Characterization of O-acetyl-(4-O-methylglucurono)xylan isolated from birch and beech

The structures of water-soluble birch and beech xylans, extracted from holocellulose using dimethyl sulfoxide, were determined employing 1H and 13C NMR spectroscopy together with chemical analysis. These polysaccharides were found to be O-acetyl-(4-O-methylglucurono)xylans containing one 4-O-methylglucuronic acid substituent for approximately every 15 D-xylose residues. The average degree of acetylation of the xylose residues in these polymers was 0.4. The presence of the structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1--> was demonstrated. Additional acetyl groups were present as substituents at C-2 and/or C-3 of the xylopyranosyl residues. Utilizing size-exclusion chromatography in combination with mass spectroscopy, the weight-average molar masses (and polydispersities) were shown to be 8000 (1.09) and 11,100 (1.08) for birch and beech xylan, respectively.     

NMR structural determination of dissolved O-acetylated galactoglucomannan isolated from spruce thermomechanical pulp

Water-soluble O-acetylated galactoglucomannan (GGM) isolated from spruce thermomechanical pulp (TMP) by hot-water extraction was characterized by 1D and 2D (homo- and heteronuclear) NMR analysis. The backbone was found to consist of (1-->4)-linked mannopyranosyl and glucopyranosyl units in a ratio of 10:1.9-2.6. The mannopyranosyl units were acetylated at C-2 and C-3 with a degree of acetylation around 0.28-0.37 as determined by NMR. A slightly larger amount of 2-O-acetylated mannopyranosyl was detected when compared to the 3-O-acetylated component. Approximately every 10th mannopyranosyl unit was substituted at C-6 by a single alpha-galactopyranosyl unit. Fine structure determination based on sequence-specific chemical shift variations showed that the distribution of glycosyl residues is random. Small amounts of other minor polysaccharide species including xylans and galactans could also be identified by NMR.     

Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill

A heteroxylan was isolated from Eucalyptus globulus wood by extraction of peracetic acid delignified holocellulose with dimethyl sulfoxide. Besides (1-->4)-linked beta-D-xylopyranosyl units of the backbone and short side chains of terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl residues (MeGlcA) in a 1:10 molar ratio, this hemicellulose contained galactosyl and glucosyl units attached at O-2 of MeGlcA originating from rhamnoarabinogalactan and glucan backbones, respectively. About 30% of MeGlcA units were branched at O-2. The O-acetyl-(4-O-methylglucurono)xylan showed an acetylation degree of 0.61, as determined by 1H NMR spectroscopy, and a weight-average molecular weight (M(w)) of about 36 kDa (P=1.05) as revealed from size-exclusion chromatography (SEC) analysis. About half of the beta-D-xylopyranosyl units of the backbone were found as acetylated moieties at O-3 (34 mol%), O-2 (15 mol%) or O-2,3 (6 mol%). Practically, all beta-D-xylopyranosyl units linked at O-2 with MeGlcA residues were 3-O-acetylated (10 mol%).     

Structural characterization of the acetylated heteroxylan from the natural hybrid Paulownia elongata/Paulownia fortunei

The heteroxylan from the hybrid Paulownia elongata/Paulownia fortunei is an O-acetyl-(4-O-methylglucurono)xylan with an acetylation degree (DS) of 0.59 and a molecular weight (M(w)) of 29 kDa. The heteroxylan backbone is composed by (1-->4)-linked beta-d-xylopyranosyl units (Xylp) partially ramified with terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl (MeGlcpA) and a small proportion of alpha-D-glucuronosyl (GlcpA) residues in a molar ratio of Xylp:(MeGlcpA+GlcpA) of 20:1. Roughly half of the beta-D-xylopyranosyl units in the backbone are acetylated: 3-O-acetylated (22 mol %), 2-O-acetylated (23 mol %) or 2,3-di-O-acetylated (7 mol %). ESI-MS and MALDI-MS studies of partially hydrolyzed heteroxylan revealed a random distribution of O-Ac and MeGlcpA within the backbone. However, the frequency of substitution with O-Ac along the backbone is not uniform and the molecular regions that did not contain MeGlcpA substituents possessed an acetylation degree significantly lower than the average DS of the xylan.     

Structural studies of the mix-linked beta-(1-->3)/beta-(1-->4)-D-xylans from the cell wall of Palmaria palmata (Rhodophyta)

The structure and organization of Palmaria palmata cell walls, which are largely involved in biological and physiological functions as well as in biotechnological and food applications of this red marine alga, are principally assumed by the interactions and linkages of major mix-linked beta-(1-->3)/beta-(1-->4)-D-xylans. These partly acidic polysaccharides are essentially held in the cell wall by H-bonds. The location of the acid groups and the distribution of 1-->3-linkage were studied following the endo-beta-(1,4)-xylanase hydrolysis of sequentially extracted xylans, and fine analysis of the oligosaccharides produced by anion exchange chromatography, high performance anion exchange chromatography (HPAEC)-PAD, nuclear magnetic resonance (NMR) and electrospray ion trap mass spectrometry (ESI-MS) techniques. The results indicate that the acidity of the xylans was related to potential linkages to sulfated and/or phosphorylated xylogalactoprotein complexes. H-bonding of the mix-linked xylans involved a regular 1,3-linkages distribution idealized in a pentameric repeating structure (one 1,3-linkage and four 1,4-linkages). Furthermore, MS analysis of the xylo-oligosaccharides revealed a substitution of the mix-linked xylans by a non-osidic component of 175 g mol(-1). The presence of this substituent and of the proposed covalent linkage between the mix-linked xylans and charged glycoproteins are discussed with regard to the polysaccharides interactions in P. palmata cell walls.     

Isolation and characterization of xylans from seed pericarp of Argania spinosa fruit

Hemicellulose-type polysaccharides were isolated from the pericarp of seeds of Argania spinosa (L.) Skeels fruit by sequential alkaline extractions and fractionated by precipitation. Water soluble and water insoluble fractions were obtained, purified and characterized by sugar analysis and 1H and 13C NMR spectroscopy. The water soluble fractions were assumed to be (4-O-methyl-D-glucurono)-D-xylans, with 4-O-methyl-D-glucopyranosyluronic acid groups linked to C-2 of a (1-->4)-beta-D-xylan. The 1H NMR spectrum showed that the water soluble xylans have, on average, one non-reducing terminal residue of 4-O-methyl-D-glucuronic acid for every seven xylose units. The water insoluble fractions consisted of a neutral xylan with linear (1-->4)-beta-D-xylopyranosyl units.     

Water stress and cell wall polysaccharides in the apical root zone of wheat cultivars varying in drought tolerance

Glycosyl composition and linkage analysis of cell wall polysaccharides were examined in apical root zones excised from water-stressed and unstressed wheat seedlings (Triticum durum Desf.) cv. Capeiti ("drought-tolerant") and cv. Creso ("drought sensitive"). Wall polysaccharides were sequentially solubilized to obtain three fractions: CDTA+Na(2)CO(3) extract, KOH extract and the insoluble residue (alpha-cellulose). A comparison between the two genotypes showed only small variations in the percentages of matrix polysaccharides (CDTA+Na(2)CO(3) plus KOH extract) and of the insoluble residues (alpha-cellulose) in water-stressed and unstressed conditions. Xylosyl, glucosyl and arabinosyl residues represented more than 90mol% of the matrix polysaccharides. The linkage analysis of matrix polysaccharides showed high levels of xyloglucans (23-39mol%), and arabinoxylans (38-48mol%) and a low amount of pectins and (1-->3), (1-->4)-beta-d-glucans. The high level of xyloglucans was supported by the release of the diagnostic disaccharide isoprimeverose after Driselase digestion of KOH-extracted polysaccharides. In the "drought-tolerant" cv. Capeiti the mol% of side chains of rhamnogalacturonan I and II significantly increased in response to water stress, whereas in cv. Creso, this increase did not occur. The results support a role of the pectic side chains during water stress response in a drought-tolerant wheat cultivar.     

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]     

Arabinosyl and glucosyl residues as structural features of acetylated galactomannans from green and roasted coffee infusions

A good yield mild fractionation procedure was developed for the purification of mannans from green and roasted coffee infusions that included anion-exchange chromatography and phenylboronic acid immobilized Sepharose chromatography of the dialyzed and ethanol precipitated material. Enzymatic hydrolysis with endo-beta-mannanase and ESIMS allowed finding that the mannans from roasted coffee infusions, as well as those from green coffee, are acetylated (8 mol% and 11 mol%, respectively). Fragmentation pattern obtained by ESIMS/MS analysis of the acetylated oligosaccharide ions indicates that the acetylation also occurs at O-2 of the mannose residues. Doubly acetylated and contiguously acetylated hexose residues were also found. Arabinose residues, as side chains, were also found as structural features of hot water soluble green (2%) and roasted (<0.9 mol) coffee galactomannans. Methylation analysis, hydrolysis with specific glycosidases and GC-EIMS analysis of the reduced and methylated oligosaccharides allowed to conclude that beta-(1-->4)-linked glucose residues are also structural features of green and roasted coffee galactomannans (6 and 1 mol%, respectively). In hot water soluble green coffee mannans, glucose residues are a constituent of the mannan backbone, and in the roasted coffee they were detected only at the reducing end of the mannan backbone.     

Cell wall changes in immature Asparagus stem tissue after excision.

Cell wall material (CWM) was prepared from sections of fresh and aerobically-stored asparagus (Asparagus officinalis, L. cv. Connovor Collossus) stems. Polymers were solubilized from the CWM by successive extraction with cyclohexane-trans-1,2-diamine-N N N' N'-tetraacetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue which contained a significant amount of cross-linked pectic polysaccharides. The polymers were fractionated by anion-exchange chromatography and selected fractions were subjected to methylation analysis. The storage-related decrease in (1-4)-linked Galp was detected in all the fractions rich in pectic polysaccharides, particularly in the CDTA, Na2CO3, 0.5 M KOH fractions and alpha-cellulose residue. A smaller decrease in Araf was also observed. This was mainly due to a decrease in (1-5)-linked Araf in the Na2CO3-1-soluble polymers, and terminal Araf in the alpha-cellulose residue. There was evidence for the occurrence of significant amounts of complexes containing pectic polysaccharides and xylans having a relatively low degree of polymerization in the dilute alkali-soluble polymers, and some of these contained phenolic compounds; the storage-induced increase in (1-4)-linked Xylp was confined to these polymers. Interestingly, no free acidic xylans could be detected in the 1 M and 4 M KOH-soluble polymers; instead, the bulk of the hemicellulosic polysaccharides appeared to be mixtures of xyloglucans and xylans in which the ratio of xyloglucan to xylan increased with increasing strength of alkali used for extraction of the polymers. The non-degradative extraction and fractionation procedures revealed heterogeneity in pectic polysaccharides, pectic polysaccharide-xylan complexes and xyloglucans in close association with xylans. The possible relationship between pectic polysaccharide-xylan-phenolic complexes and the onset of lignification in maturing tissues is discussed.     

Purification, structure and immunobiological activity of an arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds

The structure and bioactivity of a polysaccharide extracted and purified from a 4M KOH + H3BO3 solution from Prunus dulcis seed cell wall material was studied. Anion-exchange chromatography of the crude extract yielded two sugar-rich fractions: one neutral (A), the other acidic (E). These fractions contain a very similar monosaccharide composition: 5:2:1 for arabinose, uronic acids and xylose, respectively, rhamnose and galactose being present in smaller amounts. As estimated by size-exclusion chromatography, the acidic fraction had an apparent molecular mass of 762 kDa. Methylation analysis (from the crude and fractions A and E), suggests that the polysaccharide is an arabinan-rich pectin. In all cases, the polysaccharides bear the same type of structural Ara moieties with highly branched arabinan-rich pectic polysaccharides. The average relative proportions of the arabinosyl linkages is 3:2:1:1 for T-Araf:(1-->5)-Araf:(1-->3,5)-Araf:(1-->2,3,5)-Araf. The crude polysaccharide extract and fractions A and E induced a murine lymphocyte stimulatory effect, as evaluated by the in vitro and in vivo expression of lymphocyte activation markers and spleen mononuclear cells culture proliferation. The lymphocyte stimulatory effect was stronger on B- than on T-cells. No evidence of cytotoxic effects induced by the polysaccharide fractions was found.     

Studies on the structure of a lithium-treated soybean pectin: characteristics of the fragments and determination of the carbohydrate substituents of galacturonic acid

Two galacturonic-acid-containing polysaccharide fractions (ChSS and P) were isolated from soybean meal and subjected to lithium treatment. The fragments obtained were analyzed by using monosaccharide and methylation analyses, and NMR spectroscopy. Lithium degradation of ChSS, followed by sodium borodeuteride reduction, hydrolysis, sodium borohydride reduction, and acetylation afforded alditol acetates, of which the labeled ones reflected residues linked to GalA. As followed from quantifications of the labeled and non-labeled alditols from each constituent monosaccharide by GLC-EIMS, 6 mol% of Ara, 22 mol% of Fuc, 13 mol% of Gal, 53 mol% of Rha, and 57 mol% of Xyl are glycosidically linked to GalA. Analysis of the lithium-treated polymer revealed that it contains arabinogalactan side chains linked to Rha O-4, which consist of a beta-(1 --> 4)-linked galactan substituted with highly branched arabinan chains. On average, an arabinogalactan chain contains up to 29 Gal and 25 Ara residues. Surface plasmon resonance was used to determine conditions for affinity chromatography. Furthermore, this technique confirmed the presence of terminal alpha-Fuc residues in ChSS. Polysaccharide P turned out to be relatively resistant to lithium degradation.     

13C-n.m.r. spectra of xylo-oligosaccharides and their application to the elucidation of xylan structures

¹³C-N.m.r. spectra of thirteen xylo-oligosaccharides [a complete series of α- and β-d-xylopyranosyl derivatives of methyl α-d-xylopyranoside, β-d-xylopyranosyl derivatives of methyl 4-O-β-d-xylopyranosyl-d-xylopyranoside, methyl O-α-d-xylopyranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranoside, and a branched methyl β-xylotetraoside] have been interpreted. The data obtained have been used for the carbon signal assignment in the spectra of a number of red-algal xylans. ¹³C-N.m.r. spectroscopy is shown to be a rapid and convenient method for the structural analysis of xylose-rich polysaccharides.     

Isolation and structure of D-xylans from pericarp seeds of Opuntia ficus-indica prickly pear fruits

Xylans were isolated from the pericarp of prickly pear seeds of Opuntia ficus-indica (OFI) by alkaline extraction, fractionated by precipitation and purified. Six fractions were obtained and characterized by sugar analysis and NMR spectroscopy. They were assumed to be (4-O-methyl-D-glucurono)-D-xylans, with 4-O-alpha-D-glucopyranosyluronic acid groups linked at C-2 of a (1-->4)-beta-D-xylan. The sugar composition and the 1H and 13C NMR spectra showed that their chemical structures were very similar, but with different proportions of D-Xyl and 4-O-Me-D-GlcA. Our results showed that, on average, the water soluble xylans have one nonreducing terminal residue of 4-O-methyl-D-glucuronic acid for every 11 to 14 xylose units, whereas in the water non-soluble xylans, xylose units can varied from 18 to 65 residues for one nonreducing terminal residue of 4-O-methyl-D-glucuronic acid.     

An alternative approach for the synthesis of fluorogenic substrates of endo-beta-(1-->4)-xylanases and some applications

Fluorogenic substrates of endo-beta-(1-->4)-xylanases (EXs), 4-methylumbelliferyl beta-glycosides of xylobiose and xylotriose were synthesized from fully acetylated oligosaccharides using the alpha-trichloroacetimidate procedure. A commercially available syrup containing xylose and xylo-oligosaccharides was used as the starting material. Both fluorogenic glycosides were found to be suitable substrates for EXs, particularly for sensitive detection of the enzymes in electrophoretic gels and their in situ localization on sections of fruiting bodies of some plants, such as tomato, potato and eggplant, all of the family Solanaceae.     

NMR spectroscopy and chemical studies of an arabinan-rich system from the endosperm of the seed of Gleditsia triacanthos

Exhaustive extraction of the endosperm from the seed of Gleditsia triacanthos using water at room temperature and 50 degrees C left a residue, which was further extracted at 95 degrees C. Precipitation of this extract with 2-propanol yielded major amounts of galactomannan components, while the supernatant was mainly composed of arabinose-rich constituents. Two fractions were obtained by anion-exchange chromatography. The fraction that eluted with water is an arabinan with (1-->5) alpha-L linkages and branching mainly on C-2, accompanied with equal amounts of a low-galactose galactomannan oligosaccharide, and a small proportion of a beta-(1-->4)-galactan. The fraction eluted with an increased ionic strength consists mainly of a similar arabinan, and lower proportions of a high-galactose galactomannan, galactan, and protein. The arabinan moiety in both fractions was characterized by chemical analysis and 1D and 2D NMR spectroscopic techniques.     

X4 modules represent a new family of carbohydrate-binding modules that display novel properties

The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell wall-degrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydrate-binding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.     

Preparation of Acetylated Grapefruit Peel Polysaccharide

Grapefruit peel polysaccharide has antioxidant, antitumor, hypoglycemic and other biological activities, and chemical modification can further improve the properties of the polysaccharide. Acetylation modification of polysaccharides has the advantages of simple operation, low cost and little pollution, and is widely used at present. Different degrees of acetylation modification have different effects on the properties of polysaccharides, so it is necessary to optimize the preparation technology of acetylated grapefruit peel polysaccharides. In this article, acetylated grapefruit peel polysaccharide was prepared by acetic anhydride method. With the degree of acetyl substitution as the evaluation index, combined with the analysis of sugar content and protein content in the polysaccharide before and after modification, the effects of three feeding ratios of 1:0.6, 1 : 1.2 and 1 : 1.8 (polysaccharide: acetic anhydride, mass/volume) on acetylation modification were explored through single factor experiments. The results showed that the optimum ratio of material to liquid for acetylation modification of grapefruit peel polysaccharide was 1:0.6. Under these conditions, the degree of substitution of acetylated grapefruit peel polysaccharide was 0.323, the sugar content was 59.50 % and the protein content was 1.038 %. The results provide some reference for the study of acetylated grapefruit peel polysaccharide.     

Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans

Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated xylo-oligosaccharides and complex non-soluble acetylglucuronoxylan. Both enzymes performed optimally at pH 7.0 and 40 °C.Axe2 has a clear preference for acetylated xylo-oligosaccharides (AcXOS) with a high degree of substitution and Axe3 does not show such preference. Axe3 has a preference for large AcXOS (DP 9-12) when compared to smaller AcXOS (especially DP 4-7) while for Axe2 the size of the oligomer is irrelevant. Even though there is difference in substrate affinity towards acetylated xylooligosaccharides from Eucalyptus wood, the final hydrolysis products are the same for Axe2 and Axe3: xylo-oligosaccharides containing one acetyl group located at the non-reducing xylose residue remain as examined using MALDI-TOF MS, CE-LIF and the application of an endo-xylanase (GH 10).     

Preparation and Antioxidant Activity of Acetylated Mung Bean Peel Polysaccharides

Mung bean peel polysaccharides are one of the main active components in mung bean peel. Acetylated mung bean peel polysaccharides were prepared by extracting and acetylating them, and characterized by infrared and ultraviolet methods to preliminarily understand the structural characteristics and activity of acetylated mung bean peel polysaccharides. Acetylation modification can improve the structure of polysaccharides, thereby causing changes in their properties. The product obtained after acetylation modification exhibited new characteristic absorption peaks at 1732 cm⁻¹, and the scavenging ability of hydroxyl radicals was improved. Therefore, acetylation modification of mung bean peel polysaccharides could enhance the activity by improving the structure, which provided an experimental basis for the application of mung bean peel polysaccharides.