Comparison of milk oligosaccharides pattern in colostrum of different horse breeds.

Colostrum oligosaccharides are known to exhibit prebiotic and immunomodulatory properties. Oligosaccharide composition is species-specific, and equine colostrum has been reported to contain unique oligosaccharides. Therefore, equine oligosaccharides (EMOS) from colostrum from different horse breeds were analyzed by CE-LIF, CE-MS(n), HILIC-MS(n), and exoglycosidase degradation. Sixteen EMOS were characterized and quantified, of which half were neutral and half were acidic. EMOS showed about 63% structural overlap with human milk oligosaccharides, known for their bioactivity. Seven EMOS were not reported before in equine oligosaccharides literature: neutral Gal(ß1-4)HexNAc, Gal(ß1-4)Hex-Hex, ß4'-galactosyllactose, and lactose-N-hexaose, as well as acidic 6'-Sialyl-Hex-Ac-HexNAc, sialyllacto-N-tetraose-a, and disialylacto-N-tetraose (isomer not further specified). In all colostrum samples, the average oligosaccharide concentration ranged from 2.12 to 4.63 g/L; with ß 6'and 3'- galactosyllactose, 3'-sialyllactose, and disialyllactose as the most abundant of all oligosaccharides (27-59, 16-37, 1-8, and 1-6%, respectively). Differences in presence and in abundance of specific EMOS were evident not only between the four breeds but also within the breed.

Two-step enzymatic fingerprinting of sugar beet pectin.

A two-step enzymatic fingerprinting method was introduced to analyze a highly methylesterified and acetylated sugar beet pectin having a degree of methylesterification (DM) of 62 and acetylation of 30. A cocktail of pectolytic enzymes, including endo-polygalacturonase II (endo-PGII) and pectin lyase (PL), was used for the first digestion. The endo-PGII and PL resistant pectin fragments were isolated and subjected to a second digestion using fungal pectin methylesterase and endo-PGII. After the two sequential digestions, 78% of the total GalA residues present in the parental pectin were recovered as mono- and oligomers, which were used to quantitatively describe the parental SBP. For this reason, the descriptive parameters degree of blockiness (DBabs), degree of hydrolysis by PG (DHPG) and degree of hydrolysis by PL (DHPL) were established for both digestions. The first digestion revealed the presence of short blocks of nonesterified GalA residues and blocks of partly methylesterified and acetylated GalA residues in the parental SBP, in addition to blocks of highly methylesterified and acetylated GalA residues. The second digestion revealed the presence of blocks of methylesterified, partly methylesterified and/or acetylated GalA residues in a sequence not to be degradable by neither endo-PGII nor by PL. The acetyl groups were present in an blockwise manner. Application of the method to two differently prepared DM 50 SBPs showed that the two pectins differ in the ratio of blocks of nonesterified and blocks of partly methylesterified and acetylated GalA residues.

A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3.

A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7 kDa and shows optimal activity at pH 8.0 and 50°C. It is stable in the range pH 5.0-7.0 and below 50°C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, (1)H NMR of polymer and LC-HILIC-MS(n) after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties.

Descriptive parameters for revealing substitution patterns of sugar beet pectins using pectolytic enzymes.

Enzymatic fingerprinting was applied to sugar beet pectins (SBPs) modified by either plant or fungal pectin methyl esterases and alkali catalyzed de-esterification to reveal the ester distributions over the pectin backbone. A simultaneous pectin lyase (PL) treatment to the commonly used endo-polygalacturonase (endo-PG) degradation showed to be effective in degrading both high and low methylesterified and/or acetylated homogalaturonan regions of SBP simultaneously. Using LC-HILIC-MS/ELSD, we studied in detail all the diagnostic oligomers present, enabling us to discriminate between differently prepared sugar beet pectins having various levels of methylesterification and acetylation. Furthermore, distinction between commercially extracted and de-esterified sugar beet pectin having different patterns of substitution was achieved by using novel descriptive pectin parameters. In addition to DBabs approach for nonmethylesterified sequences degradable by endo-PG, the "degree of hydrolysis" (DHPG) representing all partially saturated methylesterified and/or acetylated galacturonic acid (GalA) moieties was introduced as a new parameter. Consequently, the description DHPL has been introduced to quantify all esterified unsaturated GalA oligomers.

Enzymatic saccharification of sugar beet pulp for the production of galacturonic acid and arabinose; a study on the impact of the formation of recalcitrant oligosaccharides.

Enzymatic saccharification of sugar beet pulp was optimized on kg-scale to release the maximum amounts of monomeric galacturonic acid and arabinose with limited concomitant degradation of cellulose, using conditions that are feasible for industrial upscaling. A selected mixture of pectinases released 79% of the galacturonic acid and 82% of the arabinose as monomers from sugar beet pulp while simultaneously degrading only 17% of the cellulose. The recalcitrant structures that were obtained after hydrolysis were characterized using mass spectrometry. The most abundant structures had an average degree of polymerization of 4-5. They were identified as partially acetylated rhamnogalacturonan-oligosaccharides, mostly containing a terminal galacturonosyl residue on both reducing and non-reducing end, partially methyl esterified/acetylated homogalacturonan-oligosaccharides, mostly containing methyl and acetyl esters at contiguous galacturonosyl residues and arabinan-oligosaccharides, hypothesized to be mainly branched. It could be concluded that especially rhamnogalacturonan-galacturonohydrolase, arabinofuranosidase and pectin acetylesterase are lacking for further degradation of recalcitrant oligosaccharides.

Combined HILIC-ELSD/ESI-MS(n) enables the separation, identification and quantification of sugar beet pectin derived oligomers.

The combined action of endo-polygalacturonase (endo-PGII), pectin lyase (PL), pectin methyl esterase (fungal PME) and RG-I degrading enzymes enabled the extended degradation of methylesterified and acetylated sugar beet pectins (SBPs). The released oligomers were separated, identified and quantified using hydrophilic interaction liquid chromatography (HILIC) with online electrospray ionization ion trap mass spectrometry (ESI-IT-MS(n)) and evaporative light scattering detection (ELSD). By MS(n), the structures of galacturonic acid (GalA) oligomers having an acetyl group in the O-2 and/or O-3 positions eluting from the HILIC column were elucidated. The presence of methylesterified and/or acetylated galacturonic acid units within an oligomer reduced the interaction with the HILIC column significantly compared to the unsubstituted GalA oligomers. The HILIC column enables a good separation of most oligomers present in the digest. The use of ELSD to quantify oligogalacturonides was validated using pure GalA standards and the signal was found to be independent of the chemical structure of the oligomer being detected. The combination of chromatographic and enzymatic strategies enables to distinguish SBPs having different methylesters and acetyl group distribution.

The ferulic acid esterases of Chrysosporium lucknowense C1: purification, characterization and their potential application in biorefinery.

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6.

Characterizing plant cell wall derived oligosaccharides using hydrophilic interaction chromatography with mass spectrometry detection.

Analysis of complex mixtures of plant cell wall derived oligosaccharides is still challenging and multiple analytical techniques are often required for separation and characterization of these mixtures. In this work it is demonstrated that hydrophilic interaction chromatography coupled with evaporative light scattering and mass spectrometry detection (HILIC-ELSD-MS(n)) is a valuable tool for identification of a wide range of neutral and acidic cell wall derived oligosaccharides. The separation potential for acidic oligosaccharides observed with HILIC is much better compared to other existing techniques, like capillary electrophoresis, reversed phase and porous-graphitized carbon chromatography. Important structural information, such as presence of methyl esters and acetyl groups, is retained during analysis. Separation of acidic oligosaccharides with equal charge yet with different degrees of polymerization can be obtained. The efficient coupling of HILIC with ELSD and MS(n)-detection enables characterization and quantification of many different oligosaccharide structures present in complex mixtures. This makes HILIC-ELSD-MS(n) a versatile and powerful additional technique in plant cell wall analysis.

Chrysosporium lucknowense arabinohydrolases effectively degrade sugar beet arabinan.

The filamentous fungus Chrysosporium lucknowense (C1) is a rich source of cell wall degrading enzymes. In the present paper four arabinose releasing enzymes from C1 were characterized, among them one endoarabinanase, two arabinofuranosidases and one exoarabinanase. Combinations of these enzymes released up to 80% of the arabinose present in sugar beet arabinan to fermentable monosugars. Besides the main product arabinobiose, unknown arabinose oligomers are produced from highly branched arabinan when endoarabinanase was combined with exoarabinanase and/or arabinofuranosidase. All described arabinose releasing enzymes are temperature stable up to 50 degrees C and have a broad pH stability. This makes C1 arabinohydrolases suitable for many biotechnical applications, like co-fermentation bioethanol production.

Effects of pH and heat treatments on the structure and solubility of potato proteins in different preparations.

The soluble potato proteins are mainly composed of patatin and protease inhibitors. Using DSC and both far-UV and near-UV CD spectroscopy, it was shown that potato proteins unfold between 55 and 75 degrees C. Increasing the ionic strength from 15 to 200 mM generally caused an increase in denaturation temperature. It was concluded that either the dimeric protein patatin unfolds in its monomeric state or its monomers are loosely associated and unfold independently. Thermal unfolding of the protease inhibitors was correlated with a decrease in protease inhibitor activities and resulted in an ionic strength dependent loss of protein solubility. Potato proteins were soluble at neutral and strongly acidic pH values. The tertiary structure of patatin was irreversibly altered by precipitation at pH 5. At mildly acidic pH the overall potato protein solubility was dependent on ionic strength and the presence of unfolded patatin.

Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana.

Protease inhibitors from potato juice of cv. Elkana were purified and quantified. The protease inhibitors represent ca. 50% of the total soluble proteins in potato juice. The protease inhibitors were classified into seven different families: potato inhibitor I (PI-1), potato inhibitor II (PI-2), potato cysteine protease inhibitor (PCPI), potato aspartate protease inhibitor (PAPI), potato Kunitz-type protease inhibitor (PKPI), potato carboxypeptidase inhibitor (PCI), and "other serine protease inhibitors". The most abundant families were the PI-2 and PCPI families, representing 22 and 12% of all proteins in potato juice, respectively. Potato protease inhibitors show a broad spectrum of enzyme inhibition. All the families (except PCI) inhibited trypsin and/or chymotrypsin. PI-2 isoforms exhibit 82 and 50% of the total trypsin and chymotrypsin inhibiting activity, respectively. A strong variation within the latter activities was shown within one family and between protease inhibitor families.

Heat-induced conformational changes of patatin, the major potato tuber protein.

This paper presents a first structural characterization of isolated patatin, the major potato tuber protein, at ambient and elevated temperatures. Isolated patatin at room temperature is a highly structured molecule at both secondary and tertiary levels. It is estimated from far-ultraviolet circular dichroism data that about 33% of the residues adopts an alpha-helical and 46% a beta-stranded structure. Patatin is thermally destabilized at temperatures exceeding 28 degrees C, as was indicated by near-ultraviolet circular dichroism. It was shown that parts of the alpha-helical contributions unfold in the 45-55 degrees C region, whereas the beta-stranded parts unfold more gradually at temperatures of 50-90 degrees C. This was confirmed with Fourier-transform infrared spectroscopy. Differential scanning calorimetry indicated a cooperative transition between 50-60 degrees C, most likely reflecting the unfolding of alpha-helical parts of the molecule. Furthermore, fluorescence spectroscopy confirmed a global unfolding of the protein between 45-55 degrees C. The observed unfolding of the protein coincides with the inactivation of the patatin enzyme activity and with the precipitation as occurs in the potato fruit juice upon heating. At high temperatures, patatin still contains some helical and stranded structures. Upon cooling the protein partly refolds, it was observed that mainly alpha-helical structures were formed.

Mode of action of (1-->4)-beta-D-arabinoxylan arabinofuranohydrolase (AXH) and alpha-L-arabinofuranosidases on alkali-extractable wheat-flour arabinoxylan.

Arabinoxylan-derived oligosaccharides were treated with (1-->4)-beta-D-arabinoxylan arabinofuranohydrolase (AXH) and two types of alpha-L-arabinofuranosidase, A and B. Analysis of reaction products by high performance anion-exchange chromatography indicated the removal of arabinofuranosyl groups from singly substituted xylopyranosyl residues. In addition, differences in the specificity of these enzymes towards the various differently substituted oligosaccharides were observed. 1H NMR spectroscopy and methylation analysis of alkali-extractable wheat-flour arabinoxylan treated with AXH confirmed the specificity of AXH towards (1-->3)-linked arabinofuranosyl groups on singly substituted xylopyranosyl residues. With these techniques, alpha-L-arabinofuranosidase B was found to cause minor changes in (1-->2)- and (1-->3)-linked arabinofuranosyl groups on doubly substituted xylopyranosyl residues.

Mode of action of the xylan-degrading enzymes from Aspergillus awamori on alkali-extractable cereal arabinoxylans.

Alkali-extractable cereal arabinoxylan and oligosaccharides of known structure derived from it by enzymic hydrolysis were treated with endo-(1-->4)-beta-D-xylanases I and III from Aspergillus awamori CMI 142717 and the digests subjected to analysis by high performance anion-exchange chromatography. Clear differences in the mode of action of the two endo-(1-->4)-beta-D-xylanases were observed. When counting from the reducing end, at least one unsubstituted xylopyranosyl residue adjacent to singly substituted xylopyranosyl residues or two unsubstituted xylopyranosyl residues adjacent to doubly substituted xylopyranosyl residues cannot be removed by endo-(1-->4)-beta-D-xylanase I. At least two unsubstituted xylopyranosyl residues adjacent to singly or doubly substituted xylopyranosyl residues cannot be removed by endo-(1-->4)-beta-D-xylanase III. beta-D-Xylosidase from the same xylanolytic system was able to remove terminal xylopyranosyl residues from the nonreducing end of branched oligosaccharides only when two contiguous unsubstituted xylopyranosyl residues were present adjacent to singly or doubly substituted xylopyranosyl residues.

Characterisation by 1H NMR spectroscopy of oligosaccharides derived from alkali-extractable wheat-flour arabinoxylan by digestion with endo-(1-->4)-beta-D-xylanase III from Aspergillus awamori.

Alkali-extractable wheat-flour arabinoxylan, treated with endo-(1-->4)-beta-D-xylanase III from Aspergillus awamori CMI 142717, was fractionated by Bio-Gel P-2 size exclusion chromatography at 60 degrees C. Column fractions, corresponding to oligosaccharides with degrees of polymerisation from 5 to 10, were collected, and subfractionated by high performance anion-exchange chromatography on CarboPac PA-1. The structures of the oligosaccharides thus obtained were elucidated by 1H NMR spectroscopy, showing chains of (1-->4)-linked beta-D-xylopyranosyl residues differently substituted at O-3 and/or O-2,3 with alpha-L-arabinofuranosyl groups. The structures were different from those obtained with endo-(1-->4)-beta-D-xylanase I of the same xylanolytic enzyme system.