Mannuronic Acid Research Articles

An approach to the synthesis of α-(1-6)- C-disaccharides by tandem Tebbe methylenation and Claisen rearrangement

Uronic acids, most efficiently synthesised from the corresponding alcohols by two step Dess-Martin and sodium chlorite mediated oxidation, may be used as coupling partners for esterification with an allo glycal as substrates for the tandem Tebbe/Claisen approach to the synthesis of 1-6 linked C-disaccharides. Whilst esters of glucuronic and mannuronic acids successfully undergo Tebbe methylenation, esters derived from galacturonic acids are unreactive under these conditions. Thermal Claisen rearrangement of vinyl ethers produced by methylenation yields α- C-disaccharides with complete control of anomeric stereochemistry.

Real-time 3-D dark-field microscopy for the validation of the cross-linking process of alginate microcapsules

Alginate-based microencapsulation is a promising method for long-term maintenance of cellular and membrane function of the cells and tissue fragments required for in vitro and in vivo biosensors, for tissue engineering and particularly for immunoisolation of non-autologous transplants. Microcapsules of high mechanical strength and optimum permeability can be produced by injection of BaCl 2 crystals into alginate droplets before they come into contact with external Ba 2+. A key requirement is that the system parameters (number of crystals, speed of the crystal stream etc.) are properly adjusted according to the mannuronic and guluronic acid ratio and the average molecular mass of the alginate as well as to the diameter of the microcapsules. Robust, reliable, rapid and low-cost validation tools are, therefore, needed for assurance of the microcapsule quality. Here, we describe a novel three-dimensional (3-D) dark-field microscopy that allows the real-time measurement of the number and spatial distribution of the injected Ba 2+ ions throughout the microcapsules after treatment with sulphate. This novel method requires only a conventional microscope equipped with three polarising filters and a double aperture stop. In contrast to confocal laser scanning microscopy images, peripherally attached BaSO 4 precipitates can clearly be distinguished from internal ones. The data also demonstrate that several steps of the alginate gelling process must be improved before such immunoisolation can be used in patients.

Polysaccharides of Algae 58. The polysaccharide composition of the Pacific brown alga Alaria fistulosa P. et R. (Alariaceae, Laminariales)

Quantitative determination of mannitol, fucoidan and alginate in different parts of the thallus (the blade, the midrip, the sporophylls, and the stipe with rhizoids) of the brown alga Alaria fistulosa, collected from the coastal waters of Paramushir Island, was carried out. Sporophylls were shown to differ considerably in carbohydrate composition from all other parts of the plant. In particular, the fucoidan content in sporophylls is several times as high as that in the blade. Alginate preparations isolated from sporophylls and from blade have different proportions of monomers (mannuronic acid and guluronic acid residues) and different molecular masses. Fucoidan preparations isolated from these parts of the plant differ slightly in the sulfate content. Their specific feature is a rather high galactose content comparable with the content of fucose. The sporophylls of A. fistulosa may be regarded as a rich source for practical isolation of fucoidan.

Isolation and identification of an EPS-producing Rhizobium strain from arid soil (Algeria): characterization of its EPS and the effect of inoculation on wheat rhizosphere soil structure

The production of exopolysaccharides (EPSs) by bacterial populations in the rhizosphere has been demonstrated to contribute to water and nutrient uptake by plant roots through the modification of the physical properties of rhizosphere soil. We report here the characterization of a new EPS produced by a bacterial strain (KYGT207) isolated from an arid soil in southern Algeria (Gassi Touil), and the effect of inoculation of this strain on soil physical properties in the rhizosphere of Triticum durum L. Strain KYGT207 was assigned to the genus Rhizobium by 16S ribosomal DNA sequencing and belongs to a new species closely related to Rhizobium sullae. The EPS produced by this strain was found to be composed of glucose (Glc), galactose (Gal), and mannuronic acid (ManA) in a molar ratio of 2:1:1. The primary structure of the EPS was determined by sugar analysis, 1D and 2D NMR spectroscopy, consisting of a tetrasaccharide repeating unit with the following original structure: [Display omitted] . A rheological analysis showed that this EPS could be considered as a thickening agent with polyelectrolyte properties. Inoculation of wheat plantlets with strain KYGT207 caused significant promotion of plant growth (+85% for shoot dry mass and +56% for root dry mass), a significant increase in root-adhering soil (RAS) dry mass (dm) per root dm (RAS/RT) up to 137%, and in RAS aggregate water stability. We demonstrate that EPS-producing bacteria were present in sandy soils subjected to water stress and that EPS-producing Rhizobium populations play an important role in the rhizosphere through their contribution to soil aggregation.

Long-Term Normoglycemia in Rats Receiving Transplants with Encapsulated Islets

To follow up on previously successful transplantation of encapsulated islets in mice, the present study was performed in rats to determine the effects of several factors, including alginate composition and concentration of cross-linking agent and capsule size on the effectiveness of encapsulated islets. Highly purified alginate of either high guluronic acid or high mannuronic acid (M) with low endotoxin content was used. Regular-size (0.8-1.1 mm) or small microcapsules (0.5-0.7 mm) were produced by cross-linking with BaCl2 without additional poly-L-lysine coating and were transplanted into abdominal cavity of normoglycemic (empty capsules) or streptozotocin induced diabetic Lewis rats (islet containing capsules). Empty regular-size capsules made of different alginate compositions had similar biocompatibility and stability results. Compared with empty capsules, regular-size capsules made of high-M alginate containing syngeneic islets had inferior stability indicated with lower fractional volume retrieved. Islet-containing smaller-size microcapsules made of high-M alginate were more stable and had less cellular attachment compared with the regular-size capsules, although the normoglycemic period was comparable between two groups of rats receiving transplants with smaller-size microcapsules (48+/-8 days, n=8) or regular-size capsules (59+/-11 days, n=4) in allogeneic experiments. In syngeneic experiments, all of the rats (n=4) maintained normoglycemia up to 210 days after transplantation. These results indicate that regular-size alginate capsules do less well in rats than in our previous experiments with mice. Smaller capsules made of alginate cross-linked with barium appear to provide better stability and may be a useful strategy for use in larger recipients.

Antibacterial activity of lyase-depolymerized products of alginate

A series of mannuronic acid (M-block) and guluronic acid (G-block) fractions (M1–M5 and G1–G5) with different molecular weights were obtained by lyase depolymerization of alginate and evaluated for in vitro antibacterial activity against 19 bacterial strains. The antibacterial data revealed that both types of fractions generally showed activity against certain tested bacteria, whereas M-block fractions showed broader spectra and more potent inhibition than G-block fractions. Among these fractions, M3 (molecular weight 4.235 kDa) exhibited the broadest spectrum of inhibition and high inhibitory activity against Escherichia coli (minimal inhibitory concentration, MIC = 0.312 μg mL−1), Salmonella paratyphi B (MIC = 0.225 μg mL−1), Staphylococcus aureus (MIC = 0.016 μg mL−1) and Bacillus subtilis (MIC = 0.325 μg mL−1).

Tailor-Made Alginate Bearing Galactose Moieties on Mannuronic Residues: Selective Modification Achieved by a Chemoenzymatic Strategy

1-Amino-1-deoxygalactose (12%, mole) has been chemically introduced on a mannuronan sample via an N-glycosidic bond involving the uronic group of the mannuronic acid (M) residues. The unsubstituted M residues in the modified polymer were converted into guluronic moieties (G) by the use of two C-5 epimerases, resulting in an alginate-like molecule selectively modified on M residues. The molecular details of the newly formed polymer, in terms of both composition and molecular dimensions, were disclosed by use of (1)H NMR, intrinsic viscosity, and high-performance size-exclusion chromatography-multiple-angle laser light scattering (HPSEC-MALLS). Circular dichroism has revealed that the modified alginate-like polymer obtained after epimerization was able to bind calcium due to the introduction of alternating and homopolymeric G sequences. The gel-forming ability of this M-selectively modified material was tested and compared with an alginate sample containing 14% galactose introduced on G residues. Mechanical spectroscopy pointed out that the modified epimerized material was able to form stable gels and that the kinetics of the gel formation was similar to that of the unsubstituted sample. In contrast, the G-modified alginate samples showed a slower gel formation, eventually leading to gel characterized by a reduced storage modulus. The advantage of the selective modification on M residues was confirmed by measuring the Young's modulus of gel cylinders of the different samples. Furthermore, due to the high content in alternating sequences, a marked syneresis was disclosed for the modified-epimerized sample. Finally, calcium beads obtained from selectively M-modified alginate showed a higher stability than those from the G-modified alginate, as evaluated upon treatment with nongelling ions.

Human Monoclonal Antibodies toPseudomonas aeruginosaAlginate That Protect against Infection by Both Mucoid and Nonmucoid Strains

Two fully human mAbs specific for epitopes dependent on intact carboxylate groups on the C6 carbon of the mannuronic acid components of Pseudomonas aeruginosa alginate were found to promote phagocytic killing of both mucoid and nonmucoid strains as well as protection against both types of strains in a mouse model of acute pneumonia. The specificity of the mAbs for alginate was determined by ELISA and killing assays. Some strains of P. aeruginosa did not make detectable alginate in vitro, but in vivo protection against lethal pneumonia was obtained and shown to be due to rapid induction of expression of alginate in the murine lung. No protection against strains genetically unable to make alginate was achieved. These mAbs have potential to be passive therapeutic reagents for all strains of P. aeruginosa and the results document that alginate is a target for the proper type of protective Ab even when expressed at low levels on phenotypically nonmucoid strains.

AlgX is a periplasmic protein required for alginate biosynthesis in Pseudomonas aeruginosa.

Alginate, an exopolysaccharide produced by Pseudomonas aeruginosa, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients. Previous studies identified a gene, algX, within the alginate biosynthetic gene cluster on the P. aeruginosa chromosome. By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm. Consistent with these results is the presence of a 26-amino-acid signal sequence. To examine the requirement for AlgX in alginate biosynthesis, part of algX in P. aeruginosa strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette. The resulting algXDelta::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The algXDelta::Gm mutant was restored to the mucoid phenotype with wild-type P. aeruginosa algX provided on a plasmid. The algXDelta::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths. Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid. These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.

AlgR functions in algC expression and virulence in Pseudomonas syringae pv. syringae.

Pseudomonas syringae pv. syringae strain FF5 is a phytopathogen associated with a rapid dieback on ornamental pear trees. P. syringae and the human pathogen Pseudomonas aeruginosa produce the exopolysaccharide alginate, a copolymer of mannuronic and guluronic acid. In P. aeruginosa, the response regulator AlgR (AlgR1) is required for transcription of algC and algD, which encode key enzymes in the alginate biosynthetic pathway. In P. syringae FF5, however, algR is not required for the activation of algD. Interestingly, algR mutants of P. syringae remain nonmucoid, indicating an undefined role for this response regulator in alginate biosynthesis. In the current study, the algC promoter region was cloned from P. syringae pv. syringae strain FF5, and sequence analysis of the algC promoter indicated the presence of potential binding sites for AlgR and sigma(54), the alternative sigma factor encoded by rpoN. The algC promoter from P. syringae FF5 (PsalgC) was cloned upstream of a promoterless glucuronidase gene (uidA), and the PsalgC-uidA transcriptional fusion was used to monitor algC expression in strains FF5.32 (algR mutant of P. syringae FF5) and PG4180.K2 (rpoN mutant of P. syringae pv. glycinea PG4180). Expression of the PsalgC-uidA fusion was fourfold lower in both the algR and rpoN mutants as compared to respective wild-type strains, indicating that both AlgR and sigma(54) are required for full activation of algC transcription in P. syringae pv. syringae. AlgR from P. syringae was successfully overproduced in Escherichia coli as a C-terminal translational fusion to the maltose-binding protein (MBP). Gel shift experiments indicated that MBP-AlgR binds strongly to the algC promoter region. Biological assays demonstrated that the algR mutant was significantly impaired in both pathogenicity and epiphytic fitness as compared to the wild-type strain. These results, along with the gene expression studies, indicate that AlgR has a positive role in the activation of algC in P. syringae and contributes to both virulence and epiphytic fitness. Furthermore, the symptoms observed with wild-type P. syringae FF5 suggest that this strain can move systemically in leaf tissue, and that a functional copy of algR is required for systemic movement.

On the binding of copper and lead by water-soluble polysaccharides

Optical, chirooptical and thermodynamic techniques have been used to study the binding between copper (II) ions and carboxyethylscleroglucan, oxidised glucomannan, 6-oxychitin and reacetylated chitosan in dilute aqueous solution. For oxidised glucomannan and reacetylated chitosan, the binding of lead was also studied. The results obtained were consistent with the occurrence of a chelate that was evidenced by the existence of a charge transfer band in the near UV region and supported by other experimental data. The variations on binding of the thermodynamic functions Gibbs free energy, enthalpy and hence entropy were computed. Indications on the site(s) of binding were derived. In the case of oxidised glucomannan, the UV spectrophotometry revealed the presence of two sites of binding, the carboxyl groups of glucuronic and mannuronic acids. Finally, the possibility of avoiding misleading results with appropriate procedures is shown.

Polar components of Black sea seaweed Colpomenia peregrina (Sauv.) Hamel

GC-MS of trimethylsilyl derivatives of the compounds present in the butanolic extract of biomass of brown seaweed Colpomenia peregrina from the Black Sea aided in identification of 24 components, including aliphatic hydroxy and keto and aromatic acids, glycerol, mannitol, floridoside, and monosaccharides. The polysaccharide composition of the biomass was also studied, with high sodium alginate and laminaran contents and a comparatively low level of fucoidan being revealed. The polysaccharides were isolated from the biomass by fractional extraction and purified by precipitation or ion exchange chromatography. The structures of alginic acid and laminaran were deduced from 13C NMR spectra and confirmed, in the case of laminaran, by methylation analysis. The sodium alginate was shown to contain more guluronic (G) than mannuronic acid (M) residues, the M/G ratio being 0.48. Laminaran was demonstrated to be a beta-glucan with 1-->3 linkages in its backbone and 1-->6 linkages in its branching points, which is characteristic of brown algae. Fucoidan turned out to be a complex heteropolysaccharide containing, in addition to fucose and sulfate, other neutral monosaccharides and uronic acids. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru

Alginic acids in Lessonia vadosa: Partial hydrolysis and elicitor properties of the polymannuronic acid fraction

Extraction of Lessonia vadosa(Laminariales, Phaeophyta) collectedin three different localities near PuntaArenas in the south of Chile, with 3%aqueous sodium carbonate solutions gave asodium alginate yield in the range3.0–17.7% dry weight. There were markeddifferences in the mannuronic acid toguluronic acid ratio (M/G) (0.21–1.69) inthe alginic acids, samples collected inPuerto del Hambre in winter were composedmainly of mannuronic acid residues. Thealginate samples were characterized bypartial hydrolysis and FT-IR spectroscopy.No relationship was found between tissuetype and polyguluronic acid content. Theelicitor activity of the polymannuronicacid enriched fraction from alginic acid ofblades from Puerto del Hambre was assayedin wheat plants. The polymannuronic acidenriched fraction induced substancialelicitation of phenylalanine ammonia-lyase(PAL) and peroxidase (POD) activities.

Characterization of riboflavin-photosensitized changes in aqueous solutions of alginate by dynamic light scattering.

The effect of irradiation, in the wavelength range of 310-800 nm, on aqueous solutions (pH = 7.4) of alginate in the presence of the photosensitizer riboflavin (RF) has been investigated with the aid of dynamic light scattering (DLS). Under aerobic conditions light irradiation of RF causes scission of the polymer chains which affects the polymer dynamics. The time correlation data obtained from DLS experiments showed at all conditions the existence of two relaxation modes: one single exponential at short times, followed by a stretched exponential at longer times. The slow relaxation time revealed, over the whole considered concentration range, lower values for the alginate/RF system, whereas no effect of photochemical degradation was observed for the fast relaxation time in the semidilute regime. The results suggest that the photochemically induced fragmentation of alginate affects the slow relaxation mode, associated with disengagement relaxation of individual chains or cluster relaxation, in a similar way as the zero-shear viscosity. These findings provide detailed insight into the dynamics of the polymer matrix, and this knowledge can be useful in the context of controlled-release delivery of drugs. The chemical units of alginate (M = mannuronic acid and G = guluronic acid).

C(6)-oxidation followed by C(5)-epimerization of guar gum studied by high field NMR.

Guar gum, a beta-D-(1-->4)-linked D-mannan with alpha-D-galactopyranosyl units attached as side groups, was treated with alpha-galactosidase, an enzyme that splits off the alpha-D-galactosyl units to obtain a galactomannan with a low galactose content. The galactose-depleted polysaccharide was then selectively oxidized in C(6) position and epimerized using mannuronan C(5)-epimerases, namely AlgE1, AlgE4, AlgE6, and their mixtures, obtaining new pseudo-alginates. In this paper, we report a full high field 1D and 2D NMR study of guar gum as such and of the galactose-depleted, oxidized and epimerized compounds, respectively. From the 1H NMR spectra, the degree of epimerization, the distribution of mannuronic acid (M) and guluronic acid (G) residues and the average G-block length, N(G>1), were obtained. By means of NMR diffusion experiments, it was also shown that no significant degradation of the polysaccharide occurs as a consequence of the epimerization reactions.

A smart bioconjugate of trypsin with alginate.

Alginate is a polymer of guluronic acid and mannuronic acid residues and is an inexpensive, nontoxic polysaccharide of marine origin. Trypsin was immobilized noncovalently on alginate with 100% retention of activity. The enzyme did not leach off the polymer even in the presence of 0.01 M HCl and Triton X-100 (0.2% vv(-1)). The V(max)/K(m) values did not change significantly on immobilization. There was 22% loss of activity in first cycle of pH change and after that the conjugate could be reused upto 4 precipitation cycles without any further loss of activity. This smart bioconjugate was also found to have better operational stability in the presence of casein than free enzyme. Fluorescence studies were carried out to probe structural changes upon immobilization.

Biosynthesis of Polysaccharide Alginate by Azotobacter vinelandii in a Bubble Column

Polysaccharide alginate, which is composed of mannuronic acid (M) and guluronic acid (G), is used as a gel-former and viscosifier in a wide range of applications. The properties of gel-forming, water-binding, and immunogenicity are influenced by the fraction of M- and G-monomers in the alginate. In this study, we engineered an alginate synthesis using culture of Azotobacter vinelandii IAM 9046. The effect of growth conditions on the cell concentration, alginate synthesis, fraction of monomers, etc., were investigated. As a result, alginate with a high content of serial G-monomers was produced in a bubble column reactor. It is our goal to control the monomer fraction of alginate that is required for medical and industrial applications.

The tensile properties of alginate hydrogels

Alginate hydrogels are currently being employed and explored for a broad range of medical applications including cell encapsulation, drug delivery, and tissue engineering. In these capacities, knowledge of the mechanical and material properties of the hydrogels and the properties that govern and influence them is necessary to adequately design and effectively use these systems. Although much is known about the mechanical properties of alginate in compression and shear, little is known about the tensile characteristics. Thus, an extensive tensile assessment of alginate hydrogels was completed as a function of alginate type, formulation, gelling conditions, incubation, and strain rate. In general, the initial tensile behavior and properties of alginate hydrogels were highly dependent on the choice of the alginate polymer and how it was processed. Specifically, high guluronic acid containing alginate polymers yielded stronger, more ductile hydrogels than high mannuronic acid containing alginates. The ultimate stress, ultimate strain, and tensile modulus were decreased by increased phosphate concentrations, solution reconstitution with phosphate buffered saline instead of culture media, and peptide modification. Incubation of hydrogels for at least 7 days diminished many of the initial tensile property differences associated with formulation and gelling conditions. Overall, by controlling the specific alginate polymer and processing methods, a wide range of tensile properties are available from these hydrogels.

The role of the CaCl 2–guluronic acid interaction on alginate encapsulated βTC3 cells

Previously we demonstrated that alginate composition has a significant effect on the growth of encapsulated βTC3 cells and consequently on the overall metabolic and secretory activities of the encapsulated cultures. Based on these results we postulated that the mechanical properties of alginate were not responsible for the observed effects but rather, changes in the strength of the alginate gel network caused by changes in the number of alginate strands held together in the “egg-box” model are responsible for the observed effects. In this study we address this hypothesis with a series of experiments in which the strength of this interaction is manipulated by varying the calcium concentration either at the time of gelation or during culture maintenance. Our data show that increasing the concentration of the CaCl 2 solution used at the time of gelation, thus increasing the strength of the alginate gel network, impedes the growth characteristics of βTC3 cells encapsulated in a high guluronic acid content alginate. This effect is amplified by maintaining a constant supply of calcium ions during culture thus sustaining the interaction between guluronic acid residues and calcium ions. However, preparations of βTC3 cells encapsulated in an alginate with high mannuronic acid content are not affected by changes in CaCl 2 concentration due to the low percentage of consecutive guluronic acid residues. Therefore, the present data show that the strength of the alginate gel network is an important factor that influences the growth characteristics of encapsulated cell preparations.

Affinity precipitation of Aspergillus niger pectinase by microwave-treated alginate

Affinity precipitation is a simple, single plate separation process in which the complex of a smart macroaffinity ligand with the target protein (from a crude broth) can be selectively precipitated by application of a suitable stimulus. Alginate is a copolymer of guluronic acid and mannuronic acid residues and precipitates with Ca 2+ ions. It was found to bind to pectinase present in a commercial preparation of Aspergillus niger, Pectinex Ultra-SPL. Microwave pretreatment of alginate at 75 °C was found to enhance the selectivity of the affinity precipitation. Using microwave-treated alginate, 83% of the enzyme activity with 20-fold purification could be recovered. SDS–PAGE upon silver staining confirmed the enhanced selectivity of affinity precipitation when microwave-treated alginate was used.