Spatial and temporal variations of Chondrus crispus (Gigartinaceae, Rhodophyta) carrageenan content in natural populations from Galicia (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes and tetrasporophytes of Chondrus crispus were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν- and μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 50% of the total carrageenans). In tetrasporophytes, the presence of λ-carrageenan was confirmed. Carrageenan content in gametophytes (37.4?±?1.68% DW) was higher than in tetrasporophytes (29.13?±?0.76% DW). Spatial and temporal variation in carrageenan content in both life cycle phases appears to be related mainly to seawater and air temperatures, insolation, water movement and desiccation. The highest values of carrageenan content were recorded in those localities where higher values of precipitation, wind speed or water movement occurred. A bimodal temporal pattern on carrageenan content was observed. Fronds showed a high carrageenan content in spring and autumn. During these seasons, the content was over 40% in gametophytes and 30% in the tetrasporophytes. In summer and winter, these values down in both life cycle phases below 30%. In general the highest carrageenan contents were related to highest seawater temperatures. On the contrary, high air temperature and high insolation appeared to be unfavourable for carrageenan production. GLM models were obtained to predict carrageenan production from natural C. crispus populations, along Galician coast.     

Controlling Carrageenan Structure Using a Novel Formylglycine-Dependent Sulfatase, an Endo-4S-iota-Carrageenan Sulfatase

Carrageenans are sulfated polysaccharides that are found in the cell walls of red algae. These polysaccharides have gelling and texturizing properties that are widely appreciated in industrial applications. However, these functional properties depend strongly on the sulfation of the moieties of the carrabiose repetition unit. Here we aimed to monitor the sulfate composition of gelling carrageenan. To do so, we screened and purified from Pseudoalteromonas atlantica a 4S-iota carrageenan sulfatase that converts ι-carrabiose into α-carrabiose units. The sequence of this protein matched the annotated Q15XH3 (Uniprot databank) formylglycine-dependent sulfatase found in the P. atlantica genome. With pure enzyme, ι-carrageenan could be transformed into a hybrid ι-/α-carrageenan or pure α-carrageenan. Analysis of the distribution of the carrabiose moieties in hybrid carrageenan chain using enzymatic degradation with Alteromonas fortis ι-carrageenase, coupled with chromatography and NMR spectroscopy experiments, showed that the sulfatase has an endo mode of action. The endo-character and the specificity of the sulfatase made it possible to prepare hybrid κ-/ι-/α-carrageenan and κ-/α-carrageenan starting from κ-/ι-carrageenan.     

Effect of pressure on the sol-gel transition of carrageenans

The effects of pressure on the sol-gel transition of κ- and ι-carrageenans were studied in KCl solutions under high pressures up to 3000 kg/cm². The carrageenan gels were destabilized by pressure: the pressure depression of melting temperature, (dT/dP)_m, was ?5.7 × 10^?3 and ?4.0 × 10^?3 K cm²/kg independent of KCl concentration for κ- and ι-carrageenans, respectively. The enthalpy, entropy and volume changes accompanying the gel formation were calculated from the Eldridge-Ferry's plots and the Clausius-Clapeyron equation. The volume change per unit cross-link (two disaccharide residues) was estimated to be (2.5 ～ 4.9) and (1.7 ～ 3.4) ml/mol for κ- and ι-carrageenans, respectively. The compressibility of both carrageenan molecules appeared to be larger by (1.6 ～ 2.6) × 10^?12 (κ-form) and by (0.8 ～ 1.3) × 10^?12cm²/dyn (i-form) in gel state as compared with in sol state These increases in volume and compressibility on gelation were attributed to a reduction of water of hydration from the carrageenan molecules, which is mainly due to a replacement of the polymer-water hydrogen bond by the polymer-polymer hydrogen bond. These results seemed not inconsistent with the idea that a double helix structure of carrageenan gels may persist in solution as well as in the solid state.     

Qualitative and quantitative analysis of carrageenan content in gametophytes of Mastocarpus stellatus (Stackhouse) Guiry along Galician coast (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes of the rhodophyte Mastocarpus stellatus (Gigartinales) were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν-, μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 80 % of the total carrageenans). Mean total carrageenan content in gametophytes was of 37.32?±?1.21 % DW. Spatial and seasonal variations were observed, mainly related to changes on environmental and oceanographic factors and the role of carrageenans in adapting the fronds to these changes. Maximum values in carrageenan content were observed for San Román (Biscay Bay) in May and for Laxe and Mougás (Atlantic coast) in June. The results of this study indicated that spatial differences in carrageenan content were due to interactions of different factors, rather than the effect of a single factor. Fronds from San Román had higher carrageenan content (43.23?±?1.87 % DW) than those collected at two sites of the Atlantic coast, Mougás and Laxe (32.20?±?1.14 % DW). San Román is exposed to the open sea, windy and oriented to the north, and the water temperature is higher in summer than in the Atlantic coast. However, seasonal variations in carrageenan content resulted to be more related to other factors directly correlated with the input of energy in the ecosystems (irradiance, sunshine hours and insolation). Thus, carrageenan content began to increase in early spring when the number of sunlight hours increased. Maximum values were reached in late spring or early summer, just before maximum values of irradiance and air temperature were achieved.     

Carrageenan patterns in the Gigartinaceae

Carrageenans of known reproductive phases of gigartinacean species are shown to differ: gelling κ (τ)-type carrageenan occurs in all instances in gametophytes, and viscous non-gelling λ-carrageenan or its variant ξ- or π-carrageenan in tetrasporophytes. Multiple analyses demonstrate that the carrageenan type is a species characteristic, with little difference between individual samples. No difference exists between the carrageenans of male and female plants of each species. Carrageenan chemistry combined with morphological, reproductive and life history feature is useful for defining the taxonomic limits of this family and these genera.     

DIFFERENTIAL LOCALIZATION OF CARRAGEENAN GELLING SEQUENCES IN KAPPAPHYCUS ALVAREZII VAR. TAMBALANG (RHODOPHYTA) WITH FITC-CONJUGATED CARRAGEENAN OLIGOSACCHARIDES1

The ability of kappa (κ) and iota (ι) carrageenans to form gels is dependent upon the regular repeat of disaccharide units along the carbohydrate chain. Short, chemically- and enzymatically-purified fragments of κ and ι carrageenan were conjugated to fluorescein and used as specific hybridization probes for localization of κ and ι carrageenan gelling sequences within the cells walls and intercellular matrices of Kappaphycus alvarezii (Doty) Doty. The probes label cell walls and intercellular matrices under ionic conditions appropriate for gelation of κ and ι carrageenans. The distribution of κ and ι carrageenans in the matrix and cell walls of K. alvarezii was determined with respect to cell type (epidermis, cortex, medulla, and central axis) and age. The κ-probe labels the cell walls of all cell types except epidermal in both young and old tissues. In contrast, the ι-probe labels the cell walls of the epidermis in both young and old tissue and the cell walls of the thylles only in old tissue. Both probes label intercellular matrix material; however, ι-probe labelling is very much weaker than κ-probe labelling. The results indicate that FITC-conjugated carrageenan oligosaccharides are useful tools that provide information on gelling subunit distribution.     

Compressibility,densimetric and calorimetric studies of hydration of carrageenans in the random form

The partial molal volume and adiabatic compressibility were measured, as well as their counterion activity, for sodium and potassium salts of three types of carrageenan (κ-, ι- and λ-components) in aqueous solutions at 25°C. Furthermore, the amount of related unfreezable water was estimated by the differential scanning calorimetry. On the basis of these results, the hydration states of carrageenans in the random form were comparatively discussed in relation to their chemical structure, counterion binding and polymer concentration. The sodium salt of each component showed a larger amount of hydration when compared with the corresponding potassium salt. The amount of hydration estimated from molal volume and compressibility data (in dilute solution) increased in the order of κ < ι < λ, while the amount of unfreezable water (in concentrated solution) decreased in the same order. These characteristics hydration behaviours of carrageenans seemed to be reasonably explained in terms of the effects of the charge density and counterion dissociation of these polyions.     

Carrageenans from Cystocarpic and Sterile Plants of Chondrus Pinnulatus (Gigartinaceae, Rhodophyta) Collected from the Russian Pacific Coast

The chemical structure, gel properties and biological activity of the carrageenans isolated from cystocarpic and sterile plants of Chondrus pinnulatus were investigated. The total carrageenan content of the sterile plant was observed to be twice that of the cystocarpic plants. According to data obtained by ¹³C-NMR and FT IR, the gelling polysaccharides from cystocarpic and sterile plants of C. pinulatus have similar structures and were identified as κ/ι-carrageenans. The difference between these polysaccharides was in the ratio of the κ- and ι-segments, with a predominant content of κ-segments in cystocarpic plants (80%). Moreover, KCl-insoluble fractions possibly contain hetero-disperse μ/ν precursor: amounts of this in the polysaccharide from sterile plants were more than that extracted from the cystocarpic plants. The KCl-soluble fractions (non gelling) were λ-carrageenans with another carrageenan type that had a low amount of 3,6-anhydrogalactose. Carrageenans from cystocarpic stages showed good gelling properties, whereas those from sterile plants formed a very weak gel. Structural differences and molecular weight of carrageenans obviously determine the biological activity of the polysaccharides. Non gelling-carrageenans from both types of ιt C. pinnulatus plants showed high macrophage-phosphatase activity and κ/ι-carrageenan from cystocarpic plant possessed a potent anti-coagulant activity, which was extremely strong in a low concentration of 100 μg ml⁻¹.     

Structural and conformational investigation of carrageenans

A detailed assignment of the ¹³C chemical shifts of κ- and ι-carrageenans in their Na⁺ and K⁺ forms in D₂O and dimethylsulfoxide is given. Evidence of the conformational transition induced by temperature variation in the absence of any gel formation on κ-carrageenans is also presented. This evidence is based on ¹³C-nmr and optical rotation experiments.     

Chemical structure and gel properties of carrageenans from algae belonging to the Gigartinaceae and Tichocarpaceae, collected from the Russian Pacific coast

The chemical and gel characteristics of carrageenans isolated from the most abundant algal species growing on the Russian Pacific coast – Chondrus pinnulatus, C. armatus and Iridaea cornucopiae belonging to the Gigartinaceae and Tichocarpus crinitus from the Tichocarpaceae were investigated. The polysaccharides were identified by FTIR and NMR spectroscopy as predominantly κ-carrageenans with traces of ι-type (Gigartinaceae) and κ / β-type repeating structures (Tichocarpaceae) together with a small quantity of λ-carrageenan (10%). The chemical structure and the hydrodynamic properties play a determinant role on the rheology of these carrageenans. κ-Carrageenans from the Gigartinaceae displayed good gelling properties. The highest gel strength was obtained from C. pinnulatus (1232.7 Pa) at a 2.5% polymer concentration, while carrageenans from the Tichocarpaceae formed very weak gels (77.4 Pa) at the same concentration. Optimum gel characteristics were found with 1.0–2.0% KCI concentrations for kappa- carrageenans from Gigartinaceae and 0.75% for T. crinitus. The flow curves of λ-carrageenans solutions from the Gigartinaceae were similar, all between 20 and 65 °C, and characteristic of conformational disordered ‘random coil’ polysaccharides. Carrageenans from T. crinitus displayed the properties of ’random coil' only at high temperatures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carrageenans from <Emphasis Type="Italic">Sarcothalia crispata</Emphasis> and <Emphasis Type="Italic">Gigartina skottsbergii</Emphasis>: Structural Analysis and Interpolyelectrolyte Complex Formation for Drug Controlled Release

The aims of the present study were to characterize for the first time the carrageenan extracted from cystocarpic stage of S. crispata collected in the Patagonian coast of Argentina, and to prepare interpolyelectrolytic complexes (IPECs) between the polysaccharide extracted from cystocarpic stage of Sarcothalia crispata and Gigartina skottsbergii thalli, and basic butylated methacrylate copolymer (Eudragit E), in order to test their potential for the controlled release of ibuprofen as model drug. The structural determination revealed that the polysaccharides extracted from S. crispata and G. skottsbergii were mainly constituted by κ-carrageenan, particularly in the case of G. skottsbergii; however, significant amounts of ι- and ν-carrageenan were also detected in both polygalactans. The differences in diad composition and possibly in their distribution along the polysaccharide chain of both carrageenans would favor a different arrangement in the resulting IPEC structure. The smaller pores observed by scanning electron microscopy in the IPEC of S. crispata suggest that the kinks in the polysaccharide backbone are evenly distributed, resulting in a slower ibuprofen release compared to the IPEC of G. skottsbergii.     

Genetic and biochemical characterization of the Pseudoalteromonas tetraodonis alkaline κ-carrageenase

An alkaline κ-carrageenase, Cgk-K142, was found in the culture broth of a deep-sea bacterium, Pseudoalteromonas tetraodonis JAM-K142. A gene for the enzyme was cloned and expressed. Purified recombinant Cgk-K142 (rCgk-K142) showed an optimal pH of about 8.8 in glycine-NaOH buffer at 30 °C and of about 8.0 in MOPS buffer at 50 °C. The optimal temperature for the enzyme was 55 °C at pH 8.0. rCgk-K142 was unstable, but λ- and ι-carrageenans, non-degradative substrate homologs, extensively enhanced its stability. The nucleotide sequence of the gene for Cgk-K142 comprised 1,194 bp, and the deduced amino acid sequence (397 amino acids) showed a high level of similarity to the κ-carrageenase of P. carrageenovora, with 94% identity. Another gene for a κ-carrageenase-like protein was found downstream of the gene for Cgk-K142. The nucleotide sequence of that gene consisted of 966 bp (321 amino acids), and it showed the highest similarity, at 64% identity, to protein CgkB of P. carrageenovora, which has been reported as an incomplete 57-amino acid sequence.     

Studies on carrageenans and effects of seawater phosphorus concentration on carrageenan content and growth ofAgardhiella subulata (C. Agardh) Kraft and Wynne (Rhodophyceae,Solieriaceae)

Gas liquid chromatography, chemical analyses, and infrared and¹³C-NMR spectroscopies indicated that phycocolloids extracted fromAgardhiella subulata had a dominant ι-carrageenan feature with less deviant ι-carrageenan and υ-carrageenan. The presence of methylated galactose and a small contamination by xylose were registered. Unattached plants were cultivated for 4 weeks in tanks receiving seawater enriched with 53.5 µM nitrate and 0 to 20 µM phosphate (P_i) week^?1. The growth was phosphorus (P)-limited up to a tissue P content of 0.14 ± 0.03% dry weight. Maximal specific growth rate and carrageenan content were observed with enrichments of 6 µM P_i and 3 µM P_i, respectively. Hence carrageenan production was promoted in the range of 3–6 µM P_i. Further P_i enrichment was useless. This phenomenon, observed with P nutrition, is comparable to the ‘Neish effect’ in nitrogen nutrition studies.     

Structure and thermal stability of pyruvated carrageenans from the red alga Coccotylus truncatus

The composition, structure, and thermal stability of carrageenans from unattached Coccotylus truncatus (the Baltic Sea, Estonia) were investigated. The complex polysaccharide was characterized by ¹³C NMR and FTIR spectroscopy, ICP-OES and gel permeation chromatography methods. The main components of C. truncatus galactan are 3,6-anhydro-α-d-galactose-2-sulfate (30 ± 1.5%) and β-d-galactose-4-sulfate (45.3%), indicating a ι-carrageenan backbone. As the minor components, α-d-galactose-2,6-disulfate (12 ± 2%) from ν-carrageenan and 4′,6′-pyruvated β-d-galactopyranosyl residues (1.4%) from pyruvated α-carrageenan are found to be present, latter being responsible for the undersulfated nature of the galactan. The native polysaccharide with the average molecular weight of about 1500 kDa is highly susceptible to thermal degradation. The high-temperature treatment of this galactan gives products with 3,6-anhydro-α-d-galactose units predominantly at the reducing end. The carrageenan extraction from C. truncatus gives characteristically low yields (12-17%); weak gelling ability of the polysaccharides from this seaweed species (gel strength 30-40 g/cm²) does not depend significantly on extraction conditions.     

Synthesis of Some Isocoumarin Derivatives

An alkaline κ-carrageenase, Cgk-K142, was found in the culture broth of a deep-sea bacterium, Pseudoalteromonas tetraodonis JAM-K142. A gene for the enzyme was cloned and expressed. Purified recombinant Cgk-K142 (rCgk-K142) showed an optimal pH of about 8.8 in glycine-NaOH buffer at 30 °C and of about 8.0 in MOPS buffer at 50 °C. The optimal temperature for the enzyme was 55 °C at pH 8.0. rCgk-K142 was unstable, but λ- and ι-carrageenans, non-degradative substrate homologs, extensively enhanced its stability. The nucleotide sequence of the gene for Cgk-K142 comprised 1,194 bp, and the deduced amino acid sequence (397 amino acids) showed a high level of similarity to the κ-carrageenase of P. carrageenovora, with 94% identity. Another gene for a κ-carrageenase-like protein was found downstream of the gene for Cgk-K142. The nucleotide sequence of that gene consisted of 966 bp (321 amino acids), and it showed the highest similarity, at 64% identity, to protein CgkB of P. carrageenovora, which has been reported as an incomplete 57-amino acid sequence.     

The λ-components of the “intermediate” fractions of the carrageenan form Iridaea undulosa

The “intermediate” fractions of the carrageenan of Iridaea undulosa, with are precipitated at 1.20–1.25m, 1.35–1.40m, and 1.55–1.65m concentrations of potassium chloride, are mixtures of μ/ν-carrageenans and λ-carrageenans. The λ-carrageenans contain mainly 3-linked, 2-sulfated (33.5–39.0%) and 2,6-disulfated (5.6–11.8%) galactose units, together with 4-linked, 2-sulfated (1.8–5.9%), 6-sulfated and/or 2,6-disulfated (39.0–40.1%), and other 2,6-disubstituted (3.7–4.3%) galactose residues. The presence of this last unit suggests that some C-6 atoms must be either branching points or blocked by glycosidic linkages. When the compositions of these gel-forming λ-carrageenans are compared with those of the “soluble” λ-carrageenans from the same seaweed, it is clear that in the latter ones there is a higher diversification of the structural units and more “solubility-promoting” residues.     

Production and characteristics of some new β-agarases from a marine bacterium,Vibrio sp. strain JT0107

Vibrio sp. strain JT0107 is one of the marine bacteria that secrete β-agarases which catalyze the hydrolysis of agarose. The optimum culture conditions for the production of some β-agarases have been determined. To increase agarase activity, aeration and a sufficient concentration of agarose are needed. One of the enzymes that the bacteria secreted into the culture medium was isolated and purified 39-fold using a combination of ultrafiltration and subsequent anion exchange column chromatography. The purified protein migrated as a single band (72 kDa) on sodium dodecyl sulfate polyacrylamide gel electrophoresis and its isoelectric point was 4.7. Amino acid sequence analysis revealed a single N-terminal sequence that had no sequence identity to other marine bacterial agarases. This novel enzyme was found to be an endo-type β-agarase (EC 3.2.1.81) that catalyzes the hydrolysis of the β-1,4 linkage of agarose to yield neoagarotetraose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-O-β-d-galactopyranosyl(1→4)-O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d -galactose] and neoagarobiose [O-3,6-anhydro-α-l-galactopyranosyl(1→3)-d-galactose]. The optimum pH and temperature for obtaining high activity of the enzyme were at around 8 and 30°C, respectively. The enzyme did not degrade sodium alginate, λ-carrageenan, ι-carrageenan or κ-carrageenan.     

Effects of Coexistence of β-Casein on Gelation of κ-Carrageenan

The effects of caseins on the rheological properties of κ-carrageenan-calcium gel was investigated by measuring the gel breaking strength. The existence of β-casein in the system promoted the gelation of κ-carrageenan in the presence of calcium ion. Beta-casein increased the strength of calcium gels of κ-carrageenan with increasing NaCl concentration up to 80 mM and strengthened the κ-carrageenan-calcium gel at neutral pH. The values obtained from the slopes of the logarithmic plots of the gel strength versus concentration were 2.15 for κ-carrageenan gel and 2.27 for a β-casein-κcarrageenan mixture gel, suggesting that β-casein may participate in the gelation of κ-carrageenan through the mediation of calcium ions.     

Investigation of the ability of several naturally occurring and synthetic polyanions to bind to and potentiate the biological activity of acidic fibroblast growth factor

The ability of several animal, plant, and bacterial derived polyanions (PAs) as well as synthetic PAs to compete with heparin for the binding of acidic fibroblast growth factor (aFGF) was correlated with their ability to potentiate the mitogenic and neurotrophic actions of this factor. Dextran sulphate, K-carrageenan, pentosan sulphate, polyanethole sulfonate, heparin, and fucoidin competed for the heparin binding site on aFGF at relatively low concentrations (≤50 μg/ml). λ-carrageenan, ι-carrageenan, and polyvinyl sulphate exhibited lower affinity for aFGF, whereas hyaluronic acid, dermatan sulphate, chondroitin-6-sulphate, chondroitin-4-sulphate, and uncharged dextran displayed very low or no demonstrable affinity. Potentiation of the mitogenic action of aFGF for Balb/c 3T3 fibroblasts tended to be in general agreement with the aFGF binding affinity of the PAs. However, polyanethole sulfonate, the carrageenans, polyvinyl sulphate, fucoidin, and pentosan sulphate exerted a mitogenic action on the 3T3 cells that was independent of, and in addition to, the ability of these GAGs to potentiate the action of aFGF. The ability to potentiate the neurotrophic action of aFGF for E8 chick ciliary neurons was a general property of those PA with low or no activity in the mitogen assay. Thus hyaluronic acid, dermatan sulphate, chondroitin-4-sulphate, chondroitin-6-sulphate, and even uncharged dextran all potentiated aFGF induced neuronal survival. The differential effects of these PA in potentiating the biological activities of aFGF are discussed in relation to their ability to compete for the heparin-binding site of aFGF. © 1993 Wiley-Liss, Inc.     

Inhibition of the soluble guanylate cyclase from rat lung by sulfated polyanions

Soluble guanylate cyclase was partially purified from rat lung homogenates, and shown to be inhibited by the following sulfated polyanions, with the I₅₀ in μg/ml in parentheses: Polyvinyl sulfate (0.33), 40,000-dalton dextran sulfate (0.45), polyanetholesulfonate (0.63) 500,000-dalton dextran sulfate (1.8), λ-carrageenan (2.9), τ-carrageenan (6.1), κ-carrageenan (48.0), heparin (68.0). There was a good correlation between inhibitory potency and sulfate content (as total sulfur). Inhibition by heparin and the carrageenans (but not the others) was potentiated by Mn²⁺, but not Ca²⁺ or Mg²⁺, when [Mn²⁺] exceeded [GTP]. Mn²⁺-potentiation could be blocked by high Na⁺. Heparin-agarose shows promise as an affinity matrix for guanylate cyclase.