded to smaller oligomers, which are often analyzed by high-performance anion exchange chromatography (HPAEC). However, the high salt concentration necessary to elute pectin oligomers by HPAEC is incompatible with online mass detection. To overcome such a disadvantage, a CE-IT-MS system was set up to further elucidate the fine structure of charged oligosaccharides. An effective separation of differently substituted galacturonic acid containing oligomers was obtained by low-pH CE-LIF analysis. By adapting the buffer and capillary online MS detection was enabled. Moreover, with MS/MS it was possible to localize sugar residues' substitutions. With this combined CE-MS approach LIF electropherograms of xylogalacturonan and rhamnogalacturonan I digests could be annotated. The method was further exemplified by a complex oligomer mixture of acid hydrolyzed apple pectin, which was separated and characterized by CE-MSn. Oligomers present in low amounts could be localized by their corresponding m/z, as was demonstrated by selected mass range representation.

In flaxseed hulls, lignans are present in an oligomeric structure. Secoisolariciresinol diglucoside (SDG), ester-linked to hydroxy-methyl-glutaric acid (HMGA), forms the backbone of this lignan macromolecule. The hydroxycinnamic acids p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) are also part of the lignan macromolecule. However, their position and type of linkage are still unknown. The aim of this study was to investigate how CouAG and FeAG are linked within the lignan macromolecule from flaxseed hulls. Fragments of the lignan macromolecule were obtained by partial saponification. After isolation of the fragments by preparative RP-HPLC, several key structures were identified by MS and NMR. Within the lignan macromolecule, CouAG is attached to the C-6 position of a glucosyl moiety of SDG. FeA is linked to the C-2 position of a glucosyl moiety of SDG. FeAG is ester-linked within the lignan macromolecule with its carboxyl group, but it remains unclear whether FeAG links to the C-2 or C-6 position of SDG. Attachment of HMGA to the glucosyl moiety of CouAG or FeAG was not observed. The results clearly show that within the lignan macromolecule, the hydroxycinnamic acids are linked directly via an ester bond to the glucosyl moiety of SDG.

There is an increasing interest to positively influence the human intestinal microbiota through the diet by the use of prebiotics and/or probiotics. It is anticipated that this will balance the microbial composition in the gastrointestinal tract in favor of health promoting genera such as Bifidobacterium and Lactobacillus. Carbohydrates like non-digestible oligosaccharides are potential prebiotics. To understand how these bacteria can grow on these carbon sources, knowledge of the carbohydrate-modifying enzymes is needed. Little is known about the carbohydrate-modifying enzymes of bifidobacteria. The genome sequence of Bifidobacterium adolescentis and Bifidobacterium longum biotype longum has been completed and it was observed that for B. longum biotype longum more than 8% of the annotated genes were involved in carbohydrate metabolism. In addition more sequence data of individual carbohydrases from other Bifidobacterium spp. became available. Besides the degradation of (potential) prebiotics by bifidobacterial glycoside hydrolases, we will focus in this review on the possibilities to produce new classes of non-digestible oligosaccharides by showing the presence and (transglycosylation) activity of the most important carbohydrate modifying enzymes in bifidobacteria. Approaches to use and improve carbohydrate-modifying enzymes in prebiotic design will be discussed.

The effect of process conditions used for wheat straw pretreatments on the liquor- and residue-composition was studied. Hereto, the pretreatment conditions were expressed in a 'combined severity R(0)(')-factor'. The higher the combined severity factor (R(0)(')) the more xylan was released from the wheat straw, but the more xylan decomposed and furfural formation occurred. The percentage of residual xylan present after pretreatment appeared to be a good indicator concerning cellulose degradability or bio-ethanol production. Namely, cellulose degradation by using commercial enzymes was higher at higher severities corresponding to a lower amount of residual xylan. The xylan release and degradation was studied in more detail by using HPSEC and MALDI-TOF mass spectrometry. The more severe the treatment the more (acetylated) xylose oligomers with a DP lower than nine were analysed. The presence of (acetylated) xylans with a DP of 9-25 increased slightly from low to medium severity. The quantification of the DP-distribution of the (acetylated) xylans released proved to be a good tool to predict cellulose degradability.

Lignans in flaxseed are known to be part of a macromolecule in which they are connected through the linker-molecule hydroxy-methyl-glutaric acid (HMGA). In this study, the lignan macromolecule was extracted from flaxseed hulls and degraded to its monomeric constituents by complete saponification. Besides secoisolariciresinol diglucoside (SDG), the phenolic compounds p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) were isolated, which was expected based on indications from the literature. Also the flavonoid herbacetin diglucoside (HDG) was found. The presence of HDG was confirmed by NMR following preparative RP-HPLC purification. Also the presence of the three other constituents (CouAG, FeAG and SDG) was confirmed by NMR. To prove that HDG is a substructure of the lignan macromolecule, the macromolecule was fragmented by partial saponification. A fragment consisting of HDG and HMGA was indicated. This fragment was isolated by preparative RP-HPLC and its identity was confirmed by NMR. It is concluded that the flavonoid HDG is a substructure of the lignan macromolecule from flaxseed hulls and that it is incorporated in the macromolecule via the same linker-molecule as SDG.

A large number of proteins are glycosylated, either in vivo or as a result of industrial processing. Even though the effect of glycosylation on the aggregation of proteins has been studied extensively in the past, some reports show that the aggregation process is accelerated, whereas others found that the process is inhibited by glycosylation. This paper investigates the reasons behind these controversial results as well as the potential mechanism of the effect of glucosylation on aggregation using bovine beta-lactoglobulin as a model. Glucosylation was found to inhibit denaturant-induced aggregation, whereas heat-induced aggregation was accelerated. It was also found that the kinetic partitioning from an unfolded state was driven toward refolding for glucosylated protein, whereas aggregation was the preferred route for the nonglucosylated protein.

Bilberries are known to have one of the most complex xyloglucan structures described in the plant kingdom until now. To characterise this structure, xyloglucans were enzymatically degraded and the oligosaccharides obtained were analysed. More than 20 different building blocks were found to make up the xyloglucan polymer. Bilberry xyloglucan was of XXXG-type, but some XXG-type oligomers were present, as well. The building blocks contain galactose-xylose (L) and fucose-galactose-xylose (F) side chains. In both side chains, the galactose units can be acetylated. In addition, beta-xylose-alpha-xylose (U) side chains were shown. This U chain was present in three building blocks described before (XUXG, XLUG and XUFG) and four novel blocks that have not been described in the literature previously: XUG, XUUG, XLUG and XXUG.

Various plant polysaccharide derived mono- and oligosaccharides were derivatized with the fluorescent 9-aminopyrene-1,4,6-trisulfonate (APTS) and subjected to capillary electrophoresis (CE) in combination with laser induced fluorescence (LIF) detection. CE-LIF was suitable for mol-based quantification of various APTS-monosaccharides. CE-LIF of APTS-oligosaccharides showed high resolutions, while analysis times were at maximum 15 min. The coupling of CE to electrospray-iontrap mass spectrometery (MS) with online UV detection showed to be a powerful technique in the identification of APTS-oligosaccharides. For the first time, various APTS-xylo-oligosaccharides, having either no, O-acetyl, arabinosyl or xylosyl substitutions at varying positions, were identified by using CE-LIF and CE-MS(n).

The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.

Different separation (HPAEC, RP-HPLC, CE) and identification (MALDI-TOF-MS, ESI-MS(n)) techniques were compared to analyse oligosaccharides obtained after incubation of xyloglucan with endo-glucanase. It was possible to analyse xyloglucan oligosaccharides with each technique. Several techniques, including off line (HPAEC-MALDI-TOF-MS) or online (CE-ESI-MS(n), RP-HPLC-ESI-MS(n)) connection provided complementary information on xyloglucan structure. Online CE-MS and RP-HPLC-MS are described for the first time in xyloglucan analysis. Advantages and disadvantages of the techniques for different purposes such as structural characterisation of oligosaccharides or oligosaccharide profiling are discussed. Black currant xyloglucans had a rather simple XXXG-type structure with galactose and fucose containing side chains.

In the present study emulsions were made with various potato protein preparations, which varied in protease inhibitor and patatin content. These emulsions were characterized with respect to average droplet size, plateau surface excess, and the occurrence of droplet aggregation. Droplet aggregation occurred only with potato protein preparations that contained a substantial amount of protease inhibitors and could be prevented only at pH 3. The average droplet size of the emulsions made with potato proteins appeared to be related to the patatin content of the preparation used. Average droplet size was found to be dominated by the patatin-catalyzed lipolytic release of surface active fatty acids and monoglycerides from the tricaprylin oil phase during the emulsification process. Addition of monoglycerides and especially fatty acids, at concentrations representative of those during emulsification, was shown to cause a stronger and much faster decrease of the interfacial tension than that with protein alone and to result in a drastic decrease in droplet size. The patatin used was shown to have a lipolytic activity of 820 units/g with emulsified tricaprylin as the substrate. Because of the droplet aggregating properties of the protease inhibitors, the patatin-rich potato preparations seem to be the most promising for food emulsion applications over a broad pH range, provided the lipolytic activity can be diminished or circumvented.

Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends on the rate of adsorption to the interface, which can be controlled by bulk concentration. Therefore, the adsorption of proteins with varying structural stabilities at several protein concentrations was studied using ellipsometry and surface tensiometry. For beta-lactoglobulin the adsorbed amount (Gamma) needed to reach a certain surface pressure (Pi) decreased with decreasing bulk concentration. Ovalbumin showed no such dependence. To verify whether this difference in behavior is caused by the difference in structural stability, similar experiments were performed with cytochrome c and a destabilized variant of this protein. Both proteins showed identical Pi-Gamma, and no dependence on bulk concentration. From this work it was concluded that unfolding will only take place if the kinetics of adsorption is similar or slower than the kinetics of unfolding. The latter depends on the activation energy of unfolding (which is in the order of 100-300 kJ/mol), rather than the free energy of unfolding (typically 10-50 kJ/mol).

The aim of this work is to evaluate the impact of sulfhydryl groups on ovalbumin aggregation and gelation. Ovalbumin was chemically modified to add sulfhydryl groups in various degrees. The rate of aggregation was not affected by the introduction of sulfhydryl groups, and disulfide bond formation was preceded by physical interactions. Hence, disulfide interactions may not be the driving force for the aggregation of ovalbumin. Investigation of the aggregates and gels by electron microscopy and rheology suggested that a critical number of sulfhydryl groups can be introduced beyond which the microstructure of the aggregates transforms from fibrillar into amorphous. Rheological studies further suggested that covalent networks, once formed, do not have the possibility to rearrange, reducing the possibility to attain a stronger network. These results show that, even though aggregation of ovalbumin may be primarily driven by physical interactions, formed disulfide bonds are important to determine the resulting aggregate morphology and rheological properties.

The intermolecular distributions of amide groups within two commercial LMA pectins was studied after removal of the methyl esters followed by fractionation of the different populations by anion exchange chromatography. The populations obtained had almost equal degrees of amidation while the values of the degree of blockiness were not the same, indicating also a different intramolecular distribution of the substituents considered as semirandom. Populations from the methyl-esterified amidated pectins showed a rather random distribution for almost all populations. A striking difference between these different populations was that, despite the same level of substitution, the ratio between amide groups and methyl esters varied significantly, indicating a heterogeneous amidation process.

The major cationic peroxidase in sorghum grain (SPC4) , which is ubiquitously present in all sorghum varieties was purified to apparent homogeneity, and found to be a highly basic protein (pI approximately 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34,227 and 35,629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w/w) in glycoform I and II, respectively, and a mass of the apoprotein of 33,246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal fragment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca2+ binding increased the protein conformational stability by raising the melting temperature (Tm) from 67 to 82 degrees C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aromatic compounds above pH 5.

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.

Thickening and gelling properties of commercial amidated pectins depend on the degree of amidation and methyl-esterification, but also the distribution of these groups is of great importance. Methods have been developed during the last few years to determine the distribution of methyl esters over the pectic backbone. We applied the strategies developed for the analysis of high methyl-esterified pectins for studying the distribution of amide groups in amidated pectins. Low methyl-esterified amidated (LMA) pectins were digested before and after removal of methyl esters by an endo-polygalacturonase to determine the degree of blockiness of the substituents. The nature of the substituents (amide groups compared to methyl esters) did not modify the behavior of the enzyme. Oligomers released were separated by using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD) at pH 5. Fractions collected after on-line desalting were identified by using MALDI-TOF mass spectrometry. Oligomers were found to elute from the column as a function of their total charge. For the same overall charge and size, oligomers with methyl esters eluted before oligomers with amide groups. Both amide groups and methyl esters of the LMA pectins studied were found to be semirandomly distributed over the pectic backbone, but this may vary according to the amidation process used., (Copyright (c) 2006 Wiley Periodicals, Inc.)

A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Ber e 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1.

The stability of adsorbed protein layers against deformation has in literature been attributed to the formation of a continuous gel-like network. This hypothesis is mostly based on measurements of the increase of the surface shear elasticity with time. For several proteins this increase has been attributed to the formation of intermolecular disulfide bridges between adsorbed proteins. However, according to an alternative model the shear elasticity results from the low mobility of the densely packed proteins. To contribute to this discussion, the actual role of disulfide bridges in interfacial layers is studied. Ovalbumin was thiolated with S-acetylmercaptosuccinic anhydride (S-AMSA), followed by removal of the acetylblock on the sulphur atom, resulting in respectively blocked (SX) and deblocked (SH) ovalbumin variants. This allows comparison of proteins with identical amino acid sequence and similar globular packing and charge distribution, but different chemical reactivity. The presence and reactivity of the introduced, deblocked sulfhydryl groups were confirmed using the sulfhydryl-disulfide exchange index (SEI). Despite the reactivity of the introduced sulfhydryl groups measured in solution, no increase in the surface shear elasticity could be detected with increasing reactivity. This indicates that physical rather than chemical interactions determine the surface shear behaviour. Further experiments were performed in bulk solution to study the conditions needed to induce covalent aggregate formation. From these studies it was found that mere concentration of proteins (to 200 mg/mL, equivalent to a surface concentration of around 2 mg/m(2)) is not sufficient to induce significant aggregation to form a continuous network. In view of these results, it was concluded that the adsorbed layer should not be considered a gelled network of aggregated material (in analogy with three-dimensional gels formed from heating protein solutions). Rather, it would appear that the adsorbed proteins form a highly packed system of proteins with net-repulsive interactions.

The effect of high-pressure processing (HPP) on cell wall polysaccharides in berries was investigated. HPP decreased the degree of methyl esterification (DM), probably by activation of pectin methyl esterase (PME), and improved the extractability of pectins. When commercial enzyme mixtures were added to mashed berries, a synergistic effect was observed between treatment with commercial enzymes and HPP. Compared to treatment at atmospheric pressure, pectic polysaccharides were degraded to a larger extent when HPP was used. In contrast, hemicelluloses were hardly affected by the added enzymes when HPP was included, although they were degraded during similar treatment at atmospheric pressure. Additionally, the activity of rhamnose-releasing enzymes present in minor quantities might be enhanced after HPP, resulting in a decrease of rhamnose in the polymeric cell wall material. These results exploring the effect of HPP at representative conditions clearly point out the potential of HPP for polysaccharide modification.

Commercial cellulase preparations are potentially effective for processing biomass feedstocks in order to obtain bioethanol. In plant cell walls, cellulose fibrils occur in close association with xylans (monocotyls) or xyloglucans (dicotyls). The enzymatic conversion of cellulose/xylans is a complex process involving the concerted action of exo/endocellulases and cellobiases yielding glucose and xylanases yielding xylooligomers and xylose. An overview of commonly measured cellulase-, cellobiase-, and xylanase-activity, using respectively filter paper, cellobiose, and AZCL-dyed xylan as a substrate of 14 commercially available enzyme preparations from several suppliers is presented. In addition to these standardized tests, the enzyme-efficiency of degrading native substrates was studied. Grass and wheat bran were fractionated into a water unsoluble fraction (WUS), which was free of oligosaccharides and starch. Additionally, cellulose- and xylan-rich fractions were prepared by alkaline extraction of the WUS and were enzymatically digested. Hereby, the capability of cellulose and xylan conversion of the commercial enzyme preparations tested was measured. The results obtained showed that there was a large difference in the performance of the fourteen enzyme samples. Comparing all results, it was concluded that the choice of an enzyme preparation is more dependent on the characteristics of the substrate rather than on standard enzyme-activities measured.

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.

An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105 degrees C and a temperature optimum of 80 degrees C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 U.mg(-1)). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed.

Contents of phenolic compounds and related enzymes before and after sorghum grain germination were compared between varieties either resistant or susceptible to biotic (sooty stripe, sorghum midge, leaf anthracnose, striga, and grain molds) and abiotic (lodging, drought resistance, and photoperiod sensitivity) stresses. Independent of grain germination, sorghum varieties resistant to biotic and abiotic stresses had on average higher contents of proanthocyanidins (PAs), 3-deoxyanthocyanidins (3-DAs), and flavan-4-ols than susceptible varieties. Results show that content of 3-DAs is a good marker for sorghum resistance to both biotic and abiotic stresses because it correlates with resistance to all stresses except for photoperiod sensitivity. The second good marker for stress resistance is content of PAs. Total phenolic compounds and the activities of related enzymes are not good markers for stress resistance in sorghum grains.

Arabinogalactan type I from potato was partially degraded by endo-galactanase from Aspergillus niger. High-performance anion-exchange chromatography revealed that several of the oligomeric degradation products eluted as double peaks. To investigate the nature of these products, the digest was fractionated by Bio-Gel P2 chromatography. The pool that contained tetramers was treated with a beta-D-Galp-(1-->4)-specific galactosidase from Bifidobacterium adolescentis to obtain a dimer with deviating linkage type, which was further purified by BioGel P2 chromatography. By obtaining all (1)H and (13)C chemical shifts and the presence of intra residual scalar coupling (HMBC) it could be concluded that the dimer contained a beta-(1-->3)-linkage instead of the expected beta-(1-->4)-linkage. Using the same NMR techniques as for the dimer, it was found that the pool of tetramers consisted of the following two galactose tetramers: beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->4)-alpha/beta-Galp-OH and beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->3)-alpha/beta-Galp-OH. The fact that the deviating beta-(1-->3)-linked galactose was found at the reducing end of the dimer showed that this deviating linkage is present within the backbone. The beta-(1-->3)-galactosyl interruption appeared to be a common structural feature of type I arabinogalactans with a frequency ranging from approximately 1 in 160 (potato, soy, citrus) to 1 in 250 (onion).

In this study a set of chemically engineered variants of ovalbumin was produced to study the effects of electrostatic charge on the adsorption kinetics and resulting surface pressure at the air-water interface. The modification itself was based on the coupling of succinic anhydride to lysine residues on the protein surface. After purification of the modified proteins, five homogeneous batches were obtained with increasing degrees of modification and zeta-potentials ranging from -19 to -26 mV (-17 mV for native ovalbumin). These batches showed no changes in secondary, tertiary, or quaternary structure compared to the native protein. However, the rate of adsorption as measured with ellipsometry was found to decrease with increasing net charge, even at the initial stages of adsorption. This indicates an energy barrier to adsorption. With the use of a model based on the random sequential adsorption model, the energy barrier for adsorption was calculated and found to increase from 4.7 kT to 6.1 kT when the protein net charge was increased from -12 to -26. A second effect was that the increased electrostatic repulsion resulted in a larger apparent size of the adsorbed proteins, which went from 19 to 31 nm2 (native and highest modification, respectively), corresponding to similar interaction energies at saturation. The interaction energy was found to determine not only the saturation surface load but also the surface pressure as a function of the surface load. This work shows that, in order to describe the functionality of proteins at interfaces, they can be described as hard colloidal particles. Further, it is shown that the build-up of protein surface layers can be described by the coulombic interactions, exposed protein hydrophobicity, and size.

A putative endogalactanase gene classified into glycoside hydrolase family 53 was revealed from the genome sequence of Bifidobacterium longum strain NCC2705 (Schell et al., Proc. Natl. Acad. Sci. USA 99:14422-14427, 2002). Since only a few endo-acting enzymes from bifidobacteria have been described, we have cloned this gene and characterized the enzyme in detail. The deduced amino acid sequence suggested that this enzyme was located extracellularly and anchored to the cell membrane. galA was cloned without the transmembrane domain into the pBluescript SK(-) vector and expressed in Escherichia coli. The enzyme was purified from the cell extract by anion-exchange and size exclusion chromatography. The purified enzyme had a native molecular mass of 329 kDa, and the subunits had a molecular mass of 94 kDa, which indicated that the enzyme occurred as a tetramer. The optimal pH of endogalactanase activity was 5.0, and the optimal temperature was 37 degrees C, using azurine-cross-linked galactan (AZCL-galactan) as a substrate. The K(m) and V(max) for AZCL-galactan were 1.62 mM and 99 U/mg, respectively. The enzyme was able to liberate galactotrisaccharides from (beta1-->4)galactans and (beta1-->4)galactooligosaccharides, probably by a processive mechanism, moving toward the reducing end of the galactan chain after an initial midchain cleavage. GalA's mode of action was found to be different from that of an endogalactanase from Aspergillus aculeatus. The enzyme seemed to be able to cleave (beta1-->3) linkages. Arabinosyl side chains in, for example, potato galactan hindered GalA.

A novel colanic acid-degrading enzyme was isolated from a mixed culture filtrate obtained by enrichment culturing of a compost sample using colanic acid as carbon source. The enzyme was partially purified resulting in a 17-fold increase in specific activity. Further purification by Native PAGE revealed that the enzyme is part of a high-molecular weight multi protein complex of at least six individual proteins. The enzyme showed a temperature optimum at 50 degrees C while after 5h at 50 degrees C and pH7 still 70% of the total activity was left. The pH optimum was found to be pH7. Analysis of the degradation products showed that the enzyme is a novel 1,4-beta-fucoside hydrolase that liberates repeating units of colanic acid with varying degrees of acetylation. Km and Vmax of the enzyme were determined against the native substrate as well as its de-O-acetylated and depyruvated forms. Compared to the native substrate the affinity of the enzyme for the modified substrates was much lower. However, for the de-O-acetylated sample a dramatic increase in catalytic efficiency was observed. The native form of the substrate showed substrate inhibition at high concentrations, probably due to the formation of nonproductive substrate complexes. Removal of the acetyl groups probably prevents this effect resulting in a higher catalytic efficiency.