STRUCTURE OF EXTRACELLULAR POLYSACCHARIDES PRODUCED BY A SOIL CRYPTOMONAS SP. (CRYPTOPHYCEAE)1

The Hindak strain of a Cryptomonas species (Cryptophyceae) produces extracellular polysaccharides. Because there is no information on the structure of these compounds in the Cryptophyceae we conducted structural studies. Gas–liquid chromatographic analyses showed that the polysaccharide is composed of fucose, rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, glucuronic acid, and traces of 3-O-methyl galactose. The polysaccharide was separated into two subtractions by ion-exchange chromatography. Fraction A consisted mainly of 1,3-linked galactose units and 1,4-linked galacturonic acid. Unlike fraction B, fraction A did not have xylose, 3-O-methyl galactose, or glucuronic acid. Also, its degree of branching was low compared to that of fraction B. Only traces of sulfate were present infraction A, but fraction B was 10–15% sulfated. Protein was approximately 1% in both fractions. These polysaccharides appear to be a novel type of polymer in algae.     

Analytical and structural features of the gum exudate from Combretum hartmannianum schweinf.

The gum exudate from Combretum hartmannianum is water-soluble, forms very viscous solutions, and contains galactose (22%), arabinose (43%), mannose (10%), xylose (6%), rhamnose (4%), glucuronic acid (6%), 4-O-methylglucuronic acid (2%), and galacturonic acid (7%). The acidic components produced on hydrolysis of the gum were 6-O-(β-D-glucopyranosyluronic acid)-D-galactose, and two saccharides that had the same chromatographic mobility, and contained mannose and galacturonic acid, and galactose and 4-O-methylglucuronic acid, respectively. Methylation and methanolysis of the gum indicated the presence of terminal uronic acid, rhamnose, xylose, galactose, arabinofuranose, and arabinopyranose. Controlled, acid hydrolysis indicated the presence of (1→3)-linked arabinopyranose side-chains and (1→6)-linked galactose residues. C. hartmannianum gum, when subjected to two Smith-degradations, yielded Polysaccharides I and II, both of which contained galactose, arabinose, and mannose. Insufficient crude gum was available for a complete structural study, but the molecule was shown to contain long, sparsely branched chains of (1→6)-linked galactose residues, to which are attached (1→3)-linked arabinose and (1→3)-linked mannose side-chains.     

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.     

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.     

Structural studies of a specific polysaccharide isolated from non-agglutinable Vibrio

The purified, specific polysaccharide from Vibrio cholera type NAG, NV 384, O-antigen, 2A, 2Bhuman, contains glucose (5.14%), galactose (4.21%), mannose (64.8%), xylose (3.16%), arabinose (1.98%), fucose (1.50%), mannuronic acid (14.3%), phosphate (0.32%), 2-amino-2-deoxy-D-glucose (2.9%), and 2-amino-2-deoxy-D-galactose (1.0%). Various reactions have shown that the material comprises a phosphoric diester-linked polysaccharide containing mainly (1→2)-linked mannopyranose residues that are highly branched with other sugar residues.     

Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans

Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3–37%), nucleic acids (9–50%), and carbohydrates (3–21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.     

Structural features of the sulphated polysaccharide from a green seaweed,Caulerpa taxifolia

A sulphated heteropolysaccharide was isolated from a green seaweed, Caulerpa taxifolia, by extraction with acid and purified via its copper complex. Methylation analysis of both the sulphated and desulphated polysaccharides revealed the presence of 1,4-linked xylose, 1,6-linked galactose, 1,4,6-linked mannose and non-reducing galactose end group units which are all devoid of sulphate groups. In addition 1,4-linked galactose units sulphated at C-3 are also present. Quantitative periodate oxidation showed a consumption of 1.30 and 1.60 moles of oxidant per anhydrosugar unit in the sulphated and desulphated polysaccharides respectively. The oxo-polysaccharides after reduction and hydrolysis revealed the presence of glycerol, erythritol and unoxidized galactose in the mol ratio 11.6:5.1:4.9 and 11.2:5.0:1.0 respectively, besides threitol (3.9 mol) in the desulphated polysaccharide.     

A cellulosic exopolysaccharide produced from sugarcane molasses by a Zoogloea sp.

An extracellular polysaccharide producing bacterium Zoogloea sp. was isolated from an agro-industrial environment in the north-eastern region of Brazil. The extracellular polysaccharide produced from sugarcane molasses was hydrolysed with trifluoroacetic acid (mild and strong conditions) giving 88% of soluble material. The main monosaccharides present in the soluble fraction were glucose (87.6%), xylose (8.6%), mannose (0.8%), ribose (1.7%), galactose (0.1%), arabinose (0.4%) and glucuronic acid (0.8%). Methylation analysis of the polysaccharide showed mainly 2,3,6-tri-O-methylhexitol (74.7%) and 2,3,-di-O-methylhexitol (17.7%). Enzyme hydrolysis of the polysaccharide with a cellulase confirmed the presence of (1→4)-β- -glucopyranosyl residues.     

Characterization of a neutral polysaccharide with antioxidant capacity from red wine

A neutral fraction (PS-SI) (0.3 g/L) with MW of 74 kDa, which contained galactose, arabinose, mannose, and glucose in the molar ratio of 1.0:0.6:0.4:0.2 was obtained by treatment of the whole polysaccharide extracted from red wine with cetrimide, followed by gel permeation chromatography. Spectroscopic and methylation analyses indicated that PS-SI is a mixture of neutral polysaccharides, consisting mainly of β (1→3)-linked galactopyranosyl residues, with side chains of galactopyranosyl residues at positions O-6. Arabinofuranosyl residues linked α (1→5), α-mannopyranosyl and glucosyl residues appear to be components of different polysaccharides. The in vitro antioxidant capacity of fractions of wine polysaccharide was studied by hydroxyl radical scavenging and ORAC assays. Fraction PS-SI presented the strongest effect on hydroxyl radicals (IC₅₀ = 0.21).     

Structure of polysaccharides from the Polyporus tumulosus cell wall

Cell walls of the Basidiomycete fungus Polyporus tumulosus (Cooke) were fractionated, and the polysaccharide content of the fractions investigated. The major constituents of the cell wall include four polysaccharides, chitin, a β-1, 3-glucan and the alkali soluble α-glucan and xylomannan.The glucan is highly dextrotatory with an [α]_D²¹ of + 221° and gave on partial acid hydrolysis and acetolysis an homologous series of oligosaccharides. The disaccharide was shown to be nigerose 3-0-α-D-glucopyranosyl-D-glucose. Periodate oxidation and methylation studies provided supporting evidence that the polysaccharide is an essentially unbranched polymer of 1,3-linked glucose residues.The other alkali-soluble polysaccharide, a xylomannan, is a polymer of mannose and xylose in the approximate molar proportions of 1.2:1. It has an [α]_D = + 56° and on partial acid hydrolysis and acetolysis gave an homologous series of 1,3-linked mannodextrins but no oligosaccharides containing xylose were obtained. An α-1,3-linked mannan was prepared from the xylomannan by degradation with mild acid or by degradation of the periodate-oxidased and reduced xylomannan. The structure therefore is visualised as having a backbone of 1,3-linked mannan, to which xylose residues are attached. Methylation studies showed that branching occurs at C-4 of the mannopyranose units; the presence of 2,3-di-o-methyl-d-xylose in the hydrolysate of the methylated polysaccharide indicated that some of the xylose residues are 1,4-linked. The possible structure of the fungal cell wall is discussed in the light of the results obtained.     

Some structural features of a new sulphated heteropolysaccharide from Padina pavonia

An acid-extractable, water-soluble, polysaccharide sulphate, isolated from Padina pavonia, comprised variable proportions of glucuronic acid, galactose, glucose, mannose, xylose, and fucose in addition to a protein moiety. Partial acid hydrolysis and autohydrolysis of the free acid polysaccharide yielded several oligosaccharides. Evidence from periodate oxidation studies indicated that the inner polysaccharide portion is composed of (1 → 4)-linked β-D-glucuronic acid, (1 → 4)-linked β-D-mannose and (1 → 4)-linked β-D-glucose residues. The heteropolymeric partially sulphated exterior portion is attached to the inner part and comprises various ratios of (1 → 4)-linked β-D-galactose, β-D-galactose-3-sulphate residues, (1 → 4)-linked β-D-glucose residues, (1 → 2)-linked α-L-fucose 4-sulphate residues and (1 → 3)-linked β-D-xylose residues.     

Highly sulphated galactan from Halymenia durvillei (Halymeniales,Rhodophyta), a red seaweed of Madagascar marine coasts

Halymenia durvillei is a red seaweed with a great potential as sulphated galactan producer collected in the coastal waters of small island of Madagascar (Nosy-be in Indian Ocean). To elucidate the structure of its polysaccharide, NMR (¹H and ¹³C), FTIR, HPAEC and different colorimetric methods were carried out. It has been shown that this polysaccharide, consisted mainly of galactose, was branched by xylose and galactose in minor amounts. Arabinose and fucose were also detected. This galactan was found highly sulphated (42%, w/w) and pyruvylated (1.8%, w/w). Analysis of glycosidic linkages by CPG-MS and ¹³C NMR indicated that the polysaccharide has the defining linear backbone of alternating 3-β-d-galactopyranosyl units and 4-linked α-l/d-galactopyranosyl residues. 3,6-Anhydrogalactose units have been also detected in minor quantity. This λ-carrageenan like polysaccharide has shown original sulphatation patterns with 2-O (26%) or 2/6-O (58%) sulphated 3-linked β-d-galactopyranosyl units and 6-O (19%) or 2/6-O (47%) 4-linked α-l/d-galactopyranosyl residues.     

Water-soluble glycoproteins from Cannabis sativa (Thailand)

Glycoproteins were extracted with water from leaves of Cannabis sativa grown from seeds of Thailand origin. By ion exchange chromatography the material was separated into a neutral and an acidic fraction. Both glycoprotein fractions contained arabinose, galactose, glucose, mannose and xylose, and in addition rhamnose and galacturonic acid were present in the acidic fraction. The carbohydrate moieties were investigated by methylation analysis and Smith-degradation, whereas the glycopeptide linkage was studied by alkaline hydrolysis in the presence of NaBH₄ and Na₂SO₃, respectively. This linkage was shown to be of the serine-O-galactoside type. The carbohydrate structure is highly branched, the majority of branches terminating in arabinofuranose end groups. Arabinose is also present in the chain, predominantly (1 → 4)- and/or (1 → 5)-linked. Galactose makes up most of the main chain as (1 → 3)-linked residues but also constitutes end groups and branch points, as do mannose and/or glucose. Xylose and rhamnose are present as (1 → 4)- and (1 → 2)-linked units, respectively. Galacturonic acid is assumed to be (1 → 4)- linked with some branching at 3 position. The amino acid hydroxyproline, present in the glycoprotein of South African Cannabis leaves, was absent in the corresponding Thailand material.     

Structure of Hemicellulose Isolated from Rice Endosperm Cell Wall : Mode of Linkages and Sequences in Xyloglucan, β-Glucan and Arabinoxylan

A hemicellulosic polysaccharide, which was homogeneous on sedimentation analysis and also on electrophoresis, was isolated from the rice endosperm cell walls by the combination of alkaline extraction, ion exchange chromatography and iodine complex formation. It is composed of arabinose, xylose and glucose (molar ratio, 1.0: 2.0: 5.7) together with a small amount of galactose and rhamnose. Methylation analysis, Smith degradation and fragmentation with cellulase showed that this polysaccharide is composed of three distinct polysaccharide moieties i.e., xyloglucan, β-glucan and arabinoxylan. The xyloglucan consists of β-(1→4)-linked glucan back bone and short side chains of single xylose units or galactosylxylose both attached to C-6 of the glucose residues. The β-glucan contains both (1 →3)-and (1→4)-linkages similarly to the other cereal β-glucans, but differ from them in containing the blocks of (1→3)-linked glucose residues in the chain. The arabinoxylan has a highly branched structure, in which 78% of (1→4)-linked xylose residues have short side chains of arabinose at C-3 position.

On the basis of these findings, the interconnection of these polysaccharide moieties is discussed.     

Water-soluble polysaccharides from Ginkgo biloba leaves.

The water-soluble polysaccharides from dried Ginkgo biloba leaves were isolated after exhaustive extraction with organic solvents. The polysaccharide mixture could be separated into a neutral (GF1) and two acidic (GF2 and GF3) polysaccharide fractions by ion exchange chromatography. According to the Mr distribution GF1 and GF3 seemed to be homogenous, whereas GF2 could be further fractionated into two subfractions (GF2a and GF2b) by gel permeation chromatography. GF1 (Mr 23,000) showed the structural features of a branched arabinan. The main chain was composed of 1,5-linked arabinose residues and three in 12 arabinose molecules were branched via C-2 or C-3. GF2a (Mr 500,000) consisted mainly of 1,2,4-branched mannose (29%), 1,4-linked glucuronic (32%) and galacturonic (8%) acid as well as terminal rhamnose (25%). After removal of ca 70% of the terminal rhamnose the remaining polysaccharide showed a decrease in 1,2,4-branched mannose and an increase in 1,2-linked mannose indicating that at least half of the rhamnose residues were linked to mannose via C-4. GF3 (Mr 40,000) consisted of 1,4-linked galacturonic (30%) and glucuronic (16) acid, 1,3,6-branched galactose (15%), 1,2-linked (5%) and 1,2,4-branched (3.5%) rhamnose as well as 1,5-linked arabinose (11%). Rhamnose (5%) and arabinose (10%) were present as terminal groups. Mild acid hydrolysis selectively cleaved arabinose and the remaining polysaccharide showed an increased amount of 1,6-linked and terminal galactose and a decreased quantity of 1,3,6-branched galactose. These results indicated that the terminal as well as the 1,5-linked arabinose were mainly connected to galactose via C-3. The GF3 polysaccharide appeared to be a rhamnogalacturonan with arabinogalactan side chains.     

Purification and chemical characterization of polysaccharides obtained from Lampteromyces japonicus by concanavalin A-sepharose affinity chromatography

Two classes of neutral polysaccharide which could not be separated from each other by conventional methods were isolated from the fungus, Lampteromyces japonicus, by affinity chromatography using concanavalin A-Sepharose. The polysaccharide retained on the concanavalin A-Sepharose column was eluted with 0.05 M methyl α-d-mannopyranoside and appeared to be α-mannan, while that which passed through the column was virtually all β-glucan.Both polysaccharides were subjected to Smith-type degradation, methylation, acetolysis and glucosidase treatment. The results indicated that the α-mannan contained predominantly α-(1 → 2)-linked side chains branching from an α-(1 → 6)-linked backbone at the (1 → 2,6)-linked mannopyranosyl residues. Galactose was attached to approximately one-quarter of the non-reducing mannose terminals. The β-glucan seemed to contain mainly (1 → 6)-linked side chains branching from a (1 → 3)-linked backbone at the (1 → 3,6)-linked glucopyranosyl residues.     

In vitro anti-herpetic activity of sulfated polysaccharide fractions from Caulerpa racemosa

A sulfated polysaccharide fraction was isolated from the hot water extract of the green alga Caulerpa racemosa and designated HWE. This polymer, which contained galactose, glucose, arabinose and xylose as the major component sugars, had [alpha](D)(30) + 46.2 degrees in water and contained 9% sulfate hemiester groups. Sugar linkage analysis indicates that HWE was branched and mainly contained 1,3- and 1,3,6-linked galactose, 1,3,4-linked arabinose, 1,4-linked glucose and terminal- and 1,4-linked xylose residues. Sulfation was deduced from infrared spectroscopy and methylation analysis to occur on O-6 of galactose and O-3 of arabinose. The native polysaccharide could be fractionated by size exclusion chromatography into two overlapping fractions and the major fraction has a hydrodynamic volume similar to that of 70 kDa dextran. HWE was a selective inhibitor of reference strains and TK(-) acyclovir-resistant strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in Vero cells, with antiviral effective concentration 50% (EC(50)) values in the range of 2.2-4.2 microg/ml and lacking cytotoxic effects. Furthermore, HWE did not exhibit anticoagulant properties at concentrations near the EC(50).     

EXTRACELLULAR POLYSACCHARIDES FROM ANKISTRODESMUS DENSUS (CHLOROPHYCEAE)

The dissolved extracellular compounds produced by the Chlorococcalean Ankistrodesmus densus Kors. were investigated for their molecular masses and chemical composition. Gel permeation chromatography showed apparent relative molecular masses of 2 × 10⁶ and 10⁴, respectively, for two distinct fractions, termed A and B. The higher molecular weight fraction (A) contained mainly fucose and 3- O -methylgalactose, whereas galactose, glucose, mannose, and rhamnose were present in smaller amounts. Methylation analyses showed that the main structural features are fucose as a highly branched part of polymer A with substitutions in both position 2 and position 4, the substitutions on one of those positions being primarily terminal 3- O -methylgalactose and the other one involved as the linkage of the main chain of the polymer. Because of the presence of both fucose and 3- O -methylgalactose, this polymer is highly hydrophobic. In fraction B, mannose represented more than 60% of the carbohydrate material present, whereas the remaining part contained rhamnose, fucose, xylose, and glucose in almost equal amounts. 3- O -methylgalactose and galactose were present as minor elements. Fraction B is basically a mannose-containing polymer in which the mannose units are either 1→4 or 1→2 linked. Traces of glucuronic acid and protein were present in both fractions; neither sulfate nor phosphate was detected.     

Comparing the sugar profiles and primary structures of alkali-extracted water-soluble polysaccharides in cell wall between the yeast and mycelial phases from <Emphasis Type="Italic">Tremella fuciformis</Emphasis>

To gain insights into dimorphism, cell wall polysaccharides from Tremella fuciformis strains were obtained from alkali-extracted water-soluble fractions PTF-M38 (from the mycelial form), PTF-Y3 and PTF-Y8 (from the yeast form) of T. fuciformis strains were used to gain some insights into dimorphism study. Their chemical properties and structural features were investigated using gel permeation chromatography, gas chromatography, UV and IR spectrophotometry and Congo red binding reactions. The results indicated that the backbones of PTF-M38, PTF-Y3 and PTF-Y8 were configured with α-linkages with average molecular weights of 1.24, 1.08, and 1.19 kDa, respectively. PTF-M38 was mainly composed of xylose, mannose, glucose, and galactose in a ratio of 1:1.47:0.48:0.34, while PTF-Y3 and PTF-Y8 were mainly composed of xylose, mannose and glucose in a ratio of 1:1.65:4.06 and 1:1.21:0.44, respectively. The sugar profiles of PTF-M38, PTF-Y3 and PTF-Y8 were also established for further comparison. These profiles showed that all three polysaccharides contained the same sugars but in different ratios, and the carbon sources (xylose, mannose, glucose, and galactose) affected the sugar ratios within the polysaccharides.