Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.     

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-α-d-glucopyranosyluronic acid groups linked at C-2 of a (1→4)-β-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.     

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.     

Structural characterization of the cell wall D-glucans isolated from the mycelium of Botryosphaeria rhodina MAMB-05

Three D-glucans were isolated from the mycelium of the fungus Botryosphaeria rhodina MAMB-05 by sequential extraction with hot-water and hot aqueous KOH (2% w/v) followed by ethanol precipitation. Following their purification by gel permeation chromatography on Sepharose CL-4B, the structural characteristics of the D-glucans were determined by FT-IR and 13C NMR spectroscopy and, after methylation, by GC-MS. The hot-water extract produced a fraction designated Q1A that was a beta-(1-->6)-D-glucan with the following structure: [Formula: see text] The alkaline extract, when subjected to repeated freeze-thawing, yielded two fractions: K1P (insoluble) that comprised a beta-(1-->3)-D-glucan with beta-D-glucose branches at C-6 with the structure: [Formula: see text] and K1SA (soluble) consisting of a backbone chain of alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with alpha-D-glucopyranosyl residues: [Formula: see text]     

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of -->2)-alpha-D-Man(1--> and -->4)-beta-D-GlcA(1-->, with a minor portion of -->4)-beta-D-Gal(1--> units. The branch points were at C-3 of -->2)-Man-(1-->, C-2 of -->4)-Gal-(1--> and C-2 of -->6)-Gal-(1-->. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1-->4)-, (1-->3)- and (1-->2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1-->4)-linked. Most of the mannose units were (1-->2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1-->6)-linked. The absolute configurations of the sugar residues were alpha-D-Manp, alpha-L-Fucp, alpha-D-Xylp, beta-D-Galp and beta-D-GlcpA. Sulfate groups in F32 were at C-6 of -->2,3)-Man-(1-->, C-4 and C-6 of -->2)-Man-(1-->, C-3 of -->6)-Gal-(1-->, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.     

Structural analysis of the alpha-D-glucan (EPS180) produced by the Lactobacillus reuteri strain 180 glucansucrase GTF180 enzyme

The neutral exopolysaccharide EPS180 produced from sucrose by the glucansucrase GTF180 enzyme from Lactobacillus reuteri 180 was found to be a (1-->3,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis, periodate oxidation, and 1D/2D 1H and 13C NMR spectroscopy of the intact EPS180, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis of EPS180, a composite model, that includes all identified structural features, was formulated as follows: [Formula: see text].     

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.     

Structural characterization of a polysaccharide and a beta-glucan isolated from the edible mushroom Flammulina velutipes

Two polysaccharides were isolated from the basidiomycete Flammulina velutipes, via successive hot extraction with water, 2% and 25% aq. KOH, and then submitted to freeze-drying. The precipitate formed by repeated freeze-thawing from the 2% aq. KOH extraction PK2 was analyzed by determination of its monosaccharide composition, as well as by methylation analyses using GC-MS, mono- ((13)C, (1)H NMR) and bidimensional ((1)H (obs.), (13)C HMQC) spectroscopy, and controlled Smith degradations. It was established to be a branched beta-glucan, with a main chain of (1-->3)-linked-Glcp residues, substituted at O-6 by single-unit beta-Glcp side chains. The precipitate formed by repeated freeze-thawing from the 25% KOH extraction PK25 contained Xyl, Man, and Glc and was heterogeneous by HSPEC and extraction with DMSO gave a soluble xylomannan (XM). It was homogeneous with a molar mass 30.8 x 10(4)g/mol (dn/dc=0.186). Using the above chemical analyses, it was a xylomannan with Man and Xyl in a 3:2 molar ratio. Its main chain consisted of (1-->3)-linked alpha-Manp units, mainly substituted at O-4 by beta-Xylp units or with some beta-Xylp-(1-->3)-beta-Xylp groups.     

Structural determination of the O-antigenic polysaccharide from the Shiga toxin-producing Escherichia coli O171

The structure of the O-antigenic part of the lipopolysaccharide (LPS) obtained from the verotoxin-producing Escherichia coli O171 has been determined. (1)H and (13)C NMR spectroscopy techniques in combination with component analysis were used to elucidate the O-antigen structure of O-deacylated LPS. Subsequent NMR analysis of the native LPS revealed acetylation at O-7/O-9 of the sialic acid residue. The sequence of sugars was determined by inter-residue correlations in (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation spectra. The O-antigen is composed of pentasaccharide repeating units with one equivalent of O-acetyl groups distributed over two positions: -->4)-alpha-Neu5Ac7,9Ac-(2-->6)-beta-D-Galp-(1-->6)-beta-DGlcp-->(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1--> Based on biosynthetic considerations, this should also be the biological repeating unit.     

Purification and characterization of dermatan sulfate from the skin of the eel,Anguilla japonica

Glycosaminoglycans were isolated from the eel skin (Anguilla japonica) by actinase and endonuclease digestions, followed by a beta-elimination reaction and DEAE-Sephacel chromatography. Dermatan sulfate was the major glycosaminoglycan in the eel skin with 88% of the total uronic acid. The content of the IdoA2Salpha1-->4GalNAc4S sequence in eel skin, which shows anticoagulant activity through binding to heparin cofactor II, was two times higher than that of dermatan sulfate from porcine skin. The anti-IIa activity of eel skin dermatan sulfate was determined to be 2.4 units/mg, whereas dermatan sulfate from porcine skin shows 23.2 units/mg. The average molecular weight of dermatan sulfate was determined by gel chromatography on a TSKgel G3000SWXL column as 14 kDa. Based on 1H NMR spectroscopy, the presence of 3-sulfated and/or 2,3-sulfated IdoA residues was suggested. The reason why highly sulfated dermatan sulfate does not show anticoagulant activity is discussed. In addition to dermatan sulfate, the eel skin contained a small amount of keratan sulfate, which was identified by keratanase treatment.     

Structure of a heteroxylan of gum exudate of the palm Scheelea phalerata (uricuri)

The polysaccharide isolated from the gum exudate of palm Scheelea phalerata (SPN) was water-insoluble and composed of Fuc, Ara, Xyl, and uronic acid moieties in a 5:34:54:7 molar ratio: 12% of phenolics were also present. A soluble polysaccharide (SPNa) was obtained after alkaline treatment, which contained Fuc, Ara, Xyl and uronic acid in a 7:44:42:7 molar ratio, with only 2% phenolics. SPNa had an M(W) approximately 1.04 x 10(5) g mol(-1) and was almost monodisperse (M(W)/M(N) : 1.25 +/-0.22). It had a branched structure with side chains of 2-O-substituted Xylp (approximately 8%) and 3-O-substituted Araf (12%) units, and a large proportion of nonreducing end-units of Araf (15%), Fucp (10%), Xylp (4%), and Arap (6%). The (1 --> 4)-linked beta-Xylp main-chain units were 3-O- (9%), 2-O- (13%), and 2,3-di-O- (13%) substituted. Its (13)C NMR spectrum contained at least 9 C-1 signals, those at delta 108.6 and 107.7 arising from alpha-Araf units. Others were present at delta 175.4 from C-6 of alpha-GlcpA and delta 15.6 from C-6 of Fucp units. The main chain of SPNa was confirmed by analysis of a Smith-degraded polysaccharide (SPDS): methylation analysis provided a 2,3-Me(2)-Xyl (65%) derivative and its (13)C NMR spectrum showed five main signals typical of a (1 --> 4)-linked beta-Xylp units. Methylation analysis of a carboxy-reduced polysaccharide (SPN-CR) revealed a 2,3,4,6-Me(4)-Glc derivative (4%) arising from nonreducing end-units of GlcpA. Alpha-GlcpA-(1 --> 2)-alphabeta-Xy1p and alpha-GlcpA-(1 --> 2)-beta-Xylp-(1 --> 4)-alphabeta-Xylp were obtained via partial acid hydrolysis of SPN, showing the structure of side-chain substituents on O-2 of the main-chain units.     

Structural analysis of the alpha-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme

The neutral exopolysaccharide EPS35-5 (reuteran) produced from sucrose by the glucansucrase GTFA enzyme from Lactobacillus reuteri 35-5 was found to be a (1-->4,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis and 1D/2D 1H and 13C NMR spectroscopy of intact EPS35-5, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis and enzymatic hydrolysis, using pullulanase M1 (Klebsiella planticola), of EPS35-5, a composite model, that includes all identified structural elements, was formulated as follows: [Formula: see text].     

Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy

Alkali extraction and methylation analyses in the 1970s revealed that the cell walls of the yeast Schizosaccharomyces pombe contain a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, a (1-->6)-beta-d-glucan, and a alpha-galactomannan. To refine the structures of these polysaccharides, cell-wall glucans of S. pombe were extracted, fractionated, and analyzed by NMR spectroscopy. S. pombe cells were treated with 3% NaOH, and alkali-soluble and insoluble fractions were prepared. The alkali-insoluble fraction was treated with 0.5M acetic acid or Zymolyase 100T to yield an alkali-insoluble, acetic acid-insoluble fraction, an alkali-insoluble, Zymolyase-insoluble fraction, and an alkali-insoluble, Zymolyase-soluble fraction. (13)C NMR and 2D-NMR spectra disclosed that the cell wall of S. pombe is composed of three types of glucans, specifically, a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, which may either be linear or slightly branched, and a highly branched (1-->6)-beta-d-glucan, in addition to alpha-galactomannan. The highly branched (1-->6)-beta-d-glucan was identified by selective periodate degradation of side-chain glucose as a highly (1-->3)-beta-branched (1-->6)-beta-d-glucan with more branches than that of Saccharomyces cerevisiae. Flexibility of these polysaccharides in the cell wall was analyzed by (13)C NMR spectra in D(2)O. The data collectively indicate that (1-->3)-alpha- and (1-->3)-beta-d-glucans are rigid and contribute to the cell shape, while the highly branched (1-->6)-beta-d-glucan and alpha-galactomannan are flexible.     

Structures of two O-polysaccharides of the lipopolysaccharide of Citrobacter youngae PCM 1538 (serogroup O9)

Mild acid degradation of the lipopolysaccharide of Citrobacter youngae O9, strain PCM 1538 released a homopolysaccharide of 4-acetamido-4,6-dideoxy-D-mannose (D-Rha4NAc, N-acetyl-D-perosamine). Studies by methylation analysis and (1)H and (13)C NMR spectroscopy, using two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and H-detected (1)H,(13)C HSQC experiments showed the presence of two structurally different polysaccharides consisting of the following units: -->)-alpha-D-Rhap4NAc-(1 --> and --> 3)-alpha-D-Rhap4NAc-(1 --> 3)-beta-D-Rhap4NAc-(1 -->.     

Structure of the O-polysaccharide from the lipopolysaccharide of Providencia stuartii O43 containing an amide of D-galacturonic acid with L-serine

The O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia stuartii O43:H28 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 2D ROESY, and H-detected 1H, 13C HSQC and HMBC experiments, as well as a NOESY experiment in a 9:1 H2O/D2O mixture to reveal correlations for NH protons. It was found that the polysaccharide is built up of linear tetrasaccharide repeating units containing an amide of D-galacturonic acid with L-serine [D-GalA6(L-Ser)] and has the following structure:[3)-beta-D-GalpA6(L-Ser)-(1-->3)-beta-D-GlcpNAc-(1-->2)-alpha-D-Rhap4NAc-(1-->4)-beta-D-GlcpA-(1-->]n.     

Chemical characterization of the immunomodulating polysaccharide of Aloe vera L

The polysaccharide isolated by alcohol precipitation of Aloe vera mucilaginous gel was found to have a Man:Glc:Gal:GalA:Fuc:Ara:Xyl ratio of 120:9:6:3:2:2:1 with traces of Rha and GlcA. Linkage analysis of the endo-(1-->4)-beta-d-mannanase-treated sample yielded Manp-(1--> (approximately 26%), 4-Manp (approximately 53%), 2,4-Manp (approximately 3%), 3,4-Manp (approximately 1%), 4,6-Manp (approximately 1%), 4-Glcp (approximately 5%), 4-Xylp (approximately 1%), Xylp-(1--> (approximately 2%), Galp-(1--> (approximately 5%), and traces of 4,6-Galp and 3,6-Galp. Hydrolysis with strong acids produced a mixture of short oligosaccharides and an acid-resistant fraction containing greater relative fractions of Manp-(1-->, Araf-(1-->, Xylp-(1-->, and 4-Xylp than the bulk polysaccharide. NMR analysis of oligosaccharides generated by endo-(1-->4)-beta-D-mannanase and acid hydrolysis showed the presence of di-, tri-, and tetrasaccharides of 4-beta-Manp, beta-Glcp-(1-->4)-Man, beta-Glcp-(1-->4)-beta-Manp-(1-->4)-Man, and beta-Manp-(1-->4)-[alpha-Galp-(1-->6)]-Man, consistent with a backbone containing alternating -->4)-beta-Manp-(1--> and -->4)-beta-Glcp-(1--> residues in a approximately 15:1 ratio. Analysis of the sample treated sequentially with endo-(1-->4)-beta-d-mannanase and alpha-D-galactosidase showed that the majority of alpha-Galp-(1--> residues were linked to O-2, O-3, or O-6 of -->4)-beta-Manp-(1--> residues, with approximately 16 -->4)-beta-Manp-(1--> residues between side chains. Our data provide direct evidence of a previously proposed glucomannan backbone, but draw into question previously proposed side-chain structures.     

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%).     

The structure of the O-specific polysaccharide chain of the Shewanella algae BrY lipopolysaccharide

An acidic O-specific polysaccharide was obtained by mild acid degradation of the Shewanella algae strain BrY lipopolysaccharide and was found to contain L-rhamnose, 2-acetamido-4-[D-3-hydroxybutyramido)]-2,4,6-trideoxy-D-glucose (D-BacNAc4NHbu), and 2-amino-2,6-dideoxy-L-galactose, N-acylated by the 4-carboxyl group of L-malic acid (L-malyl-(4-->2)-alpha-L-FucN) in the ratio 2:1:1. 1H and 13C NMR spectroscopy was applied to the intact polysaccharide, and the following structure of the repeating unit was established:-3)-alpha-D-BacNAc4NHbu-(1-->3)-alpha-L-Rha-(1-->2)-alpha-L-Rha-(1-->2)-L-malyl-(4-->2)-alpha-L-FucN-(1-. The repeating unit includes linkage via the residue of malic acid, reported here for the first time as a component of bacterial polysaccharides.     

Structural studies of water-soluble polysaccharides of an edible mushroom, Termitomyces eurhizus. A reinvestigation

The structure of two polysaccharides isolated from the hot aqueous extract of fruiting bodies of the mushroom, Termitomyces eurhizus, have been reinvestigated. These consist of two homogeneous fractions PS-I and PS-II. PS-I contains only D-glucose as the monosaccharide constituent. From methylation analysis and periodate oxidation studies, followed by GLC-MS analysis the linkages, the sugar units in PS-I were identified as (1-->3)-D-Glcp and (1-->6)-D-Glcp. PS-II contains D-glucose, and the mode of linkage of d-glucose was identified as (1-->6)-D-Glcp. Finally, the following possible structures of the polysaccharides were assigned using 1H, 2D-COSY, TOCSY, NOESY and 13C NMR spectral analysis: [carbohydrate structure: see text].     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.