Influence of growth conditions on production of capsular and extracellular polysaccharides byRhizobium leguminosarum

The influence of growth rate and medium composition on exopolymer production byRhizobium leguminosarum was studied. When grown in medium containing 10g/l mannitol and 1g/l glutamic acid,Rhizobium leguminosarum biovartrifolii TA-1 synthesized up to 2.0g/l of extracellular polysaccharide (EPS), and up to 1.6g/l of capsular polysaccharide (CPS). Under non-growing cell conditions in medium without glutamic acid, CPS synthesis by strain TA-1 could proceed to 2.1g/l, while EPS-production remained relatively low (0.8g/l). Maximal CPS-yield was 2.9g CPS/l medium in a medium containing 20g/l mannitol and 2g/l glutamic acid. TheEPS-deficient strain R. leguminosarum RBL5515,exo4::Tn5 was able to produce CPS to similar levels as strain TA-1, but CPS-recovery was easier because of the low viscosity of the medium and growth of the cells in pellets. With strain TA-1 in nitrogen-limited continuous cultures with a constant biomass of 500mg cell protein/l, EPS was the most abundant polysaccharide present at every dilution rate D (between 0.12 and 0.02 h^–1). The production rates were 50–100mg/g protein/h for EPS and 15–20mg/g protein/h for CPS. Only low amounts of cyclic -(1,2)-glucans were excreted (10–30 mg/l) over the entire range of growth rates.Abbreviations bv biovar - CPS capsular polysaccharide - EPS extracellular polysaccharide - HM_r high molecular mass - LM_r low molecular mass - YEMCR Yeast Extract-Mannitol-Congo Red agar     

Structural studies of the antigen III cell wall polysaccharide of Trichosporon domesticum

Cell wall and soluble polysaccharides that reacted with Trichosporon domesticum factor III serum were isolated from the type strain of T. domesticum. The fractions contained O-acetyl groups, which contributed to the serological reactivity. The antigenic structure was characterized by chromatographic and spectroscopic methods. The polysaccharide has an α-(1→3)- -mannan backbone with hetero-oligosaccharide side chains consisting of a 2-O-substituted β- -glucuronic acid residue bound to O-2 of the mannose residue, β- -xylopyranosyl residues located in the middle of the side chain, and a nonreducing terminal α- -arabinopyranosyl residue bound to O-4 of xylose. The mannan backbone is O-acetylated at O-6 of the mannose residues.     

Acidic heteroxylans from medicinal plants and their anti-ulcer activity

Xylans are the main hemicelluloses found in higher plants, and are often present in phytotherapic medicines. An acidic heteroxylan was obtained from Maytenus ilicifolia leaves by hot 10% aqueous KOH extraction. This was subjected to freeze-thawing process, giving insoluble and soluble fractions and the latter treated with Fehling solution. Its insoluble fraction (MI-HX) was further examined. The acidic heteroxylan gave xylose, galactose, glucose, and 4-O-methylglucuronic acid in a 76:6:9:9 molar ratio and methylation analyses and ¹³C NMR spectroscopy showed its main chain consists of 4-O-linked β-d-Xylp units. This polysaccharide and another acidic heteroxylan from Phyllanthus niruri had anti-ulcer activity and were able to reduce gastric lesions induced by ethanol by 65% and 78%, with ED₅₀ = 40.0 and 20.4 mg/kg, each respectively. These results suggest that this class of polysaccharide has a protective anti-ulcer effect, and that there is a relation between its chemical structure and biological function.     

Presence of rhamnogalacturonan II in the juices produced by enzymatic liquefaction of Agave pulquero stem (Agave mapisaga)

Rhamnogalacturonan II (RG-II), a small complex pectic polysaccharide is released from Agave pulquero stems (Agave mapisaga), after the production period of aguamiel. RG-II was obtained by treatment with two commercial liquefying enzyme preparations, it was isolated by size-exclusion chromatography and characterized. RG-IIs contains diagnostic sugars such as apiose, 2-O-methyl-l-fucose, 2-O-methyl-d-xylose, aceric acid, Kdo and Dha. Glycosyl-linkage compositions of the Agave pulquero RG-II like structures are similar to the theoretical model described through sycamore RG-II structure. The presence of 3′-linked apiose indicates that the obtained juice from Agave pulquero plant contains the free RG-II dimer. Thus, when pectinolytic enzyme preparations are used to process Agave pulquero (A. mapisaga), RG-II is released as one of the main soluble polysaccharide fraction.     

Cyanidin 3-[6-(p-coumaroyl)-2-(xylosyl)-glucoside]-5-glucoside and other anthocyanins from fruits of sambucus canadensis

From the fruits of Sambucus canadensis four anthocyanin glycosides have been isolated by successive application of an ion-exchange resin, droplet-counter chromatography and gel filtration. The structure of the novel, major (69.8%) pigment, cyanidin 3-O-[6-O-(E-p-coumaroyl-2-O-(β- -xylopyranosyl)-β- -glucopyranoside]-5-O-β- -glucopyranoside, was determined by means of chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques. The other anthocyanins were identified as cyanidin 3-sambubioside-5-glucoside (22.7%), cyanidin 3-sambubioside (2.3 %) and cyanidin 3-glucoside (2.1 %).     

Stimulation of extracellular polysaccharide synthesis in oat protoplasts by the host-specific phytotoxin victorin

The host-specific phytotoxin victorin (HV-toxin) stimulates mesophyll protoplasts of susceptible but not of resistant oat (Avena sativa L.) to produce an amorphous, ethanol-insoluble extracellular material which stains with Calcofluor white and aniline blue. Over a 24-h period incorporation of [¹⁴C]glucose into ethanol-insoluble products is maximally stimulated by 60 pg victorin/ml, whereas at 6 ng/ml initial rates of incorporation are higher but the protoplasts collapse. The extracellular material produced in response to victorin is solubilized by cold 4.4 N NaOH and by commercial laminarinase and pectinase. Incorporation of [¹⁴C]glucose into cellulose (material resistant to Updegraff's acetic-nitric acid reagent) is stimulated as much as incorporation into other wall polysaccharides, but cellulose constitutes less than 15% of the total victorin-stimulated incorporation. Synthesis of ethanol-insoluble material that can be digested by pronase, i.e. protein, is inhibited by victorin above 60 pg victorin/ml. Formation of extracellular polysaccharide is stimulated at concentrations of victorin which cause almost complete inhibition of protein synthesis, indicating that de-novo protein synthesis is not involved. Preincubation of protoplasts with inhibitors of RNA or protein synthesis prevents both extracellular polysaccharide synthesis and cell death in response to victorin. Although previous studies have indicated a link between calcium and the action of victorin, several compounds which interact with calcium do not influence this response to victorin.Abbreviations EPS extracellular polysaccharide - SCM 0.5 M sorbitol +10 mM CaCl₂+5 mM 2-(N-morpholino)-ethanesulfonic acid (Mes), pH 5.8 This paper is dedicated to the memory of our teacher and colleague, Martin H. Zimmermann (1926–1984)     

Biochemical properties of pigeon milk and its effect on growth

Summary The avian juvenile food pigeon milk was studied for its chemical composition and effect on growth in vivo and in vitro. Pigeon milk on a wet weight basis consisted of 9–13% protein, 9–11% fat, 0.9–1.5% carbohydrate, 0.8–1.1% ash, 0.10–0.12% non-protein nitrogen, energy content 5.6–6.8 kcal·g^-1. Except for proteins there was little or no decrease in pigeon milk constitutents during the first week of secretion. Pigeon milk proteins consisted of trichloroacetic acid (precipitable), trichloroacetic acid (soluble), and free amino acid components in the ranges 8.4–12.1%, 0.5–0.7% and 1.4–2.5%, respectively; whereas the level of trichloroacetic acid (precipitable) and trichloroacetic acid (soluble) components decreased by about 30%, that of the free amino acids increased by 9% in the first week. About 0.6–1.0% of pigeon milk sugar was found in the trichloroacetic acid (soluble) fraction and increased by 67% in the first week. The remainder was found in the trichloroacetic acid (precipitable) fraction and did not change during this period. Major lipids of pigeon milk were the neutral lipids (7.8–8.4%); the minor lipids were glycolipids (0.9–1.6%), phospholipids (0.5–1.4%) and cholesterol (0.5–0.6%). Squabs fed pigeon milk increased their body weight by 22-fold in the first 3 weeks after hatching, and crude extracts of pigeon milk stimulated the growth of cultured hamster ovary cells. These results reflect the ability of pigeon milk to stimulate growth both in vivo and in vitro.Abbreviations AOAC association of official analytical chemists - BRIT board of radiation and isotope technology - CHO chinese hamster ovary - DNA deoxyribonucleic acid - EDTA ethylenediaminetetra-acetic acid - FCS foetal calf serum - GF growth factor - GS goat serum - MEM minimum essential medium - NPN nonprotein nitrogen - PBS phosphate-buffered saline - PM pigeon milk - TCA(P) trichloroacetic acid precipitable fraction - TCA(S) trichloroacetic acid soluble fraction     

Iridoid glucosides from Thunbergia laurifolia

Two iridoid glucosides, 8-epi-grandifloric acid and 3′-O-β-glucopyranosyl-stilbericoside, were isolated from the aerial part of Thunbergia laurifolia along with seven known compounds, benzyl β-glucopyranoside, benzyl β-(2′-O-β-glucopyranosyl) glucopyranoside, grandifloric acid, (E)-2-hexenyl β-glucopyranoside, hexanol β-glucopyranoside, 6-C-glucopyranosylapigenin and 6,8-di-C-glucopyranosylapigenin. Strucural elucidation was based on the analyses of spectroscopic data.     

A polysaccharide from Lichina pygmaea and L. confinis supports the recognition of Lichinomycetes

The lichen-forming order Lichinales, generally characterized by prototunicate asci and the development of thalli with cyanobacteria, has recently been recognized as a separate class of ascomycetes, Lichinomycetes, as a result of molecular phylogenetic studies. As alkali and water-soluble (F1SS) polysaccharides reflect phylogeny in other ascomycetes, a polysaccharide from Lichina pygmaea and L. confinis was purified and characterized to investigate whether these F1SS compounds in the Lichinomycetes were distinctive. Nuclear magnetic resonance (NMR) spectroscopy and chemical analyses revealed this as a galactomannan comprising a repeating unit consisting of an α-(1→6)-mannan backbone, mainly substituted by single α-galactofuranose residues at the O-2- or the O-2,4- positions linked to a small mannan core. With the exception of the trisubstituted mannopyranose residues previously described in polysaccharides from other lichens belonging to orders now placed in Lecanoromycetes, the structure of this galactomannan most closely resembles those found in several members of the Onygenales in Eurotiomycetes. Our polysaccharide data support molecular studies showing that Lichina species are remote from Lecanoromycetes as the galactofuranose residues are in the α-configuration. That the Lichinomycetes were part of an ancestral lichenized group can not be established from the present data because the extracted polysaccharide does not have the galactofuranose residue in the β configuration; however, the data does suggest that an ancestor of the Lichinomycetes contained a mannan and was part of an early radiation in the ascomycetes.     

Production of a hypoglycemic, extracellular polysaccharide from the submerged culture of the mushroom, Phellinus linteus

Mycelial growth and extracellular polysaccharide production of Phellinus linteus were optimal at pH 5 and 25 °C. Maximum biomass production (14.2 g l^–1) was after 15 d of cultivation, whereas, extracellular polysaccharide was maximal (3.5 g l^–1) after 21 d. The hypoglycemic effect of the polysaccharide, investigated in streptozotocin-induced diabetic rats, decreased plasma glucose, total cholesterol and triacylglycerol concentrations by 49%, 32%, and 28%, respectively, and aspartate aminotransferase activity by 20%. The results indicate the potential of this polysaccharide to prevent hyperglycemia in diabetic patients.     

An extracellular sulfated fucose-rich polysaccharide produced by a tropical strain of Cryptomonas obovata (Cryptophyceae)

A tropical strain of Cryptomonas obovata Skuja, isolated from a shallow oxbow lake,releaseda sulfated fucose-rich polysaccharide. The polysaccharide is composed mainly offucose (42%), N-acetyl-galactosamine (26%) and rhamnose (15%), with smallquantities of glucuronic acid, mannose, galactose, xylose and glucose. Sulfateaccounted for 1.7% total polysaccharide. Quantitative release was studied withcells exposed to optimal culture conditions contrasted with high irradiance andnitrate depletion. This latter set of conditions could simulate stresssituations usually found in the place from which this strain was isolated. Themonosaccharide composition of the polysaccharide was evaluated using PAD-HPLCand gas chromatography. The two irradiances tested (165 molm^–2 s^–1 and 2000 molm^–2 s^–1) had no significant effect onamounts of polysaccharide released by the cells. Differences were observed whenthe nitrate availability was varied. In the nitrate-depleted situation,extracellular polysaccharide production was 2.5 times higher than replete cellsafter 6 h at 165 mol m^–2s^–1, and 2.25 times higher at 2000 molm^–2 s^–1.     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

Exopolysaccharide production by a unicellular cyanobacterium isolated from a hypersaline habitat

The unicellular cyanobacterial strain 16Som2, isolated from a Somaliland saltpan and identified asCyanothece sp., is characterized by cells surrounded by a thick polysaccharidic capsule, the external part of which dissolves into the medium during growth, causing a progressive increase in culture viscosity. In spite of this, the thickness of the capsule remained almost constant under all the culture conditions tested, demonstrating that the processes of its synthesis and solubilization occurred at a similar rate. The synthesis of carbohydrates was neither enhanced by increasing salinity (sea-water enriched with NaCl in the range 0 to 2.0 M) nor by Mg²⁺, K⁺ or Ca²⁺ deficiencies. In contrast, N-limitation and, to a lesser extent, P-limitation induced a significant enhancement of carbohydrate synthesis; in particular, N-deficiency stimulated the synthesis of all the carbohydrate fractions (intracellular, capsular and soluble). The soluble polysaccharide, separated from the culture medium and hydrolyzed with 2N trifluoroacetic acid, showed a sugar composition consisting of glucuronic acid: galacturonic acid: galactose: glucose: mannose: xylose: fucose in a molar ratio of 1: 2: 2.4: 6.8: 4.8: 2.9: 1.6.Cyanothece sp. culture subjected to nitrogen starvation synthesized polysaccharide with a mean productivity of 115 mg (EPS) l^–1d^–1, for the polymer solubilized into the medium, and of 15 mg (CPS) l^–1d^–1 for the capsular polysaccharide.     

Acylated flavonol diglucosides from Lotus polyphyllos

Three acylated flavonol diglucosides, kaempferol 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; quercetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; isorhamnetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside were isolated from the whole plant aqueous alcohol extract of Lotus polyphyllos. The known 3,7-di-O-glucosides of the aglycones kaempferol, quercetin and isorhamnetin were also characterized. All structures were established on the basis of chemical and spectral evidence.     

Kaempferol triosides from Silphium perfoliatum

Two apiose-containing kaempferol triosides, together with nine known flavonoids were isolated from the leaves of Silphium perfoliatum L. Their structures were elucidated by acid hydrolysis and spectroscopic methods including UV, LSI MS, FAB MS, CI MS, ¹H, ¹³C and 2D-NMR, DEPT, HMQC and HMBC experiments. The two new compounds were identified as kaempferol 3-O-β- -apiofuranoside 7-O-α- -rhamnosyl-(1′→6)-O-β- -galactopyranoside and kaempferol 3-O-β- -apiofuranoside 7-O-α- -rhamnosyl-(1→ 6)-O-β- (2-O-E-caffeoylgalactopyranoside).     

Structural investigation of a polysaccharide released by the cyanobacterium <Emphasis Type="Italic">Nostoc insulare</Emphasis>

The structural investigation of an extracellular polysaccharide released during photoautotrophic growth by the cyanobacterium Nostoc insulare is reported. After 60 days of cultivation, an average yield of purified, desalted, and freeze-dried released polysaccharide (RPS) of 0.9 g L⁻¹ medium was obtained. The apparent hydrodynamic volume, determined for RPS, was 1.1 × 10⁶ Da, and the average molecular weight was 2.8 × 10⁶ Da. No sulfate and only traces of pyruvate and acetate groups were detectable. A protein content of only 0.7% indicates a high degree of purity of RPS. The following constituent uronic acids and sugars were identified: glucuronic acid (GlcA), glucose (Glc), arabinose (Ara), and for the first time, cyanobacterial RPSs 3-O-methyl-arabinose (3-O-Methyl-Ara). Adapted from linkage analyses of untreated RPS and of RPS treated by means of reduction of uronic acids, mild acid hydrolysis with oxalic acid, or lithium degradation, respectively, the following partial structure of RPS is proposed, which possesses an arborisation built by 1,3,4-Glcp and a side chain built by 3-O-Methyl-Araf: →1)-Glcp-(3→1)-Glcp-[(3→1)-3-O-Methyl-Araf](4→1)-GlcAp-(4→).     

The capsule and slime polysaccharides of the wild type and a phage resistant mutant ofRhodopseudomonas capsulata St. Louis

A rhamnose, galactose and pyruvic acid containing polysaccharide (capsule) together with the peptidoglycan was isolated fromRhodopseudomonas capsulata St. Louis as the insoluble sediment after sodium dodecyl sulfate extraction of cell envelope fractions. Treatment with pronase E separated the soluble polysaccharide from the insoluble peptidoglycan. After lysozyme-digestion, both the capsule polysaccharide and peptidoglycan were soluble.The capsule was also accumulated in the combined interphase/phenol-phase of hot phenol-water extracts of whole cells. Again, the capsule and peptidoglycan were sedimented together as long as no pronase E-treatment was performed. With the phage-resistant mutant (R. capsulata St. Louis RC1^-), no capsule polysaccharide was obtained in the combined interphase/phenol phase.An acidic polysaccharide (slime) different from the capsule in composition and serology was obtained by Cetavlon fractionation of hot phenol/water extracts of cells of both the wild-type and the mutant cells. It was shown to consist mainly of rhamnose, glucosamine and galacturonic acid.The use of O/K-antisera and of capsule polysaccharideantisera allowed a separate visualization of the capsule and slime layers.This paper is dedicated to Professor Hans G. Schoegel on the occasion of his 60th birthday     

Biosynthesis of heparin

The formation of labeled heparin-precursor polysaccharide (N-acetylheparosan) from the nucleotide sugars, UDP-[¹⁴C]glucuronic acid and UDP-N-acetylglucosamine, in a mouse mastocytoma microsomal fraction was abolished by the addition of 1% Triton X-100. In contrast, the detergent-treated microsomal preparation retained the ability to convert such preformed polysaccharide into sulfated products during incubation with 3-phosphoadenylylsulfate (PAPS). However, as shown by ion-exchange chromatography of these products, the detegent treatment changed the kinetics of sulfation from the rapid, repetivive process characteristic of the unperturbed system to a slow, progressive sulfation, which involved all polysarccharide molecules simultaneously and yielded, ultimately, a more highly sulfated product. The detergent effect was attributed to solubilization of sulfotransferases from the microsomal membranes, along with other polymer-modifying enzymes and the polysaccharide substrate. The resulting product showed an apparently random distribution ofN-acetyl andN-sulfate groups, instead of the predominantly block-wise arrangement achieved through membrane-associated biosynthesis.O-Sulfation occurred mainly at C2 of the iduronic acid units in the membrane-bound polysaccharide but at C6 of the glucosamine residues in the presence of detergent.A capsular polysaccharide fromEscherichia coli K5, previously found to have a structure identical to that of the nonsulfated heparin-precursor polysaccharide, was sulfated in the solubilized system in a fashion similar to that of the endogenous substrate, but was not accessible to the membrane-bound enzymes.These findings suggest that the regulation of the polymer-modification process, and hence the structure of the final polysaccharide product, depends heavily on the organization of the enzymes and their proteoglycan substrate in the endoplasmic membranes of the cell.Abbreviations PAPS 3-phosphoadenylylsulfate - Hepes 4-(2-hydroxy-ethyl)piperazineethanesulfonic acid - GlcUA glucuronic acid This is Paper XIV of a series in which the preceeding reports are refs 10 and 12. A preliminary report has appeared [28].     

Improved synthesis and the crystal structure of methyl 4,6-dideoxy-4-(3-deoxy- -glycero-tetronamido)-α- -mannopyranoside, the methyl α-glycoside of the intracatenary repeating unit of the O-polysaccharide of Vibrio cholerae O:1

The crude product of deamination of the commercially available -homoserine was acetylated and the 2-O-acetyl-3-deoxy- -glycero-tetronolactone (18) formed was used to N-acylate methyl perosaminide (methyl 4-amino-4,6-dideoxy-α- -mannopyranoside, 12) and its 2,3-O-isopropylidene derivative. The major product isolated from the reaction was the crystalline methyl 4-(4-O-acetyl-3-deoxy- -glycero-tetronamido)-4,6-dideoxy-α- -mannopyranoside (1, 70–75%) resulting from acetyl group migration in the initially formed 2'-O-acetyl derivative. O-Deacetylation of 1 gave the title amide 2. Compound 2, obtained crystalline for the first time, was fully characterized, and its crystal structure was determined. Deoxytetronamido derivatives diastereomeric with 1 and 2, respectively, were obtained by the acylation of 12 with 2-O-acetyl-3-deoxy- -glycero-tetronolactone (prepared from -homoserine), and subsequent deacetylation. Structures of several byproducts of the reaction of 12 with 18 have been deduced from their spectral characteristics. Since these byproducts were various O-acetyl derivatives of 2, the title compound could be obtained in ≈ 90% yield by deacetylating (Zemplén) the crude mixture of N-acylation products, followed by chromatography.     

Xanthone O-glycosides from Polygala tenuifolia

Four xanthone O-glycosides, polygalaxanthones IV–VII were isolated from the roots of Polygala tenuifolia Willd., together with eight known compounds. The structures of the four xanthone O-glycosides were established as 6-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1-hydroxy-3,7-dimethoxyxanthone (polygalaxanthone IV), 6-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1,3-dihydroxy-7-methoxyxanthone (polygalaxanthone V), 6-O-(β- -glucopyranosyl)-1,2,3,7-tetramethoxyxanthone (polygalaxanthone VI), and 3-O-[α- -rhamnopyranosyl-(1→2)-β- -glucopyranosyl]-1,6-dihydroxy-2,7-dimethoxyxanthone (polygalaxanthone VII), respectively, on the basis of analysis of spectroscopic evidence.