Mannuronic Acid Research Articles

Structurally unrelated algal oligosaccharides differentially stimulate growth and defense against tobacco mosaic virus in tobacco plants

Tobacco plants were treated with structurally unrelated oligosaccharides obtained from Chilean marine macroalgae. These oligosaccharides were prepared by chemical depolymerization of native polysaccharides extracted from brown and red algae and correspond to pure polymers of around 20 units of guluronic acid (Poly-Gu), mannuronic acid (Poly-Ma) and sulphated galactan (Poly-Ga). These oligosaccharides were solubilized in water, at a final concentration of 500 μg mL−1, and sprayed on tobacco leaves, once a week for a month. Their effects on the stimulation of growth and defense against tobacco mosaic virus (TMV) were determined 7 and 15 days after the final spraying, respectively. The activities of several defense and antioxidant enzymes and the levels of water-soluble antioxidant compounds were determined. Plants treated with Poly-Ga and Poly-Ma showed an increase in height of 23% and 49%, respectively, whereas Poly-Gu did not stimulate growth. Plants treated with Poly-Gu, Poly-Ma and Poly-Ga showed an increase in defense against TMV corresponding to decreases in the number of necrotic lesions of 9%, 22% and 74%, respectively. The stimulation of plant growth correlates with activation of the antioxidant enzyme ascorbate peroxidase (AP) and with a decrease in ascorbate level. On the other hand, the stimulation of defense against TMV is correlated with the activation of the defense enzyme phenylalanine ammonia-lyase (PAL). These results indicate that algal oligosaccharides differentially stimulate growth and defense against TMV in tobacco plants and that these processes involve the activation of the enzymes AP and PAL, respectively.

Alginate from the macroalgae Sargassum sinicola as a novel source for microbial immobilization material in wastewater treatment and plant growth promotion

Alginate extracted from the macroalgae Sargassum sinicola was used as the raw material for co-immobilization of the microalgae Chlorella sorokiniana and growth-promoting bacterium Azospirillum brasilense for wastewater treatment and as an inoculant carrier of A. brasilense for plant growth promotion. The composition, structure, viscosity, color, and phenolic compound content of the alginate were analyzed and compared with commercially available alginate produced from the macroalgae Macrocystis pyrifera. From 1H NMR analysis of alginate, S. sinicola was found to have more guluronic acid (FG=0.64) than it had mannuronic acid (FM=0.38) and had a viscosity of 13.5 m Pa s compared to 50 m Pa s for M. pyrifera. The S. sinicola alginate had dark brown color, reducing light penetration, with more phenolic compounds than M. pyrifera alginate. Nonetheless, growth of C. sorokiniana and A. brasilense in S. sinicola alginate was not significantly different than the growth in M. pyrifera alginate beads. Nutrient removal from wastewater by the co-immobilized microorganisms was similar for both types of alginate beads, and so was the growth enhancement of tomato plants inoculated with microbeads containing A. brasilense. This study shows the potential use of S. sinicola alginate as a raw material for cell immobilization for wastewater treatment and plant growth promotion.

Stereocontrolled Synthesis of β-d-Mannuronic Acid Esters: Synthesis of an Alginate Trisaccharide

A facile synthesis route toward beta-linked mannuronic acid oligomers using the corresponding 1-thiomannuronic acid esters in combination with the Ph2SO/Tf2O or NIS/TMSOTf reagent combinations is presented. The presence of the remotely attached carboxylic ester sufficiently influences the electronic environment to allow good to excellent beta-selectivities.

Protective effects of an acidic polysaccharide isolated from fruiting bodies of Ganoderma lucidum against murine hepatic injury induced by Propionibacterium acnes and lipopolysaccharide

Fruiting bodies of Ganoderma lucidum were extracted with chloroform followed by hot water, from which an acidic polysaccharide named GLAa was separated and purified using 10% CCl3COOH followed by column chromatography on DE-52 cellulose and Toyopearl HW-65. Chemical and spectroscopic analyses showed that GLAa was composed of Glc, Gal, Man and Fuc (1:0.013:0.009:0.024), in which β-d-(1→6)-Glc, β-d-(1→3)-Glc and β-d-(1→4)-Glc linkages comprised 34.9, 33.9, and 28.7% of the total linkages, respectively. The average molecular weight of GLAa was 12.5×105 Da and the optical rotation was $$ [\alpha ]^{{25^\circ {\text{C}}}}_{{\text{D}}} = - 9.1^\circ $$ ; c =0.55, H2O. Uronic (mannuronic) acid accounted for 14.8% of the molecule, protein, 0.14% and nitrogen, 0.75%. The hepatoprotective effect of GLAa was evaluated using a mouse model in which hepatic injury was induced with Propionibacterium acnes and lipopolysaccharide (LPS). GLAa (0.4 and 0.8 g kg−1 day−1, b.w., p.o.), significantly prevented increases in serum aspartate aminotransferase and alanine aminotransferase levels in mice exposed to P. acnes–LPS, indicating that GLAa has hepatoprotective activity in vivo.

Evaluation of β-(1 → 3, 1 → 6)-glucans and High-M alginate used as immunostimulatory dietary supplement during first feeding and weaning of Atlantic cod ( Gadus morhua L.)

Stimulation of the non-specific defence enhances the disease resistance and growth, and has good potentials as a measure for increased microbial control in juvenile production of marine fish and shellfish. So far, the most commonly used immunostimulants are β-(1 → 3, 1 → 6)-glucans, and in this study the stimulatory potential of a β-(1 → 3, 1 → 6)-glucan of marine origin, the storage polysaccharide from the marine diatom Chaetoceros mülleri, was examined. The glucan (chrysolaminaran) was extracted from cultures of C. mülleri, and used as a dietary supplement in two first feeding experiments with larvae of Atlantic cod Gadus morhua L. In one experiment the microalgal glucan was compared to the commercial yeast-glucan product MacroGard®, and in the other to an alginate with a high content of mannuronic acid (High-M alginate) isolated from Durvillaea antarctica. The stimulants were given via rotifers, and weaning to formulated feed was initiated at day 17 or 18 after hatching. The survival ± SEM at day 27 after hatching was 24.5 ± 2.0%, 14.8 ± 4.5% and 13.1 ± 1.4% for the groups fed C. mülleri-glucan, yeast glucan and for the control, respectively, in the first experiment. The group fed C. mülleri-glucan group had higher survival compared to the control ( P < 0.05) group, whereas the yeast glucan had no positive effect on the survival ( p > 0.05). The dry weights of the groups at day 27 were low, with 203.2 ± 52.2, 165.2 ± 43.4 and 198.5 ± 58.1 μg per larva for the C. mülleri-glucan, yeast glucan and control groups, respectively. In the second experiment the survival in the period of feeding formulated feed (days 18–30) were 44.6 ± 4.3%, 44.7 ± 1.3%, and 33.8 ± 4.1% survival for the C. mülleri-glucan, High-M alginate and control group, respectively. The cod larvae fed C. mülleri-glucan reached an average weight of 531.6 ± 17.2 μg at day 30, which was significantly higher ( p < 0.05) than the control group that had an average of 473.6 ± 3.5 μg. The larvae fed High-M alginate had an average weight of 470.3 ± 31.6 μg per larva at day 30, and not significantly different from the control ( p > 0.05). The early weaning to formulated diet had detrimental effect on the growth of the larvae. In both experiments the C. mülleri-glucan group was the only group showing a positive growth rate in the period of weaning to dry feed. The microbial conditions in larval gut and water were monitored with respect to total colony forming units on Marine agar, and Vibrio- and Pseudomonas-like species on selective agars (TCBS and marine Pseudomonas Agar with CFC-supplement). The larvae were rapidly colonised after hatching, but no or weak effects of the stimulants were observed on the colonisation rates or the composition. The total CFU varied from 10 1 to 10 2 CFU per μg larva after initiation of the first feeding. The percentages of Pseudomonas-like bacteria increased throughout the period, whereas the levels of Vibrio-like bacteria were low and stable. The chrysolaminaran from the diatom C. mülleri was shown to be a promising candidate for use as an immunostimulatory feed additive, and which should be further explored.

Development of polysaccharide gel-coated pellets for oral administration: 2. Calcium alginate

Calcium alginate gel-coated pellets were developed by forming an insoluble gel coat on extruded–spheronized pellets by interfacial complexation. Experiments were designed to investigate the effect of pellet size, alginate type, alginate concentration, and dissolution medium on swelling and drug release behavior. Low swelling in acidic media was related to proton–calcium ion exchange forming insoluble acid gels. In contrast, partial formation of soluble sodium alginate in 0.1 M NaCl induced water uptake, resulting in greater swelling. Drug release from coated pellets showed a lag time when the gel coat hydrated and swelled, followed by a zero-order release. Significantly slower release was observed when either the pellet size or the alginate concentration was increased. Alginate with high guluronic acid content gave the slowest release. Different types of alginate with high mannuronic acid content showed different release behaviors that are probably due to the different monomer sequences and botanical sources. The faster drug release in acidic media and 0.1 M NaCl compared to water is probably due to reduced calcium cross-linking in the gel. These results suggest that the pellet size, alginate type and concentration and dissolution medium influenced the swelling and drug release behavior of calcium alginate gel-coated pellets.

Characterization of the Hydrolysis Mechanism of Polyalternating Alginate in Weak Acid and Assignment of the Resulting MG-Oligosaccharides by NMR Spectroscopy and ESI−Mass Spectrometry

Alginate with long strictly alternating sequences of mannuronic (M) and guluronic (G) acid residues, F(G) = 0.47 and F(GG) = 0.0, was prepared by incubating mannuronan with the recombinant C-5 epimerase AlgE4. By partial acid hydrolysis of this PolyMG alginate at pH values from 2.8 to 4.5 at 95 degrees C, alpha-L-GulpA-(1-->4)-beta-D-ManpA (G-M) linkages were hydrolyzed far faster than beta-D-ManpA-(1-->4)-alpha-L-GulpA (M-G) linkages in the polymer chain. The ratio of the rates (kG-M/kM-G) decreased with increasing pH. The dominant mechanism for hydrolysis of (1-->4)-linked PolyMG in weak acid was thus proved to be an intramolecular catalysis of glycosidic cleavage of the linkages at C-4 by the undissociated carboxyl groups at C-5 in the respective units. The higher degradation rate of G-M than M-G glycosidic linkages in the polymer chain of MG-alginate at pH 3.5 and 95 degrees C was exploited to make oligomers mainly consisting of M on the nonreducing and G on the reducing end and, thus, a majority of oligomers with an even number of residues. The ratio of the rate constants kG-M/kM-G at this pH was 10.7. The MG-hydrolysate was separated by size exclusion chromatography and the MG oligosaccharide fractions analyzed by electrospray ionization-mass spectrometry together with 1H and 13C NMR spectroscopy. Chemical shifts of MG-oligomers (DP2-DP5) were elucidated by 2D 1H and 13C NMR.

Cell-compatible covalently reinforced beads obtained from a chemoenzymatically engineered alginate

A chemoenzymatic strategy has been exploited to make covalently linked alginate beads with high stability. This was achieved by grafting mannuronan (alginate with 100% mannuronic acid (M)) with methacrylate moieties and then performing two enzymatic steps converting M to guluronic acid (G) in alternating sequences (MG-blocks) and in G-blocks. In this way a methacrylate grafted alginate with better gel-forming ability was achieved. Covalent bindings were introduced into the beads by using a photoinitiating system that initiated polymerization of the methacrylate moieties. The covalent links were demonstrated by beads remaining intact after treatment with EDTA. The new chemoenzymatic photocrosslinked (CEPC) beads were compatible with cells with low post-encapsulation ability like C2C12 myoblasts and human pancreatic islets. The islets continued secreting insulin after encapsulation. On contrary, cells with a high post-encapsulation proliferative ability like 293-endo cells died within 2-week post-encapsulation. The exceptional stability and the cell compatibility of the new CEPC beads make them interesting as bioreactors for delivering therapeutic proteins in future applications.

Heavy metal sorption by calcium alginate beads from Laminaria digitata

Alginate with a high M/G ratio, extracted from Laminaria digitata, was evaluated for Cu 2+, Cd 2+ and Pb 2+ sorption in acidic solutions, in the form of calcium cross-linked beads. The high M/G ratio of alginate extracted from this algal species is most likely the determining factor for the increased adsorption capacity of the investigated metals, indicating that the mannuronic acid is responsible for the ion exchange mechanism. The data obtained from the batch experiments have been interpreted with Langmuir, Freundlich and Sips models. The Sips equation provided the best fit with the experimental results, indicating sorption sites heterogeneity for the material. The pH was found to have a significant effect on the process, with sorption capacity reaching a maximum at pH 4.5, indicating a competition mechanism between H + and metal ions. Kinetic experiments were performed at the optimum pH. For the interpretation of the kinetic experiments the Linear Adsorption Model was employed and diffusion coefficients were determined. The model fits the experimental data at higher concentrations, where the adsorbed quantity remains almost constant. Finally, a simplified expression of the batch kinetic adsorption model was employed. The model, predicts adequately, not only the diffusivity values, but also the concentration profiles inside the spherical particles.

Seasonal studies on alginate and its composition II: Turbinaria conoides (J.Ag.) Kütz. (Fucales, Phaeophyceae)

The alginates extracted from ‘leaf’, ‘stem’ and entire thallus of Turbinaria conoides (J.Ag.) Kutz. were investigated for their viscosity and biochemical constituents namely, β-D-mannuronic acid (M-block}), α-L-guluronic acid (G-Block) and alternating sequences of β–D-mannuronic acid and α-L-guluronic acid (MG-block). Substantial seasonal variation was recorded with high yield of alginate during premonsoon. The yield of alginate was maximum in ‘leaf’ region. In contrast to this, viscosity and G-block were maximum in the ‘stem’ region. A significant positive correlation was observed between viscosity and G-block}. The ratio of guluronic: mannuronic acid was also assessed. Low levels of M/G ratio were recorded in the ‘stem’ region followed by ‘leaf’ and entire thallus.

Countercurrent Chromatographic Separation of Biotic Compounds with Extremely Hydrophilic Organic‐Aqueous Two‐Phase Solvent Systems and Organic‐Aqueous Three‐Phase Solvent Systems

Highly polar organic‐aqueous two‐phase and three‐phase solvent systems were applied to the countercurrent chromatographic (CCC) separation of biotic compounds. The ethanol/2 M ammonium sulfate (3∶5) system was used for protein separation between human serum albumin and lysozyme, and also for separations of sugars including D‐(+)‐glucose and L‐(−)‐fucose, D‐(+)‐xylose and α‐L‐rhamnose, and α‐D‐galacturonic acid, mannuronic acid lactone and D‐(+)‐glucoronolactone. When using the acetonitrile/1 M sodium chloride (5∶4) system for applying water‐soluble carboxylic acids, 2‐naphthoic acid, p‐methyl hippuric acid and p‐amino hippuric acid were well separated with upper phase mobile, and maleic acid and fumaric acid were resolved with lower phase mobile. An organic‐aqueous three‐phase solvent system composed of n‐hexane/methyl t‐butyl ether/acetonitrile/water (5∶5∶7.5∶5) was also applied to the simultaneous CCC separation of fat‐soluble vitamins and water‐soluble vitamins. Using the middle phase as the stationary phase, the separation of thiamine hydrochloride and nicotinamide was achieved first with the lower mobile phase and then vitamin K1 and K3 were eluted after the mobile phase was switched to the upper phase reversing the direction of elution. The overall results demonstrated that both extremely hydrophilic organic‐aqueous two‐phase solvent systems and organic‐aqueous three‐phase solvent systems are useful for the CCC separations of polar compounds.

Alginate polyelectrolyte complexes with cationic hydroxyethyl cellulose derivatives: Role of the block structure

It was shown that the block structure of alginate significantly affects the mechanism of formation and properties of its water-soluble polyelectrolyte complexes with cationic derivatives of hydroxyethyl cellulose. Alginate that contains residues of only mannuronic acid remains in solution in the form of coils, and the incorporation of guluronic acid residues gives rise to formation of a network structure. A difference in the structure of polyelectrolyte complexes is explained by different rigidities of blocks formed by residues of mannuronic and guluronic acids having different conformations of the pyranose ring.

Applications of Alginate in Bioseparation of Proteins

Alginate is a polysaccharide that is a block polymer consisting of block units of guluronic acid and mannuronic acid. It shows inherent biological affinity for a variety of enzymes such as pectinase, lipase, phospholipase D, a and ss amylases and glucoamylase. Taking advantage of its precipitation with Ca2+ and the above-mentioned property, alginate has been used for purification of these enzymes by affinity precipitation, aqueous two phase separation, macroaffinity ligand facilitated three phase partitioning, immobilized metal affinity chromatography and expanded bed affinity chromatography. Thus, this versatile marine resource has tremendous potential in bioseparation of proteins.

A shape-persistentd,l-dipeptide building block for the assembly of rigidified oligopeptides

Mannuronic acid (5) was transformed into the densely functionalised D,L-dipeptide mimic 1 and subsequently inserted into the cyclic hexapeptide 3. The gulo-configurated seven-membered lactam 1 exhibits an inverted ring conformation compared to the previously described L,L-dipeptide mimic 2. In spite of this considerable difference, both prefer the same i to i + 1 positions of a beta-turn within a cyclic hexapeptide.

Uronic acids functionalized polyethyleneimine (PEI)–polyethyleneglycol (PEG)- graft-copolymers as novel synthetic gene carriers

In this study, we investigated galacturonic (GalAc)- and mannuronic (ManAc) acids as novel targeting ligands for receptor-mediated gene delivery. GalAc and ManAc were coupled to either polyethyleneimine (PEI) or PEI–polyethyleneglycol (PEG). Furthermore, lactobionic acid (LacAc), which comprises a GalAc-related carbohydrate ring, was coupled to each of the polymers through its open-chain gluconic acid moiety. The molar mass distributions of the polymers were characterized by analytical ultracentrifugation and size exclusion chromatography. PEI-conjugate–pDNA complexes were transfected into HepG2-, HeLa-, and 16HBE14o −-cells. Gene expression mediated by GalAc- and LacAc-functionalized PEI-conjugates was lower than for PEI. In contrast, gene expression mediated by ManAc-functionalized PEI-conjugates was up to three orders of magnitude higher than for the other tested PEI-conjugates, in particular for negatively charged gene vectors at low N/P ratios, independent of the cell line. Pre-incubation of cells with an excess of ManAc before transfection significantly inhibited transfection rates only for ManAc-functionalized PEI-conjugates. Coupling of methyl- α- d-mannuronic acid to PEI resulted in significantly lower transfection rates than for ManAc-PEI based complexes. Together with fluorescence microscopy images of fluorescein-labelled ManAc-functionalized dextrans and FACS analyses of cells, these results demonstrate that receptor-mediated endocytosis of ManAc–PEI-conjugate–pDNA complexes via ManAc-specific receptors was involved in gene transfer. In conclusion, ManAc-modification of PEI-polymers represents a novel strategy for receptor-mediated gene delivery which could be promising for in vivo application.

Role of the Pseudomonas fluorescens Alginate Lyase (AlgL) in Clearing the Periplasm of Alginates Not Exported to the Extracellular Environment

Alginate is an industrially widely used polysaccharide produced by brown seaweeds and as an exopolysaccharide by bacteria belonging to the genera Pseudomonas and Azotobacter. The polymer is composed of the two sugar monomers mannuronic acid and guluronic acid (G), and in all these bacteria the genes encoding 12 of the proteins essential for synthesis of the polymer are clustered in the genome. Interestingly, 1 of the 12 proteins is an alginate lyase (AlgL), which is able to degrade the polymer down to short oligouronides. The reason why this lyase is associated with the biosynthetic complex is not clear, but in this paper we show that the complete lack of AlgL activity in Pseudomonas fluorescens in the presence of high levels of alginate synthesis is toxic to the cells. This toxicity increased with the level of alginate synthesis. Furthermore, alginate synthesis became reduced in the absence of AlgL, and the polymers contained much less G residues than in the wild-type polymer. To explain these results and other data previously reported in the literature, we propose that the main biological function of AlgL is to degrade alginates that fail to become exported out of the cell and thereby become stranded in the periplasmic space. At high levels of alginate synthesis in the absence of AlgL, such stranded polymers may accumulate in the periplasm to such an extent that the integrity of the cell is lost, leading to the observed toxic effects.

C(6)-Oxidation and C(5)-Epimerization of Locust Bean Galactomannan Studied by High Field NMR and Circular Dichroism

Galactose depleted locust bean gum was selectively oxidized in C(6) position and epimerized with mannuronan C(5)-epimerases to obtain the corresponding artificial uronanes. These new pseudo-alginates were characterized by NMR spectroscopy and circular dichroism (CD). Specifically, 1D and 2D NMR techniques allowed the degree of epimerization, the distribution of mannuronic and guluronic acid residues in the polysaccharidic chain, and the average G block length to be determined. In addition, NMR diffusion experiments showed that the epimerization reaction did not significantly degrade the polysaccharidic chains. Circular dichroism was used to investigate the kinetics of the epimerization reaction and to evidence the specific interaction between the epimerized locust bean samples with Ca(II) ions in dilute solution. All of the samples considered in this study form wall to wall gels in concentrated polymer solutions.

(3aS,4S,4aS,7R,9S,9aS)-Methyl 4-benzyloxy-9-methylsulfonyloxy-2,2-dimethyl-8-oxooctahydro-1,3-dioxa-5-thia-7a-azacyclopenta[f]azulene-7-carboxylate toluene solvate

The absolute configuration has been determined for the title compound, C21H27NO9S2·C7H8. The compound is a precurser in the synthesis of bicyclic dipeptide isosteres based on mannuronic acid. The seven-membered lactam ring adopts a rigid chair conformation.

Inhibition of pepsin activity by alginates in vitro and the effect of epimerization

Alginates are versatile biopolymers used extensively in the food, textile and pharmaceutical industries. One of the major uses is in the treatment of reflux disease and here we investigate whether alginates can influence pepsin activity, a major aggressor in reflux disease. The primary uronic acid structure of alginates can be altered using epimerase technology and we test tailor-made alginates to identify the optimal structure for pepsin inhibition. Pepsin activity in the presence of alginates was studied using an in vitro N-terminal assay and enzyme kinetics using a chromagenic peptide. The data described showed clearly that alginates were capable of concentration dependently reducing the activity of pepsin in a non-competitive manner, in vitro. This was variable between different alginates of wide ranging structure and size with positive correlation with alternating sequences of mannuronic and guluronic acid. We hypothesize that alginates may have a more extensive role in the treatment of reflux disease by inhibiting pepsin, a damaging component of the refluxate.

Similarities and differences between alginic acid gels and ionically crosslinked alginate gels

A Na-alginate solution can, under given conditions, undergo a sol–gel transition at pH below pKa for the uronides. These gels are presumably stabilised by intermolecular hydrogen bonds. This paper summarises the effect of alginate variables such as chemical composition and sequence and molecular weight on mechanical properties and network fine structure. Alginic acid gels resemble ionically crosslinked alginate gels in the sense that guluronic acid blocks are the building elements of most importance with respect to gel formation. The main difference is that mannuronic acid blocks, although with less efficiency compared polyguluronate, also support alginic acid gel formation. With the exception strictly alternating alginates made by treating mannuronan with the AlgE4 epimerase, the so-called alternating (random) sequence of the alginate molecule seems to destabilise alginic acid gel formation due to its lack of repeating structure. Small Angle X-ray Scattering (SAXS) reveal a scattering intensity for alginic acid gels being typically and order of magnitude higher than Ca-alginate gels, suggesting junction zones composed of random aggregates with a high degree of multiplicity. A molecular understanding of alginic acid gels is of importance in the general understanding of polyuronides at low pH as well as for the utilisation of alginates in drug delivery systems.