Bacterial antigenic polysaccharides. 12. Structure and 13C NMR spectrum of the polysaccharide chain of Shigella boydii type 8 lipopolysaccharide

A specific acidic polysaccharide was isolated from Sh. boydii type 8 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucuronic acid, D-galacturonic acid, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose and 2-amino-1,3-propanediol residues in 1:1:1:1:1 ratio. From the results of methylation analysis, partial acid hydrolysis and Smith degradation, the structure of the repeating unit of the specific polysaccharide was deduced as: (Formula: see text). The 13C NMR spectra of native, O-deacetylated and carboxyl-reduced polysaccharides, as well as the spectrum of oligosaccharide produced by Smith degradation were interpreted. The 13C NMR data fully confirmed the structure of the polysaccharide repeating unit.     

Isolation and purification of cell wall polysaccharide of Bacillus anthracis (Δ Sterne)

A polysaccharide fraction was isolated form sodium-dodecyl-sulfate (SDS) treated cell walls of Bacillus anthracis (delta Sterne) by hydrofluoric acid (HF) hydrolysis and ethanolic precipitation. The polysaccharide fraction was subsequently purified by several washings with absolute ethanol. Purity of the isolated polysaccharide was tested using the anthrone assay and amino acid analyzer. The molecular mass of the polysaccharide fraction as determined by gel filtration chromatography was about 12000 Da. Preliminary analyses of the polysaccharide was done using thin layer chromatography and amino acid analyzer, and results obtained from these analyses were further confirmed by gas liquid chromatography and 13C-NMR spectroscopy. Results showed that the polysaccharide moiety contained galactose, N-acetylglucosamine, and N-acetylmannosamine in an approximate molar ratio of 3:2:1. This moiety was devoid of muramic acid, alanine, diaminopimelic acid, glutamic acid, and lipid, thus indicating that the isolated polysaccharide was of pure quality.     

An amphiphilic polysaccharide from an adhesive Rhodococcus strain

Abstract A Rhodococcus strain possessing a capsule, but no fimbriae, was isolated from pond water by adsorption to Teflon. The strain was hydrophobic, as shown by partitioning between dodecane and buffer. A high emulsifying activity was found in the culture supernatant, from which a polysaccharide was isolated. This contained glucuronic acid, glucose, galactose and rhamnose in a molecular ratio of 1:1:1:2. One acetate residue was found per repeating unit. The polysaccharide molecules formed clusters, which disaggregated on the addition of sodium dodecyl sulphate (SDS). Rabbit antibodies against this polysaccharide aggregated the bacterial cells. Thus, it can be concluded that this polysaccharide at least contributes to the cell surface hydrophobicity, thereby mediating in the adsorption of cells to inert hydrophobic surfaces.     

Antigenic polysaccharides of bacteria of the genus Shigella. The structure of the polysaccharide chain of Shigella boydii type 14

A specific acidic polysaccharide has been isolated from the Shigella boydii type 14 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of the D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose and D-galactose residues in the ratio 1:1:3. From the results of methylation analysis and partial acid hydrolysis, the structure of the repeating unit of the specific polysaccharide was deduced as follows: (-6DGalp alpha 1-4DGlcAp beta 1-6DGalp beta 1-4DGalp beta 1-4DGlcNAcp beta 1-)n. The 13C NMR spectra of native and carboxyl-reduced polysaccharides, as well as of oligosaccharides produced by partial acid hydrolysis fully confirmed the proposed structure. The approach was suggested to determine the type of substitution of uronic acid moieties in polysaccharide chain by use of chromato-mass-spectrometry of acetylated methyl esters of partially methylated aldonic acids. Serological characteristics of Sh. boydii LPS type 14 and its modified derivatives are discussed.     

Isolation and chemical characterization of compounds isolated fromAspergillus flavus conidia

Three types of heterogenous preparations and four types of preparations of polysaccharide nature were obtained in studies aimed at the isolation of active compounds fromAspergillus flavus conidia bearing their biological stimulatory activity. Extraction with trichloroacetic acid at 0 °C yielded a preparation in which the protein component predominated over the polysaccharide moiety at a ratio of 3: 1. In the preparation isolated from the phenolic phase of the phenol—water mixture at 68 °C the protein polysaccharide ratio was 1 : 1. In the material extracted in the aquoues phase and in that obtained by extraction with acetic acid at 100 °C the polysaccharide portion highly predominated (8 : 1 and 7 : 1 respectively).     

Structural studies on colanic acid, the common exopolysaccharide found in the Enterobacteriaceae, by partial acid hydrolysis. Oligosaccharides from colanic acid

The exopolysaccharide slime colanic acid has been isolated from representative strains of Escherichia coli, Salmonella typhimurium and Aerobacter cloacae. Analysis showed that each polymer contained glucose, galactose, fucose and glucuronic acid, together with acetate and pyruvate. The molar proportions of these components were 1:1.8:1.9:1:1:1 approximately. On the basis of periodate oxidation of the natural and deacetylated polysaccharide, glucose is proposed as the site of the acetyl groups. The pyruvate is attached to galactose. Three neutral oligosaccharides and ten electrophoretically mobile oligosaccharides were isolated and partially characterized. Four of the fragments were esters of pyruvic acid. Most oligosaccharides were isolated from all three polysaccharide preparations. Three further oligosaccharides were isolated from carboxyl-reduced colanic acid and sodium borotritide was used to label the glucose derived from glucuronic acid in these fragments. One trisaccharide was obtained from periodate-oxidized polysaccharide. On the basis of these oligosaccharides a repeating hexasaccharide unit of the following structure is proposed: [Formula: see text] The significance of this structure in colanic acid biosynthesis is discussed.     

Polysaccharide-protein complexes isolated from different strains of Neisseria meningitidis serogroup B

Fractionation of the biomass of 3 serogroup B N. meningitidis strains, obtained from solid serum-free and liquid synthetic media, by increasing concentrations of cetavlone revealed that the formation of natural polysaccharide-protein complexes with the ratio of their components approaching 1:1 was possible under the conditions ensuring the intensive synthesis of capsular polysaccharide. Two strains, 125 and 1642, grown on a solid amino peptide-containing medium regularly produced two polysaccharide-protein complexes with the protein/polysaccharide ratio approaching 1:1. One of these complexes passed easily into the supernatant fluid and could be precipitated with 0.1% cetavlone. The second complex was more firmly bound to the outer membrane of the cell and could be precipitated with 1% cetavlone. In most experiments an additional fraction with high protein content in relation to sialic acid was isolated from the biomass.     

Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella aerogenes type 54[A3(S1)]

Several strains of bacteriophage have been isolated that induce the formation of a polysaccharide hydrolase after infection of Klebsiella aerogenes type 54 [A3(S1)]. The action of this enzyme on polysaccharide solutions was to decrease their viscosity and increase their reducing value. These effects were associated with the release of two oligosaccharides (O1 and O2) from the polysaccharide. These two substances are not identical with any of the four oligosaccharides isolated from autohydrolysates. The two enzymically isolated fractions have been tentatively identified as tetrasaccharides, and oligosaccharide O2 is probably an acetylated version of oligosaccharide O1. This latter oligosaccharide differs in some way, still unknown, from the tetrasaccharide cellobiosylglucuronosylfucose found in acid hydrolysates of the slime polysaccharide. The enzyme is limited in its activity to the polysaccharide excreted by the A3 strain of K. aerogenes type 54 or by similar strains. It is also active on the polysaccharides altered by acid or alkaline treatment. The enzyme has optimum activity at pH6.5. A study of the products released by enzyme action has shown it to be a fucosidase splitting the fucosylglucose linkages found in the intact polysaccharide.     

Structural investigation of a heteropolysaccharide isolated from the pods (fruits) of Moringa oleifera (Sajina)

A water-soluble polysaccharide was isolated from the aqueous extract of pods of Moringa oleifera. The polysaccharide contains d-galactose, 6-O-Me-D-galactose, D-galacturonic acid, l-arabinose, and l-rhamnose in a molar ratio of 1:1:1:1:1. On the basis of total hydrolysis, methylation analysis, periodate oxidation, and NMR ((1)H, (13)C, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC) studies, the repeating unit of the polysaccharide is established as [structure: see text].     

Extracellular acid protease of Aspergillus oryzae grown on liquid media: multiple forms due to association with heterogeneous polysaccharides.

The acid protease (EC 2.4.23.6) that is produced extracellularly when Aspergillus oryzae is grown on liquid media has been isolated and characterized. The enzyme was purified by precipitation with tannic acid, chromatography on Duolite A-2, and gel filtration on Sephadex G-100. The last step yielded four active components, with varying molecular weights ranging from 42,000 to 60,000. Two of them, designated E1 and E1a, with molecular weights of 60,000 and 55,000, respectively, were heterogeneous on isoelectric focusing, both giving at least three enzyme species with different isoelectric points, whereas the other two, E1b and E2, with molecular weights of 49,000 and 42,000, respectively, were essentially homogeneous. These four enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They had essentially the same amino acid composition and immunologically cross-reacted with each other. These catalytic, chemical, and immunological properties are similar to those of acid protease A1 and A2 from A. oryzae grown on solid bran media. Unlike acid protease from solid bran culture, which contains both carbohydrate-containing and the carbohydrate-free species, all of the four enzymes, E1, E1a, E1b, and E2, contained carbohydrate, ranging from 18.9 to 43% and comprising three hexoses, glucose, galactose, and mannose. The carbohydrate portions were polysaccharide in nature and heterogeneous with respect to both molecular weight and sugar composition, and at least a part of the carbohydrate was present in the form of homopolysaccharides such as galactan and mannan. These findings indicate that polysaccharide chains with different molecular weights and with different chemical compositions are apparently responsible for the microheterogeneity of acid protease.     

Structural studies of a methyl galacturonosyl-methoxyxylan isolated from the stem of Lagenaria siceraria (Lau)

A water-soluble polysaccharide was isolated from the aqueous extract of the stem of Lagenaria siceraria. The polysaccharide was found to be constituted of methyl d-galacturonate, 2-O-methyl-D-xylose, and d-xylose in a ratio of 1:1:1. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, NMR studies ((1)H, (13)C, 2D-COSY, TOCSY, NOESY, HSQC, and HMBC), and MALDI-TOF MS analysis, the structure of the repeating unit of the polysaccharide is determined as.     

Structural assignment of a heteropolysaccharide isolated from the gum of Cochlospermum religiosum (Katira gum)

A heteropolysaccharide isolated from the gum (Katira) of Cochlospermum religiosum was found to consist of D-galactose, D-galacturonic acid and L-rhamnose in a molar ratio 2:1:3. Structural assignment of the polysaccharide was carried out using total acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation and NMR studies (1H, 13C, DQF-COSY, TOCSY, NOESY, HMBC and HSQC) and the repeating unit of the polysaccharide was established as [Formula: see text].     

Structural investigation of a heteroglycan isolated from the fruit bodies of an ectomycorrhizal fungus Astraeus hygrometricus

A polysaccharide fraction (AQS-II) has been isolated from the hot aqueous extract of the fruits of an ectomycorrhizal fungus Astraeus hygrometricus. It was found to contain 63% polysaccharide and 35% protein. The polysaccharide part contains glucose, galactose, and fucose in a 2:1:1 molar ratio. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, DQF-COSY, TOCSY, NOESY, HMBC, and HSQC) the structure of the repeating unit of the polysaccharide was established as [structure: see text].     

Xyloglucan from the leaves of Hymenaea courbaril

A fucosylated xyloglucan was isolated from the leaves of Hymenaea courbaril by alkaline extraction, followed by ethanol precipitation and ion-exchange chromatography. The isolated polysaccharide showed Glc:Xyl:Gal:Fuc in molar ratio of 8:5:2.5:1 and (D)(25) +40.5 degrees. Composition and linkage analyses, supported by NMR spectroscopic measurements, showed that the polysaccharide has a glucan backbone which is highly substituted at O-6 with D-xylopyranose residues, about a half of which are substituted at O-2 by D-galactopyranosyl units. Some of the galactose residues are further substituted by L-fucopyranose at O-2. The M(r), as determined by HPSEC, was 49,500.     

A bacterium yielding a polysaccharide with unusual properties

A yellow-pigmented bacterium, isolated in Czapek-Dox medium, yielded copious quantities of extracellular polysaccharide. Liquid cultures became bright yellow and increasingly viscous after incubation for 4–6 d. Characterization tests indicate that it is a starch-negative (amylase-negative), gelatinase-positive strain of Pseudomonas paucimobilis , closely resembling an organism previously described as Pseudomonas elodea. The polysaccharide contained L-rhamnose and D-glucose in a molar ratio of 0.66 : 1.0, together with 16% D-glucuronic acid and 10.4% acetate. Solutions of the polysaccharide were highly viscous: certain ions tended to cause gelation of aqueous solutions. After deacetylation with mild alkali the polymer at a concentration of 0.8% yielded a strong gel.     

Properties of an extracellular polysaccharide produced by a strain of enterobacter isolated from pond water

A bacterium which was isolated from pond water and identified as Enterobacter cloacae produced a viscous extracellular polysaccharide when it was grown aerobically in a medium containing sucrose as a sole source of carbon. The maximum molecular weight of the polysaccharide was about 9.0 x 10(5). The polysaccharide was composed of fucose, galactose, glucose, and glucuronic acid in a molar ratio of 2:3:2:1, but the molecular weight and the molar ratio of the sugar component were different from those of the polysaccharide produced by the same species reported elsewhere.     

Production of a Novel Extracellular Polysaccharide by a Bacillus Strain Isolated from Soil

A bacterium that was isolated from soil and identified as Bacillus circulans was found to produce a highly viscous extracellular polysaccharide when it was grown aerobically in a medium containing glucose as a sole source of carbon. The product was characterized by TLC and GC analyses as a novel heteropolysaccharide consisted of rhamnose, mannose, galactose, and mannuronic acid as sugar components. A maximal yield of polysaccharide reached about 2 g/liter by jar-fermentor culture at 30°C for 48 hr with a medium containing 1% glucose, 0.05% asparagine, 0.005% yeast extract, and small amounts of inorganic salts. Some culture conditions for the production of polysaccharide were investigated with flask culture; an optimal production was attained with a medium containing 0.1–1 % glucose and 0.01–0.05% asparagine, pH 7–8, at 30°C under aerobic conditions.     

A novel highly acidic polysaccharide with inhibitory activity on calcification from the calcified scale "coccolith" of a coccolithophorid alga, Pleurochrysis haptonemofera

Coccolith, a calcified scale with species-specific fine structure produced by marine unicellular coccolithophorid algae, consists of calcium carbonate (CaCO(3)) crystals and a small amount of organic matrices. A novel polysaccharide named coccolith matrix acidic polysaccharide (CMAP) was isolated from the coccolith of a coccolithophorid alga, Pleurochrysis haptonemofera. The structure of CMAP was determined by chemical analysis and NMR spectroscopy including COSY, TOCSY, HMQC, and HMBC to be a polysaccharide composed of the following unit: -->4) l-iduronic acid (alpha1-->2) meso-tartaric acid (3-->1) glyoxylic acid (1-->. It has four carboxyl groups per a disaccharide unit as observed in another polysaccharide PS-2 characterized previously in Pleurochrysis carterae. CMAP showed a strong inhibitory activity on CaCO(3) precipitation. These results suggest that CMAP serves as a regulator in the calcification of the coccolith.     

Isolation and Chemical Structure of the Peptidoglycan of Spirillum serpens Cell Walls

The peptidoglycan layer of Spirillum serpens cell walls was isolated from intact cells after treatment with sodium dodecylsulfate and digestion with Pronase. The isolated peptidoglycan contained glucosamine, muramic acid, alanine, glutamic acid, and meso-diaminopimelic acid in the approximate molar ratio of 1:1:2:1:1. Aspartic acid and glycine were the only other amino acids found in significant quantities. N-terminal amino acid analyses of the tetrapeptide amino acids in the peptidoglycan revealed that 54% of the diaminopimelic acid molecules are involved in cross-linkage between tetrapeptides. This amount of cross-linkage is greater than that found in the peptidoglycan of previously studied cell walls of gram-negative bacteria. The polysaccharide backbone was isolated, after myxobacter AL-1 enzyme digestion of the peptidoglycan, by fractionation with ECTEOLA-cellulose and Sephadex G-100. An average length of 99 hexosamines for the polysaccharide chains was found (ratio of total hexosamines to reducing end groups).     

Effect of culture media on the chemical and physical characteristics of polysaccharides isolated from Poria cocos mycelia

Mycelia of a wild strain Poria cocos were cultured in two media differing in one constituent: bran extract or corn steep liquor, and are designated as wb and wc, respectively. Six polysaccharide fractions were isolated sequentially from the two mycelia by 0.9% NaCl (PCM1), hot water (PCM2), 0.5 M NaOH (PCM3-I and -II) and 88% formic acid (PCM4-I and -II). Their chemical and physical characteristics were determined by infrared spectroscopy (IR), gas chromatography (GC), 13C NMR, light scattering (LS) and viscometry. The results indicated that wb-, wc-PCM1, and PCM2 were heteropolysaccharides mainly composed of alpha-D-glucose, mannose, and galactose, whereas wb-PCM3-I and wc-PCM3-I were mainly (1-->3)-alpha-D-glucans, and wb- and wc-PCM3-II, PCM4-I and PCM4-II were (1-->3)-beta-D-glucans. Interestingly, (1-->3) alpha- and (1-->3)-beta-D-glucans co-existed in the 0.5 M NaOH fraction and were separated individually into the two fractions (PCM3-I and PCM3-II) after neutralizing with acetic acid. The polysaccharides from wc-PCM cultured in media containing corn steep liquor contained relatively more protein. The polysaccharide fractions also existed in conformations including random coil (as in PCM0 and PCM1) and expanded chain (as in PCM3), and differed molecular mass. In addition, two exo-polysaccharides isolated from the two culture media by methanol precipitation (wb- and wc-PCM0) also differed in their monosaccharide composition.