Pieter de Waard

Structural analysis of acetylated hemicellulose polysaccharides from fibre flax (Linum usitatissimum L.)

Hemicellulose polysaccharides were sequentially extracted from defatted and depectinized flax bast fibres with Me2SO (20 °C), hot water (100 °C), aq 5% NaOH (20 °C), and aq 17.5% NaOH (20 °C). The ethanol-precipitated Me2SO extract was fractionated by size-exclusion chromatography, and the two main fractions (fraction II and III), covering 85% of the total, were analyzed by HPLC for constituting monosaccharides and acetyl content, and by NMR spectroscopy (1H, 13C and 2D-heteronuclear proton detected multiple-bond coherence (HMBC)). Fraction II contained a linear β-(1 → 4)-linked xylan, O-acetylated on C-2 and C-3 with an acetylation degree of 0.5. Fraction III was enriched in Man and Glc residues. NMR spectral data gave evidence for the presence of a linear β-(1 → 4)-linked glucomannan, O-acetylated on C-2 and C-3 on Man with an acetylation degree of 0.5 (based on Man). HPLC analysis and 1H and 13C NMR spectral data showed that the ethanol-precipitated hot-water extract consists of an O-acetylated rhamnogalacturonan I (RG-I) type structure. HPLC analysis of neutral monosaccharides indicates that the alkaline extracts are composed of glucomannan, xylan, and RG-I, having similar structures as those found in the Me2SO and hot-water extracts.

Branched arabino-oligosaccharides isolated from sugar beet arabinan.

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.

Primary structure of the major O-glycosidically linked carbohydrate unit of human Von Willebrand Factor

A reduced tetrasaccharide chain was obtained from human von Willebrand factor (vWF) by mild alkaline borohydride treatment. The purification of thisO-glycosidically-linked oligosaccharide was achieved by serial affinity chromatography on immobilized concanavalin A andLens culinaris agglutinin and finally gel filtration. Its structure was determined by a combination of methylation studies and 500 MHz1H-NMR spectroscopy to be: NeuAc(α2-3)Gal(β1-3)[NeuAc(α2-6)]GalNAc-ol.

Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.

Methylobacterium sp. isolated from a Finnish paper machine produces highly pyruvated galactan exopolysaccharide

The slime-forming bacterium Methylobacterium sp. was isolated from a Finnish paper machine and its exopolysaccharide (EPS) was produced on laboratory scale. Sugar compositional analysis revealed a 100% galactan (EPS). However, FT-IR showed a very strong peak at 1611 cm(-1) showing the presence of pyruvate. Analysis of the pyruvate content revealed that, based on the sugar composition, the EPS consists of a trisaccharide repeating unit consisting of D-galactopyranose and [4,6-O-(1-carboxyethylidene)]-D-galactopyranose with a molar ratio of 1:2, respectively. Both linkage analysis and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy revealed the following repeating unit: -->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1-->. By enrichment cultures from various ground and compost heap samples a polysaccharide-degrading culture was obtained that produced an endo acting enzyme able to degrade the EPS described. The enzyme hydrolysed the EPS to a large extent, releasing oligomers that mainly consisted out of two repeating units.

Enzyme resistant feruloylated xylooligomer analogues from thermochemically treated corn fiber contain large side chains, ethyl glycosides and novel sites of acetylation

In order to use corn fiber as a source for bioethanol production the enzymatic hydrolysis of the complex glucuronoarabinoxylans present has to be improved. Several oligosaccharides present in the supernatant of mild acid pretreated and enzymatically saccharified corn fiber that resist the current available enzymes were (semi)purified for structural analysis by NMR or ESI-MS(n). The structural features of 21 recalcitrant oligosaccharides are presented. A common feature of almost all these oligosaccharides is that they contain (part of) an α-l-galactopyranosyl-(1→2)-β-d-xylopyranosyl-(1→2)-5-O-trans-feruloyl-l-arabinofuranose side chain attached to the O-3 position of the β-1-4 linked xylose backbone. Several of the identified oligosaccharides contained an ethyl group at the reducing end hypothesized to be formed during SSF. The ethyl glycosides found are far more complex than previously described structures. A new feature present in more than half of the oligosaccharides is an acetyl group attached to the O-2 position of the same xylose to which the oligomeric side chain was attached to the O-3 position. Finding enzymes attacking these large side chains and the dense substituted xylan backbone will boost the hydrolysis of corn fiber glucuronoxylan.

NMR spectroscopy of nystose

Sucrose (11, 1-kestose (21, and nystose (3) are the smallest inulin-related oligosaccharides1T2, which consist of (2 -+ l)-linked /3-D-fructofuranosyl residues, terminated by an a-D-glucopyranosyl unit in a (1 + 2) linkage. Several ‘H and 13C NMR assignments and some J,,, and J,,, values for inulin and inulin-related oligosaccharides have been published3-17. Only a partial interpretation of the spectrum of 3 has been reported16 and a complete assignment of the ‘H NMR spectrum is now presented. For purposes of comparison, NMR data on 1 have been included. The signal of Glc H-l of 3 can be identified easily, and the signals for H-2/5 can then be characterised from the 2D DQF-COSY spectrum and the 2D HOHAHA spectrum with a short mixing time (see Experimental). In the 2D HOHAHA spectrum with a long mixing time, cross-peaks can be found between H-6a,6b and H-4 of Glc, thus completing the assignment of the Glc resonances. In a similar way, the resonances of H-3/6 of the Fru units can be identified, making use of the doublet for H-3. The remaining ‘H signals are due to the isolated Fru H-la,lb spin systems. In the shift-correlation spectra, two coupled doublets for each of those CH, groups can be found. The Glc and Fru spin systems are easily distinguishable. However, the individual Fru units could not be distinguished by ‘H NMR spectroscopy. The complete ‘H assignments were obtained by application of 2D HMBC spectroscopy. Signals for Fru C-2 can be recognised by their downfield positions. The C-2 resonance at 104.54 ppm has cross-peaks in the 2D HMBC spectrum (Fig. 1) with the Glc H-l (5.431 ppm) and the Fru H-la,lb resonances (3.741 and 3.840 ppm, respectively). Therefore, these C-2 and H-la,lb resonances belong to Fru-1 (the Fru residue attached to Glc is designated Fru-1). The Fru C-2 resonance at 104.98 ppm has

Level and position of substituents in cross-linked and hydroxypropylated sweet potato starches using nuclear magnetic resonance spectroscopy.

Sweet potato starch was cross-linked using sodium trimetaphosphate and hydroxypropylated using propylene oxide. The level and position of phosphorus and hydroxypropyl groups within cross-linked and hydroxypropylated sweet potato starch was investigated by phosphorus and proton nuclear magnetic resonance spectroscopy ((31)P, (1)H NMR). The cross-linking reaction produced monostarch monophosphate and distarch monophosphate in a molar ratio of 1:1.03, indicating that only half of the introduced phosphorus resulted in a possible cross-link. One cross-link per approximately 2900 glucose residues was found. Phosphorylation leading to monostarch monophosphate mainly occurred at O-3 and O-6 (ratio 1:1). It was inferred that the majority of the cross-links formed in distarch monophosphate were between two glucose residues positioned in different starch chains, while a minor part of the cross-links may be formed between two glucose residues within the same starch chain. Hydroxypropylation under alkaline conditions resulted in the formation of intra-molecular phosphorus cross-links, subsequent hydroxypropylation following cross-linking lowered both the level of intra- and inter-molecular cross-linking. Using (1)H NMR the molar substitution of hydroxypropylation was determined to be 0.155-0.165. The hydroxypropylation predominantly occurred at O-2 (61%), and the level of substitution at O-6 (21%) was slightly higher than that at O-3 (17%). In dual modified starch, the preceding cross-linking procedure resulted in a slightly lower level of hydroxypropylation, where the substitution at O-6 decreased more compared to the substitution at O-2 and O-3.

Mannosyl-β(1-4)-N-acetylglucosaminyl-β(1-N)-urea, a compound isolated from the urine of patients with β-mannosidosis

A previously unknown substance, mannosyl-β(1–4)-N-acetylglucosaminyl-β(1-N)-urea, has been isolated from the urine of patients with β-mannosidosis in addition to the main metabolite mannosyl-β(1–4)-N-acetylglucosamine. Structural investigation was carried out by fast atom bombardment mass spectrometry and high-resolution1H-nuclear magnetic resonance spectroscopy at 500 MHz. It was postulated that the occurrence of this carbohydrate-urea conjugate in urine results mainly from urine handling.