Structural Analysis of the Extracellular Polysaccharide Produced by Sphaerotilus natans

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below.

→4)-α-D-Glcp-(1→2)-β-D-GlcAp-(1→2)-α-L-Rhap- (1→3)-β-L-Rhap-(1→     

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola

Native EPS produced by Pseudomonas syringae pv. phaseolicola in vitro was separated by ion exchange chromatography on DEAE fractogel into three different polysaccharide fractions. A neutral polysaccharide eluting with the void volume yielded only fructose upon hydrolysis and exhibited an IR spectrum similar to authentic levan. At about 300 mM KCl a mannuronan eluted. Comparison with authentic alginate by IR spectroscopy, elution behaviour during DEAE-fractogel column chromatography, and monomer composition (mannuronic acid and traces of guluronic acid) confirmed the identity of this fraction as a bacterial alginate. It contained about 56 mol% acetyl groups. A third polysaccharide eluted at about 160 mM KCl. Its monomeric composition (rhamnose, fucose, glucose, and amino sugars), elution behaviour upon DEAE-fractogel column chromatography, and TLC patterns, closely resembled the sugar moiety of lipopolysaccharides (LPS) from, Pseudomonas syringae pv. phaseolicola. The protein component of crude EPS represented a fourth macromolecular fraction. It was not covalently linked to any of the polysaccharides since it could be removed from the EPS by phenol extraction.     

Extracellular polysaccharide of Erwinia chrysanthemi CU643.

Erwinia chrysanthemi are gram-negative bacterial phytopathogens causing soft rots in a number of plants. The structure of the extracellular polysaccharide (EPS) produced by E. chrysanthemi strain CU643, pathogenic to Philodendron, has been determined using a combination of chemical and physical techniques including methylation analysis, high- and low-pressure gel-filtration and anion-exchange chromatography, high-pH anion-exchange chromatography, partial acid hydrolysis, mass spectrometry, and 1- and 2-D NMR spectroscopy. In contrast to the structures of the EPS reported for other strains of E. chrysanthemi, the EPS from strain CU643 is a linear polysaccharide containing L-Rhap, D-Galp, and D-GlcAp in the ratio 4:1:1. Evidence is presented for the following hexasaccharide repeat unit: -->3)-beta-D-Galp-(1-->2)-alpha-L-Rhap-(1-->4)-beta-D-GlcAp- (1-->2)-alpha-L- Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->(1 ).     

Structure of a polysaccharide from a Rhizobium species containing 2-deoxy-beta-D-arabino-hexuronic acid.

The structure of the extracellular polysaccharide (EPS) produced by the Rhizobium sp. B strain isolated from atypical nodules on alfalfa has been determined using a combination of chemical and physical techniques (methylation analysis, high pH-anion exchange chromatography (HPAEC), mass spectrometry and 1-D and 2-D NMR spectroscopy). As opposed to the EPS from other strains of Rhizobium, the EPS from the sp. B strain contains D-Glc together with L-Rha and 2-deoxy-D-arabino-hexuronic acid. It is a polymer of a repeating unit having the following structure: --> 4)-beta-D-Glcp-(1 --> 4)-alpha-L-Rhap -(1 --> 3)-beta-D-Glcp-(1 --> 4)-2-deoxy-beta-D-GlcpA-(1 -->. The polysaccharide also contains 0.6 O-acetyl groups per sugar which have not been located.     

STRUCTURE OF THE CAPSULAR AND EXTRACELLULAR POLYSACCHARIDES PRODUCED BY THE DESMID SPONDYLOSIUM PANDURIFORME (CHLOROPHYTA)1

The filamentous desmid Spondylosium panduriforme (Heimerl) Teiling var. panduriforme f. limneticum (West & West) Teiling (Desmidiaceae), strain 072CH-UFCAR, is surrounded by a well-defined, mucilaginous capsule consisting of a capsular polysaccharide (CPS). This microalga also produces an extracellular polysaccharide (EPS), which can be isolated from the culture medium. Analysis of the carbohydrate composition of the two polymers by gas chromatography showed that they were different. Both were composed, of galactose, fucose, xylose, arabinose, rhamnose, and glucose but in different amounts. For example, glucuronic acid accounts for 24% of the EPS material but only traces were found in the CPS. Significant differences were also found during methylation analysis. Fucose appeared to have a higher degree of branching in the EPS than in the CPS. These branches were located on C-3 and could be the position for the attachment of the glucuronic acid units in the EPS. The glucuronic acid was present as 1→4-linked and terminal units. A possible explanation for the formation of the EPS is suggested.     

An arabinogalactan from the skin of Opuntia ficus-indica prickly pear fruits

The cold-water extract from the skin of Opuntia ficus-indica fruits was fractionated by anion-exchange chromatography. The major fraction, which was purified by size exclusion chromatography, consisted of a polysaccharide composed of galactose and arabinose residues in the ratio 6.3:3.3, with traces of rhamnose, xylose and glucose, but no uronic acid. The results of methylation analysis, supported by (13)C NMR spectroscopy, indicated that this polysaccharide corresponded to an arabinogalactan having a backbone of (1-->4)-linked beta-D-galactopyranosyl residues with 39.5% of these units branched at O-3. The side-groups consisted either of single L-arabinofuranosyl units or L-arabinofuranosyl alpha-(1-->5)-linked disaccharides. This polysaccharide is thus an arabinogalactan that can be classified in the type I of the arabinogalactan family.     

淫羊藿多糖的分离纯化及结构初步分析

从淫羊藿中提取多糖并鉴定其初步结构和单糖组成.采用超声-水提醇沉法提取粗多糖、Sevag法去蛋白、DEAE-52纤维素及Sephadex G-100柱层析法纯化得到淫羊藿多糖EPSⅠ-1和EPSⅡ-1.应用紫外光谱法和红外光谱法对其结构做初步分析.采用高效液相色谱法(HPLC)测定其单糖组成及摩尔比.均一的EPSⅠ-1和EPSⅡ-1多糖在紫外和红外中具有多糖的特征吸收峰,组成中含有吡喃环结构;EPSⅠ-1的单糖组成为鼠李糖和葡萄糖,摩尔比为1:1.13;EPSⅡ-1的单糖组成为果糖、葡萄糖和一个不确定的糖,摩尔比为1:1.91.有效地分离纯化了淫羊藿多糖,这为淫羊藿多糖的广泛应用奠定了实验基础.     

Sugar determination in ulvans by a chemical-enzymatic method coupled to high performance anion exchange chromatography

The sugar determination of ulvans, the water-soluble polysaccharides from Ulva sp. and Enteromorpha sp., was optimized by combining partial acid prehydrolysis (2 mol L-1 trifluoroacetic acid, 120°C) with enzymic hydrolysis (with β-D-glucuronidase). The different constitutive sugars (rhamnose, galactose, glucose, xylose, glucuronic acid), released after hydrolysis, were separated by high performance anion-exchange chromatography and determined by pulsed amperometric detection. The ulvanobiouronic acid, β-D-GlcA-(1,4)-L-Rha, which is the main constituent of ulvans was always present after 3 h of trifluoroacetic acid hydrolysis (approx. 2% D.M.) when acid hydrolysis was performed alone but the xylose amount fell to 75% of its maximum value at this time. The optimal duration of 2 mol L⁻¹ trifluoroacetic acid hydrolysis of ulvans (i.e. without any degradation of xylose, rhamnose and glucuronic acid) was 45 min. Additionnal treatment of the partial acid hydrolysate by purified β-D-glucuronidase allowed the hydrolysis of the residual ulvanobiouronic acid in rhamnose and glucuronic acid. High performance anion exchange chromatography coupled to this chemical-enzymic hydrolysis revealed to be a high resolution chromatographic technique for monitoring the hydrolysis of the aldobiouronic acid by β-D-glucuronidase. This method allowed the simultaneous quantitative determination of neutral and acidic sugars and revealed the presence of iduronic acid inulvans. This revised version was published online in September 2006 with corrections to the Cover Date.     

Chemical modification of botryosphaeran: structural characterization and anticoagulant activity of a water-soluble sulfonated (1-->3)(1-->6)-β-D-glucan

The exopolysaccharide botryosphaeran (EPS(GLC); a (1--> 3)(1-->6)-β-D-glucan from Botryosphaeria rhodina MAMB- 05) was sulfonated to produce a water-soluble fraction (EPS(GLC)-S) using pyridine and chlorosulfonic acid in formamid. This procedure was then repeated twice to produce another fraction (EPSGLC-RS) with a higher degree of substitution (DS, 1.64). The purity of each botryosphaeran sample (unsulfonated and sulfonated) was assessed by gel filtration chromatography (Sepharose CL-4B), where each polysaccharide was eluted as a single symmetrical peak. The structures of the sulfonated and re-sulfonated botryosphaerans were investigated using ultraviolet-visible (UV-Vis), Fourier-transform infrared (FT-IR), and (13)C nuclear magnetic resonance ((13)C NMR) spectroscopies. EPS(GLC) and EPS(GLC)-RS were also assayed for anticoagulation activity, and EPS(GLC)-RS was identified as an anticoagulant.     

Structural studies on Klebsiella type 61 capsular polysaccharide

The capsular polysaccharide from klebsiella type 61 was found to contain d-galactose, d-glucose, d-mannose, and d-glucuronic acid in the ratios 1:2:1:1. Acid hydrolysis of the polysaccharide gave one aldobiouronic acid, whose structure was established. Methylation analysis of the polysaccharide provided information about the linkages in the polysaccharide. The polysaccharide is composed of a pentasaccharide repeating unit for which structures are proposed.     

Comparative studies of lipopolysaccharide and exopolysaccharide from a virulent strain of Pseudomonas solanacearum and from three avirulent mutants.

The composition of the Pseudomonas solanacearum lipolysaccharide (LPS) was found to be similar to that described for the LPS of enterobacteria. The lipid A contained fatty acids and glucosamine in a molar ratio of 5:2. The LPS fraction contained 2-keto-3-deoxyoctulosonic acid, L-glycero-D-mannoheptose, hexoses (glucose, rhamnose, and glucosamine), and a pentose (xylose). The LPSs from the wild-type strain (GMI1000), from the spontaneous rough mutant (GMI2000), and from their respective acridine orange-resistant (Acrr) mutants (GMI1178 and GMI2179) contained the same component sugars in their polysaccharide moieties, but the relative amounts of each sugar varied greatly. Spontaneous mutation to the rough type was characterized by a decrease in the ratio of rhamnose to glucose, whereas a reverse effect was seen for the acridine orange resistance mutation from the parent strains (GMI1000 and GMI2000) to the respective mutant strains (GMI1178 and GMI2179). The exopolysaccharide (EPS) from GMI1000 was found to be composed of two fractions: a heteropolysaccharide (galactosamine, glucose, and rhamnose) excluded from Sephadex G-50 and an additional glucan with a lower molecular weight. Strains GMI1000 and GMI1178 produced comparable amounts of EPS, GMI2179 synthesized less EPS, and GMI2000 produced no detectable EPS. High-pressure liquid chromatography and 13C nuclear magnetic resonance analyses revealed some differences between these EPSs. The glucan fraction seemed to be the major component of the EPS from GMI2179, whereas GMI1000 and GMI1178 EPSs contained both fractions and appeared to differ in the structures of their heteropolysaccharide fractions. Viscosity measurements confirmed differences between whole EPSs produced by the three strains.     

嗜热链球菌MGB80-7所产胞外多糖的表型结构及其抗氧化活性

【背景】胞外多糖（exopolysaccharide,EPS）是乳酸菌生长代谢过程中所产生的一种次级代谢产物,除了可以改善产品质构和品质外,其生理功能也是近年来研究人员追捧的热点。【目的】探究乳酸菌EPS的表征特性和分子结构,揭示其与EPS益生特性之间的联系。【方法】以产EPS的嗜热链球菌（Streptococcus thermophilus,S. thermophilus） MGB80-7为研究对象,利用苯酚-硫酸法测定菌株EPS产量。采用离子交换柱层析和凝胶分子筛层析对该菌株所产EPS进行分离纯化,结合凝胶色谱、红外光谱及高效液相色谱对EPS表型结构进行剖析。此外,为确定EPS表型特征对其抗氧化活性的影响,测定了EPS对超氧阴离子、羟自由基及DPPH自由基等的清除能力。【结果】S. thermophilus MGB80-7在M17培养基中EPS产量较高,为（268.25±5.36） mg/mL,分离纯化后共得到2种多糖组分,其中中性多糖（WPS-807）分子量为1.028×10⁵ Da,主要由葡萄糖、半乳糖和甘露糖组成,并含有少量的鼠李糖和阿拉伯糖,酸性多糖（...     

Structural study on lipid A and the O-specific polysaccharide of the lipopolysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease.

Bacteroides vulgatus has been shown to be involved in the aggravation of colitis. Previously, we separated two potent virulence factors, capsular polysaccharide (CPS) and lipopolysaccharide (LPS), from a clinical isolate of B. vulgatus and characterized the structure of CPS. In this study, we elucidated the structures of O-antigen polysaccharide (OPS) and lipid A in the LPS. LPS was subjected to weak acid hydrolysis to produce the lipid A fraction and polysaccharide fraction. Lipid A was isolated by preparative TLC, and its structure determined by MS and NMR to be similar to that of Bacteroides fragilis except for the number of fatty acids. The polysaccharide fraction was subjected to gel-filtration chromatography to give an OPS-rich fraction. The structure of OPS was determined by chemical analysis and NMR spectroscopy to be a polysaccharide composed of the following repeating unit: [-->4)alpha-L-Rhap(1-->3)beta-D-Manp(1-->].     

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH4)2SO4 treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100 degrees C) at pH 1.8, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.     

Structural aspects of the exudate from the fruit of Chorisia speciosa St. Hil

Mature fruit of Chorisia speciosa yield an exudate (E-I) following mechanical injury. The polysaccharide contains rhamnose, arabinose, xylose, mannose, glucose, galactose and glucuronic acid in molar ratios of 20:11:1:3:2:40:23. The main chain of the structure is composed by beta-galactopyranosyl units linked (1 --> 3) and (1 --> 6) as indicated by NMR spectra and methylation data. Arabinosef and rhamnose are terminal residues. In order to compare E-I with the polysaccharides from the fruit mesocarp, the latter was submitted to different extractions. The water fraction contains rhamnose, arabinose, xylose, mannose, glucose, galactose and uronic acid in molar ratios of 18:4:1:2:3:44:28. It was treated with CTAB yielding a precipitate which was decomplexed with NaCl, giving four fractions. The fraction obtained using 0.15 M NaCl had a quantitative composition similar that of E-I.     

Biochemical characterization of the exopolysaccharide purified from Laetiporus sulphureus mycelia

The extracellular polysaccharide (EPS) was isolated from mycelial cultures of Laetiporus sulphureus var. miniatus and purified by DEAE cellulose and Sephadex G-50 column chromatography. The purified EPS (EPS-2-1) was composed of only glucose units and its molecular mass was 6.95 kDa. The chemical structure of EPS-2-1 consisted of a main chain containing (1-->4)-Glcp units with branches at the C-6 position of the chain carrying -Glcp-(1-->4)-linked residues. The effect of purified EPS on immunomodulatory genes and proteins of the Bcl-2 family was observed using cultured U937 human leukemia cells. Of note, the levels of Bax and Bad proteins treated with the EPS (4 mg/ml) were approximately 23- and 18-times higher than those in non-treated cells, respectively. These results may suggest that the EPS purified from the mushroom L. sulphureus is associated with the activation of immunomodulatory mediators, Bax and Bad proteins.     

Culture conditions for the production of an acidic exopolysaccharide by the nitrogen-fixing bacterium Burkholderia tropica

The endophytic diazotrophic bacterium Burkholderia tropica, strain Ppe8, produced copious amounts of exopolysaccharide (EPS) on batch growth in liquid synthetic media containing mannitol and glutamate as carbon and nitrogen sources. The effect of various aeration regimes and carbon source concentrations on EPS production was determined, as well as the effects of temperature and time of growth. The degree of aeration had a great influence on the yield of EPS, in contrast with the C:N ratio of the medium. Growth temperature also affected the EPS yield after the first 24 h of culture but seemed to be irrelevant after that. After isolation and purification, the EPS was analyzed by high-performance size exclusion chromatography and multiangle laser light scattering (HPSEC-MALLS), revealing a molecular mass of 300 kDa. The acid hydrolyzate of EPS was examined by HPLC and found to contain Glc, Rha, GlcA, and an aldobiouronic acid. The latter was found to have a GlcA and a Rha unit. Carboxy-reduced EPS contained Glc and Rha (3:2). The monosaccharide composition of the native acidic EPS was calculated as GlcA, Glc, and Rha in a molar ratio of 1:2:2.     

Purification and properties of extracellular polysaccharide (EPS) antigens produced by different mould species

Extracellular polysaccharide (EPS) antigens produced by different mould species were purified and partially characterized. Purification included (NH₄)₂SO₄ treatment, Sepharose CL-4B column chromatography and Con A-sepharose chromatography. The EPS of Penicillium digitatum, Mucor racemosus and Cladosporium cladosporioides showed high antigenic capacities. Immunologically the EPS were partially genus-specific, but cross-reactivity was observed. The EPS antigens produced by species of Penicillium, Aspergillus repens and Geotrichum candidum lost their immunological activity upon heating (100C) at pH 18, while the EPS antigen of M. racemosus, Rhizopus oligosporus and C. cladosporioides were stable under the same conditions. The dominant monosaccharides present in the EPS antigen were mannose, galactose and glucose. The EPS obtained from cultures of M. racemosus and R. oligosporus also contained rhamnose. In the EPS produced by Penicillium spp. and A. repens the galactose residues were determined to be immunodominant.     

The proteinaceous, gum polysaccharide from Azadirachta indica A. Juss

The gum exuded by Azadirachta indica trees contains 35% of proteinaceous material. The most abundant amino acid is aspartic acid; there are also considerable proportions of serine and threonine, and at least 2% of amino sugars. Attempts to deproteinise the polysaccharide material were unsuccessful. Fractionation of the gum exudate on agarose columns gave a protein-depleted fraction of high molecular weight and a protein-enriched fraction of lower molecular weight. Studies involving molecular-sieve chromatography showed a close correspondence in protein and carbohydrate content, and it appears that the proportion of proteinaceous component that is free or only loosely associated with carbohydrate is small. The carbohydrate component is much more complex than was indicated by earlier investigators. In addition to galactose and arabinose (major components), mannose, xylose, fucose, and rhamnose are also present. The uronic acid content (28%) is higher than previously believed, and a relatively high methoxyl content has been found. The major aldobiouronic acid, previously undetected, is 4-O-(4-O-methyl-- -glucopyranosyluronic acid)- -galactose, with 4-O-(- -glucopyranosyluronic acid)- -galactose also present. The exudate from Azadirachta indica is therefore a complex material having several features that are unusual in gum chemistry.     

Characterization of the group-specific polysaccharide of group B Streptococcus

The group-specific polysaccharide of the group B Streptococcus was isolated by nitrous acid extraction followed by gel filtration on Sepharose 6B and chromatography on DEAE-Bio-Gel A. It was composed of rhamnose, galactose, N-acetylglucosamine, and glucitol phosphate. Mild periodate oxidation of the polysaccharide resulted in a rapid reduction in molecular weight, indicating that the glucitol was located in the backbone of the polymer. High-resolution 31P NMR showed the presence of a single type of phosphodiester bond in the molecule. Methylation analysis and several specific chemical degradations were done to determine sugar linkages. The basic structure of the group B polysaccharide consists of a backbone of 2-linked rhamnose, 2,4-linked rhamnose, and glucitol phosphate, and side chains of rhamnose(1----3)galactose(1----3)N-acetylglucosamine linked to the 4-position of a rhamnose in the backbone.