Sol–gel transition of alginate solution by the addition of various divalent cations: 13C-nmr spectroscopic study

¹³C-NMR spectroscopic studies have been made on alginate solutions undergoing sol–gel transition induced by four different divalent cations: Ca, Cu, Co, and Mn. From the analysis of nmr spectra and relaxation times, we have found different interaction modes existing between the Ca–alginate systems and the transition metal (Cu, Co, and Mn)–alginate systems. In the Ca–alginate systems, there exists a specific interaction characterized by a strong autocooperative binding between guluronate residues and calcium ions, and all functional groups in guluronate residues are considered to involve the interaction with calcium ions. On the other hand, in transition metal (Cu, Co, and Mn)–alginate systems, sol–gel transition is characterized by a complex formation in which the carboxyl groups in both mannuronate and guluronate residues are coordinated to metal ions. The other functional groups, like hydroxyl groups, do not participate in the binding to metal ions. It is suggested by relaxation time measurements that from a microscopic point of view the sol–gel transition phenomena can be explained as a dynamic process in which the low frequency molecular motions are dominant and increase their proportions with the formation of three-dimensional cross-links. © 1993 John Wiley & Sons, Inc.     

Ionic and acid gel formation of epimerised alginates; the effect of AlgE4

AlgE4 is a mannuronan C5 epimerase converting homopolymeric sequences of mannuronate residues in alginates into mannuronate/guluronate alternating sequences. Treating alginates of different biological origin with AlgE4 resulted in different amounts of alternating sequences. Both ionically cross-linked alginate gels as well as alginic acid gels were prepared from the epimerised alginates. Gelling kinetics and gel equilibrium properties were recorded and compared to results obtained with the original non-epimerised alginates. An observed reduced elasticity of the alginic acid gels following epimerisation by AlgE4 seems to be explained by the generally increased acid solubility of the alternating sequences. Ionically (Ca(2+)) cross-linked gels made from epimerised alginates expressed a higher degree of syneresis compared to the native samples. An increase in the modulus of elasticity was observed in calcium saturated (diffusion set) gels whereas calcium limited, internally set alginate gels showed no change in elasticity. An increase in the sol-gel transitional rate of gels made from epimerised alginates was also observed. These results suggest an increased possibility of creating new junction zones in the epimerised alginate gel due to the increased mobility in the alginate chain segments caused by the less extended alternating sequences.     

Interaction between alginates and manganese cations: identification of preferred cation binding sites

Algal and bacterial alginates have been studied by means of 13C NMR spectroscopy in presence of paramagnetic manganese ions in order to reveal the nature of their interaction with bivalent cations. It is found that the mannuronate blocks bind manganese cations externally near their carboxylate groups, while guluronate blocks show the capability to integrate Mn2+ into pocket-like structures formed by adjacent guluronate residues. In alternating mannuronate-guluronate blocks, manganese ions preferentially locate in a concave structure formed by guluronate-mannuronate pairs. Partial acetylation of the alginate generally reduces its capability to interact with bivalent cations, however, the selectivity of the binding geometry is conserved. The results may serve as a hint for the better understanding of the alginate gelation in presence of calcium ions.     

Carbohydrates of the seaweeds,Desmarestia ligulata and D. firma

Crystalline mannitol and some oligosaccharides were separated from ethanolic extracts of Desmarestia ligulata and D. firma. Laminaran, ‘fucans’ and alginic acid were also isolated from both species. The laminaran from D. ligulata comprised both M- and G-chains but no M-chains were found in the laminaran from D. firma. In both species the amount of ‘fucan’ was small, particularly in D. firma. Both ‘fucans’ contained glucuronic acid, galactose, xylose and fucose and that from D. ligulata also contained mannose. After sequential extraction of D. ligulata with water, acid and alkali evidence was obtained for the presence of cellulose, a uronan, and protein in the residual material.     

Mammalian-lung galactan

Exopolysaccharides of Agrobacterium tumefaciens and Rhizobium meliloti, containing d-glucose, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 6:1:1:1.5, were analysed by methylation. They were found to contain the following main structural units (all β-glycosidic): chain residues of (1→3)-linked d-glucose (24%), (1→3)-linked d-galactose (15%), (1→4)-linked d-glucose (20%), and (1→6)-linked d-glucose (18%); (1→4,1→6)-linked branching residues of d-glucose (12%), and terminal d-glucose residues substituted at positions 4 and 6 by pyruvate (11%). Uronic acid-containing exopolysaccharides of Rhizobium leguminosarum, R. phaseoli, and R. trifolii contained d-glucose, d-glucuronic acid, d-galactose, pyruvic acid, and O-acetyl groups in the approximate proportions 5:2:1:2:3. Methylation gave identical patterns of methylated sugar components, from which the following structural elements were deduced: chain residues of (1→3)-linked d-glucose substituted at positions 4 and 6 by pyruvate (13%), (1→4)-linked d-glucose (32%), and (1→4)-linked d-glucuronic acid (20%); (1→4,1→6)-linked branching residues of d-galactose and/or d-glucose (13%), and terminal d-glucose and/or d-galactose residues substituted at positions 4 and 6 by pyruvate (13%).     

Novel Alginate Lyase (Aly5) from a Polysaccharide-Degrading Marine Bacterium,Flammeovirga sp. Strain MY04: Effects of Module Truncation on Biochemical Characteristics,Alginate Degradation Patterns,and Oligosaccharide-Yielding Properties

Alginate lyases are important tools for oligosaccharide preparation, medical treatment, and energy bioconversion. Numerous alginate lyases have been elucidated. However, relatively little is known about their substrate degradation patterns and product-yielding properties, which is a limit to wider enzymatic applications and further enzyme improvements. Herein, we report the characterization and module truncation of Aly5, the first alginate lyase obtained from the polysaccharide-degrading bacterium Flammeovirga. Aly5 is a 566-amino-acid protein and belongs to a novel branch of the polysaccharide lyase 7 (PL7) superfamily. The protein rAly5 is an endolytic enzyme of alginate and associated oligosaccharides. It prefers guluronate (G) to mannuronate (M). Its smallest substrate is an unsaturated pentasaccharide, and its minimum product is an unsaturated disaccharide. The final alginate digests contain unsaturated oligosaccharides that generally range from disaccharides to heptasaccharides, with the tetrasaccharide fraction constituting the highest mass concentration. The disaccharide products are identified as ΔG units. While interestingly, the tri- and tetrasaccharide fractions each contain higher proportions of ΔG to ΔM ends, the larger final products contain only ΔM ends, which constitute a novel oligosaccharide-yielding property of guluronate lyases. The deletion of the noncatalytic region of Aly5 does not alter its M/G preference but significantly decreases the enzymatic activity and enzyme stability. Notably, the truncated protein accumulates large final oligosaccharide products but yields fewer small final products than Aly5, which are codetermined by its M/G preference to and size enlargement of degradable oligosaccharides. This study provides novel enzymatic properties and catalytic mechanisms of a guluronate lyase for potential uses and improvements.     

Monomer composition and sequence of sodium alginate extracted at pilot plant scale from three commercially important seaweeds from Mexico

The marine waters of the Baja California peninsula (Mexico) are a rich source of brown seaweeds with a great potential for exploitation. For that reason, Sargassum sinicola, Eisenia arborea, and Macrocystis pyrifera collected from different locations were subjected to extraction of sodium alginate using a pilot-plant scale process developed in our facilities. The composition and sequence parameters of the recovered alginate were studied by infrared and nuclear magnetic resonance spectroscopy. The spectral analysis of the products revealed that sodium alginate from S. sinicola contains a greater proportion of guluronate monomers (64%) than that from E. arborea (48%), and M. pyrifera (38%). Computation of the frequencies of diads and triads indicated that the alginate from S. sinicola was constructed by intercalated guluronate-blocks of 14 residues in length. In contrast, the length of the G-block in the alginates from E. arborea and M. pyrifera were 7 and 4 residues, respectively. The results show that S. sinicola, E. arborea, and M. pyrifera are sources of sodium alginate with different mannuronate/guluronate ratios, as well as a varied building-block length. In consequence, aqueous dispersions of sodium alginate from the three studied species are expected to exhibit different physical properties.     

Chemistry of the polysaccharides of the brown seaweed dictyopteris plagiogramma

Laminaran, fucose-containing polysaccharides (‘fucans’) and alginic acid were isolated from Dictyopteris plagiogramma.The laminaran comprised G- and M-chains (ratio 3: 1). The ‘fucans’ were present in four extracts of a four-step sequential extraction procedure and all contained slightly differing proportions of fucose, xylose, galactose, mannose, glucuronic acid residues and half-ester sulphate. Non-reducing chain ends as well as the positions of glycosidic linkages to fucose, xylose and glucuronic acid are the same as previously reported for other ‘fucans’. Galactose and mannose occur mainly as trisubstituted residues with substitution at 0-1, 0-3, 0-4 and at 0-1, 0-3, 0-6, respectively.     

Characterization of insoluble calcium alginates by solid-state NMR

Three series of 9 insoluble calcium alginate powders with different average calcium contents (1.5, 3.5 and 8%, w/w) are investigated by means of ¹³C solid-state NMR spectroscopy. The effect of the increased calcium content on the determination of the mannuronate (M) to guluronate (G) ratio from spectral deconvolution of the ¹³C CP/MAS spectra is discussed, and the variations observed are commented in function of possible structural modifications related to the interaction with the divalent cations. The possibility of using solid-state NMR spectroscopy for the quantification of the calcium content in unknown alginate samples is explored performing principal component analysis (PCA) of the spectra. The results obtained show that a clear separation of alginates with slightly different calcium content is possible. The proposed method relies on the sole use of the chemical shifts of the signals corresponding to pyranose carbons, suggesting that PCA of solid-state NMR data holds promises as a rapid and undestructive method for screening the calcium content of alginate-based materials with biomedical uses.     

Isolation and characterisation of the cell-wall fibres of carrot

Cell-wall fractions have been prepared from an alcohol-insoluble-residue of carrot root by treatment with (a) Pronase to remove the cytoplasmic proteins, (b) hot dilute acid and cold dilute alkali to give pectin-free residues, and (c) concentrated alkali to leave the α-cellulose and lignin. The purified cell-wall material still contained ∼ 1% protein and was composed mainly of cellulose, lignin, methyl-esterified galacturonic acid, and smaller amounts of galactose and arabinose. Methylation analysis of the insoluble residues indicated the presence, in order of decreasing concentration, of rhamnogalacturonan with the rhamnosyl residues carrying side chains at position 4, cellulose, (1→4)-linked galactan, (1→5)-linked arabinan, (1→4)-linked xylan, (1→4)-linked mannan, and xyloglucan.     

Characterization of mucoid Pseudomonas aeruginosa strains isolated from technical water systems

The occurrence of mucoid Pseudomonas aeruginosa strains was investigated in water samples and surface material from non-clinical aquatic environments. Ten of 81 environmental isolates displayed a mucoid colony type after incubation at 36°C for 24 h on Pseudomonas Isolation Agar. The mucoid strains obtained exclusively from surfaces of technical water systems were characterized in terms of medium-dependent expression of mucoid colonial phenotype, exoenzyme profile, pigment production and O-antigen type. The mucoid strains secreted substantially higher quantities of carbohydrate and uronic acid-containing material compared to non-mucoid environmental isolates. Major slime components of the mucoid strains were identified as O-acetylated alginates that contained higher proportions of mannuronate than guluronate monomer residues and were composed of blocks of poly-mannuronate and poly-mannuronate/guluronate, whereas blocks of poly-guluronate were absent. The results suggest that surfaces in aquatic environments may represent a natural habitat for mucoid (i.e. alginate-overproducing) strains of Ps. aeruginosa with properties similar to clinical mucoid strains.     

Microbial and algal alginate gelation characterized by magnetic resonance

Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca(2+) ions generates gels of different bulk homogeneities dependent on the alginate structure. Shorter spin-spin T(2) magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.     

Kinetics and specificity of alginate lyases: Part I, A case study

Purified preparations of alginate lyase from Klebsiella aerogenes and Haliotis sp. were investigated for activity and degradation patterns with alginate and alginate fragments having different compositions and sequences. With fragments approaching homopolymers of guluronate and mannuronate, Michaelis-Menten kinetics were obeyed and kinetic parameters could be obtained. Degradation of alginates containing all four possible linkages in various proportions, followed by isolation of the fragments and identification of the end groups by n.m.r. spectroscopy, indicated that the enzyme preparations can attack more than one type of linkage. The results are discussed with reference to the concept of specificity for enzymes with copolymeric substrates having non-regular distributions of units.     

Interactions of alginates with univalent cations

Interactions of alginate with univalent cations in solution have been investigated by circular dichroism (c.d.) and rheological measurements. Poly-l-guluronate chain-segments show substantial enhancement (～ 50%) of c.d. ellipticity in the presence of excess of K⁺, with smaller changes for other univalent cations: Li⁺ < Na⁺ < K⁺ > Rb⁺ > Cs⁺ > NH₄⁺. The maximum c.d. change is attained by 0.3m, with no further increase at higher concentrations of cation. No significant dependence on polymer concentration is observed. Spectral changes for poly-d-mannuronate and heterotypic chain-sequences are much smaller. For intact alginates, the magnitude of c.d. change varies almost linearly with poly-l-guluronate content. Difference spectra (c.d. with excess of univalent counterion minus c.d. in distilled water) can be fitted accurately to two Gaussian bands at 211 and 198 nm, assigned to carboxyl n → π^* and π → π^* transitions, respectively. The perturbations induced by Li⁺, K⁺, Rb⁺, Cs⁺, and NH₄⁺ show a clear family relationship, and are mainly in the π → π^* spectral region. With Na⁺, by contrast, c.d. change is largely confined to the n → π^* transition, and is similar to that previously reported for intermolecular (“egg-box”) binding of divalent cations, consistent with results of rheological studies which indicate Na⁺-induced association of poly-l-guluronate chain-sequences. These associations are further enhanced on freezing and thawing. This combined evidence is interpreted in terms of three modes of interaction between univalent cations and alginate chains in solution: (a) ion-pair formation with carboxyl groups of mannuronate and isolated guluronate residues; (b) specific site-binding to contiguous guluronate residues; and (c) co-operative “egg-box” binding, particularly of Na⁺, between poly-l-guluronate chain-sequences.     

Enzymatically Depolymerized Alginate Oligomers That Cause Cytotoxic Cytokine Production in Human Mononuclear Cells

Enzymatically depolymerized guluronate and mannuronate oligomers were prepared from polyuronates with an alginate lyase from a Pseudoalteromonas sp., and their effects on mononuclear cells from human peripheral blood were examined. Conditioned medium prepared by the incubation of cells with an untreated polyuronate had little effect on growth of human leukemic U937 cells, but a medium prepared with depolymerized uronate oligomers inhibited their growth. Inhibition was greater in a medium prepared with guluronate oligomer than one prepared with mannuronate oligomer. The cytotoxic activity of the medium was heat-labile and nondialyzable. Apoptotic nuclear morphological changes and increased caspase-3-like activity were found in U937 cells treated with a medium prepared with depolymerized uronates. The medium prepared with purified tetra-guluronate and tetra-mannuronate also was cytotoxic; these effects were inhibited by antibodies to tumor necrosis factor-α. Our results suggested that enzymatically depolymerized guluronate and mannuronate oligomers induced the production of cytotoxic cytokines in human mononuclear cells, although the uronate polymers before depolymerization had no such activity.     

Enzymatically depolymerized alginate oligomers that cause cytotoxic cytokine production in human mononuclear cells

Enzymatically depolymerized guluronate and mannuronate oligomers were prepared from polyuronates with an alginate lyase from a Pseudoalteromonas sp., and their effects on mononuclear cells from human peripheral blood were examined. Conditioned medium prepared by the incubation of cells with an untreated polyuronate had little effect on growth of human leukemic U937 cells, but a medium prepared with depolymerized uronate oligomers inhibited their growth. Inhibition was greater in a medium prepared with guluronate oligomer than one prepared with mannuronate oligomer. The cytotoxic activity of the medium was heat-labile and nondialyzable. Apoptotic nuclear morphological changes and increased caspase-3-like activity were found in U937 cells treated with a medium prepared with depolymerized uronates. The medium prepared with purified tetra-guluronate and tetra-mannuronate also was cytotoxic; these effects were inhibited by antibodies to tumor necrosis factor-alpha. Our results suggested that enzymatically depolymerized guluronate and mannuronate oligomers induced the production of cytotoxic cytokines in human mononuclear cells, although the uronate polymers before depolymerization had no such activity.     

A Novel Alginate Lyase with High Activity on Acetylated Alginate of Pseudomonas aeruginosa FRD1 from Pseudomonas sp. QD03

Summary To exploit alginate lyase which could degrade bacterial alginates, degenerate PCR and long range-inverse PCR (LR-IPCR) were used to isolate alginate lyase genes from soil bacteria. Gene algL, an alginate lyase-encoding gene from Pseudomonas sp. QD03 was cloned, and it was composed of a 1122 bp open reading frame (ORF) encoding 373 amino acid residues with the calculated molecular mass of 42.2 kDa. The deduced protein had a potential N-terminal signal peptide of 20 amino acid residues that was consistent with its proposed periplasmic location. Gene algL was expressed in pET24a (+)/E. coli BL21 (DE3) system. The recombinant AlgL was purified to electrophoretic homogeneity using affinity chromatography. The molecular weight of AlgL was estimated to be 42.8 kDa by SDS-PAGE. AlgL exhibited maximal activity at pH 7.5 and 37 °C. Na⁺, K⁺, Ca²⁺ and Ba²⁺ significantly enhanced the activity of AlgL. AlgL could degrade alginate and mannuronate blocks, but hardly degrade guluronate blocks. In particular, AlgL could degrade acetylated alginate of Pseudomonas aeruginosa FRD1 (approximately 0.54 mol of O-acetyl group per mol of alginate). It might be possible to use alginate lyase AlgL as an adjuvant therapeutic medicine for the treatment of disease associated with P. aeruginosa infection.     

A New Fungal α-D-glucan,elsinan, elaborated by Elsinoe leucospila

A new α-D-glucan, designated elsinan, has been isolated from the culture filtrate of Elsinoe leucospila grown in potato extract-sucrose medium. Acid hydrolysis of the methylated polysaccharide gave 2,3,6- and 2,4,6-tri-O-methyl-D-glucose, in the ratio of 2.5:1.0, together with small proportions of 2,3,4,6-tetra- (0.7%) and 2,4-di-O-methyl-D-glucose (0.5%), indicating that the glucan is an essentially linear polymer containing (1→4)- and (1→3)-α-D-glucosidic linkages. Periodate oxidation, followed by borohydride reduction and mild hydrolysis with acid (mild Smith degradation) yielded 2-O-α-D-glucosyl-D-erythritol and erythritol, in the molar ratio of 1.0:1.4, and a trace of glycerol. Partial acid hydrolysis, and also acetolysis, of elsinan gave nigerose, maltose, O-α-D-glucopyranosyl-(1→3)-O-α-D-glucopyranosyl (1→4)-D-glucopyranose, O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→3)-D-glucopyranose, maltotriose, and a small proportion of maltotetraose. It is concluded that elsinan is composed mainly of maltotriose residues joined by α-(1→3)-linkages, in the sequence →3)-α-D-Glcp-(1→4)-α-D-Glcp-(1→.The unique structural features of elsinan are discussed in comparison with other glucans.     

Photolytic depolymerization of alginate

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to ∼40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3 h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using ¹H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.     

Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.