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With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown that alginate from algal sources all contain a fraction of very long G-blocks (VLG), that is, consecutive sequences of guluronic acid (G) residues of more than 100 residues. By comparing the gelling properties of these materials with in vitro epimerized polymannuronic acid (poly-M) with shorter G-blocks, but comparable with the G-content, we could demonstrate that VLG have a large influence on gelling properties. Hypothesized to function as reinforcement bars, VLG prevents the contraction of the gels during formation (syneresis) and increases the Young's modulus (strength of the gel). Here we report that these VLG structures are also present in alginates from Azotobacter vinelandii and that these polymers consequently form stable, low syneretic gels with calcium, comparable in mechanical strength to algal alginates with the similar monomeric composition. The bacterium expresses seven different extracellular mannuronan epimerases (AlgE1-AlgE7), of which only the bifunctional epimerase AlgE1 seems to be able to generate the long G-blocks when acting on poly-M. The data implies evidence for a processive mode of action and the necessity of two catalytic sites to obtain the observed epimerization pattern. Furthermore, poly-M epimerized with AlgE1 in vitro form gels with comparable or higher rigidity and gel strength than gels made from brown seaweed alginate with matching G-content. These findings strengthen the viability of commercial alginate production from microbial sources.

Alginate is a promising polysaccharide for use in biomaterials as it is biologically inert. One way to functionalize alginate is by chemical sulfation to emulate sulfated glycosaminoglycans, which interact with a variety of proteins critical for tissue development and homeostasis. In the present work we studied the impact of chain length and flexibility of sulfated alginates for interactions with FGF-2 and HGF. Both growth factors interact with defined sequences of heparan sulfate (HS) at the cell surface or in the extracellular matrix. Whereas FGF-2 interacts with a pentasaccharide sequence containing a critical 2-O-sulfated iduronic acid, HGF has been suggested to require a highly sulfated HS/heparin octasaccharide. Here, oligosaccharides of alternating mannuronic and guluronic acid (MG) were sulfated and assessed by their relative efficacy at releasing growth factor bound to the surface of myeloma cells. 8-mers of sulfated MG (SMG) alginate showed significant HGF release compared to shorter fragments, while the maximum efficacy was achieved at a chain length average of 14 monosaccharides. FGF-2 release required a higher concentration of the SMG fragments, and the 14-mer was less potent compared to an equally sulfated high-molecular weight SMG. Sulfated mannuronan (SM) was subjected to periodate oxidation to increase chain flexibility. To assess the change in flexibility, the persistence length was estimated by SEC-MALLS analysis and the Bohdanecky approach to the worm-like chain model. A high degree of oxidation of SM resulted in approximately twice as potent HGF release compared to the nonoxidized SM alginate. The release of FGF-2 also increased with the degree of oxidation, but to a lower degree compared to that of HGF. It was found that the SM alginates were more efficient at releasing FGF-2 than the SMG alginates, indicating a greater dependence on monosaccharide identity and charge orientation over chain flexibility and charge density.

Sulfated glycosaminoglycans have a vast range of protein interactions relevant to the development of new biomaterials and pharmaceuticals, but their characterization and application is complicated mainly due to a high structural variability and the relative difficulty to isolate large quantities of structurally homogeneous samples. Functional and versatile analogues of heparin/heparan sulfate can potentially be created from sulfated alginates, which offer structure customizability through targeted enzymatic epimerization and precise tuning of the sulfation degree. Alginates are linear polysaccharides consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G), derived from brown algae and certain bacteria. The M/G ratio and distribution of blocks are critical parameters for the physical properties of alginates and can be modified in vitro using mannuronic-C5-epimerases to introduce sequence patterns not found in nature. Alginates with homogeneous sequences (poly-M, poly-MG, and poly-G) and similar molecular weights were chemically sulfated and structurally characterized by the use of NMR and elemental analysis. These sulfated alginates were shown to bind and displace HGF from the surface of myeloma cells in a manner similar to heparin. We observed dependence on the sulfation degree (DS) as well as variation in efficacy based on the alginate monosaccharide sequence, relating to relative flexibility and charge density in the polysaccharide chains. Co-incubation with human plasma showed complement compatibility of the alginates and lowering of soluble terminal complement complex levels by sulfated alginates. The sulfated polyalternating (poly-MG) alginate proved to be the most reproducible in terms of precise sulfation degrees and showed the greatest relative degree of complement inhibition and HGF interaction, maintaining high activity at low DS values.

This paper reports a study of the distribution and function of homopolymeric guluronic acid blocks (G-blocks) in enzymatically modified alginate. High molecular weight mannuronan was incubated with one native (AlgE6) and two engineered G-block generating mannuronan C-5 epimerases (AlgE64 and EM1). These samples were found to contain G-blocks with a DP ranging from 20 to approximately 50, lacking the extremely long G-blocks (DP > 100) found in algal alginates. Calcium gels from epimerized materials were highly compressible and exhibited higher syneresis and rupture strength but lower Youngs modulus than gels made from algal polymers of similar G-content. Addition of extremely long G-blocks to the epimerized alginate resulted in decreased syneresis and rupture strength and an increased Young's modulus that can be explained by reinforcement of the cross-linking zones at the cost of length and/or numbers of elastic segments. The presence and impact of these extremely long G-blocks found in natural alginates suggest that alginate gels can be viewed as a nanocomposite material.

Biocompatible hydrogels are very interesting for applications in, e.g., tissue engineering and for immobilization of cells, such as calcium-alginate gels where the calcium ions form specific interactions with the guluronic acid units. We here report on a new gelling system of chitosan and alginate containing only mannuronic acid (poly-M), which are prepared using the following steps: (i) mixing at a pH well above 7 where the chitosan is mainly uncharged; (ii) controlled lowering of the pH by adding the slowly hydrolyzing d-glucono-δ-lactone (GDL); (iii) formation of a homogeneous chitosan-alginate gel upon leaving the mixture at room temperature. Some properties of the new gelling system are demonstrated herein by adding controlled amounts of GDL to (i) a mixture of a polymeric and neutral-soluble chitosan with poly-M oligomers (MO) and (ii) a mixture of poly-M and neutral-soluble chitosan oligomers. The neutral-solubility of the polymeric chitosan is achieved by selecting a polymeric chitosan with an intermediate degree of acetylation of 40%, while the neutral-solubility of the fully de-N-acetylated chitosan oligomers (CO) is obtained by selecting oligomers with a chain length below 10. A proof of concept of the new gelling system is demonstrated by measuring the gel strengths of the polymeric chitosan-MO, and a poly-M-CO. The results show that the gel strength increases with decreasing the pH from neutral to 5, and that the gel strength decreases with increasing ionic strength, indicative of an ionic gel formation. Poly-M formed relatively strong gels with CO while an alginate highly enriched in Guluronic acid formed gels of very limited mechanical strength, suggesting the importance of the match in charge distances in the poly-M and chitosan, both with diequatorially linked sugar units in the (4)C1 conformation.

The polysaccharide alginate is produced by brown algae and some bacteria and is composed of the two monomers, β-D-mannuronic acid (M) and α-L-guluronic acid (G). The distribution and composition of M/G are important for the chemical-physical properties of alginate and result from the activity of a family of mannuronan C-5 epimerases that converts M to G in the initially synthesized polyM. Traditionally, G-rich alginates are commercially most interesting due to gelling and viscosifying properties. From a library of mutant epimerases we have isolated enzymes that introduce a high level of G-blocks in polyM more efficiently than the wild-type enzymes from Azotobacter vinelandii when employed for in vitro epimerization reactions. This was achieved by developing a high-throughput screening method to discriminate between different alginate structures. Furthermore, genetic and biochemical analyses of the mutant enzymes have revealed structural features that are important for the differences in epimerization pattern found for the various epimerases.

Distribution and proportion of β-D-mannuronic and α-L-guluronic acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks). This was achieved using a combination of alginate lyases with different substrate specificities, including a novel lyase that specifically cleaves diguluronic acid linkages. Reaction products and isolated fragments of alginates degraded with these lyases were subsequently analyzed with (1)H NMR, HPAEC-PAD, and SEC-MALLS. The method was applied on three seaweed alginates with large differences in sequence parameters (F(G) = 0.32 to 0.67). All samples contained considerable amounts of extremely long G blocks (DP > 100). The finding of long M blocks (DP ≥ 90) suggests that also algal epimerases act by a multiple attack mechanism. Alternating sequences (MG-blocks) were found to be much shorter than the other block types. In connection with method development, an oligomer library comprising both saturated and unsaturated oligomers of various composition and DP 2-15 was made.

The structures of guluronic-acid-rich alginate in the acid and calcium forms were investigated using fiber X-ray diffraction. Data recorded for alginate fibers in the acid form show a repeat along the chain axis of c = 0.87 nm, a value that is in agreement with the one measured by Atkins et al. (Biopolymers 1973, 12, 1865) and contradicts a repeat of 0.78 nm recently suggested by Li et al. (Biomacromolecules 2007, 8, 464). In the Ca2+ form, our observations indicate that the junction zone involves dimerization of polymer chains through Ca2+ coordination according to the egg-box model. For reasons that are not understood at present, coordination of the divalent cations reduces the ability for the lateral crystallographic packing of the dimers. A proposed model for the junction zone involves polymer chains packed on a hexagonal lattice with a lattice constant a = 0.66 nm. Random pairs of chains form dimers through coordination of Ca2+ cations. Further lateral interaction between dimers is mediated by disordered Na+ and Ca2+ cations, water molecules, and hydrogen bonding.

The present study specifically aimed at preparing and characterizing semidilute binary polymer mixtures of alginate and chitlac which might find an application in the field of cell encapsulation. A polyanion, alginate, and a polycation, a lactose-modified chitosan, were mixed under physiological conditions (pH 7.4 and NaCl 0.15) and at a semidilute concentration avoiding associative phase separation. The mutual solubility was found to be dependent on the charge screening effect of the added NaCl salt, being prevented below 0.05 M NaCl. A comparison with the behavior of the polyanion (alginate) under the same experimental conditions revealed that both the viscosity and the relaxation times of the binary polymer solutions are strongly affected by the presence of the polycation. In particular, the occurrence of electrostatic interactions between the two oppositely charged polysaccharides led to a synergistic effect on the zero-shear viscosity of the solution, which showed a 4.2-fold increase with respect to that of the main component of the solution, i.e., alginate. Moreover, the relaxation time, calculated as the reciprocal of the critical share rate, markedly increased upon reducing the alginate fraction in the binary polysaccharide solution. However, the formation of the soluble complexes and the synergistic effect are reduced upon increasing the concentration of the 1:1 electrolyte. By containing a gel-forming polyanion (alginate, e.g., with Ca(2+) ions) and a bioactive polycation (chitlac, bearing a beta-linked D-galactose), the present system can be regarded as a first step toward the development of biologically active scaffold from polysaccharide mixtures.

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.

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.

The availability of mannuronan and mannuronan C-5 epimerases allows the production of a strictly alternating mannuronate-guluronate (MG) polymer and the MG-enrichment of natural alginates, providing a powerful tool for the analysis of the role of such sequences in the calcium-alginate gel network. In view of the calcium binding properties of long alternating sequences revealed by circular dichroism studies which leads eventually to the formation of stable hydrogels, their direct involvement in the gel network is here suggested. In particular, 1H NMR results obtained from a mixed alginate sample containing three polymeric species, G blocks, M blocks, and MG blocks, without chemical linkages between the block structures, indicate for the first time the formation of mixed junctions between G and MG blocks. This is supported by the analysis of the Young's modulus of hydrogels from natural and epimerized samples obtained at low calcium concentrations. Furthermore, the "zipping" of long alternating sequences in secondary MG/MG junctions is suggested to account for the shrinking (syneresis) of alginate gels in view of its dependence on the length of the MG blocks. As a consequence, a partial network collapse, macroscopically revealed by a decrease in the Young's modulus, occurred as the calcium concentration in the gel was increased. The effect of such "secondary" junctions on the viscoelastic properties of alginate gels was evaluated measuring their creep compliance under uniaxial compression. The experimental curves, fitted by a model composed of a Maxwell and a Voigt element in series, revealed an increase in the frictional forces between network chains with increasing length of the alternating sequences. This suggests the presence of an ion mediated mechanism preventing the shear of the gel.

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.

Alginate biosynthesis involves C-5-mannuronan epimerases catalyzing the conversion of beta-D-mannuronic acid to alpha-L-guluronic acid at the polymer level. Mannuronan epimerases are modular enzymes where the various modules yield specific sequential patterns of the converted residues in their polymer products. Here, the interaction between the AlgE4 epimerase and mannuronan is determined by dynamic force spectroscopy. The specific unbinding between molecular pairs of mannuronan and AlgE4 as well as its two modules, A and R, respectively, was studied as a function of force loading rate. The mean protein-mannuronan unbinding forces were determined to be in the range 73-144 pN, depending on the protein, at a loading rate of 0.6 nN/s, and increased with increasing loading rate. The position of the activation barrier was determined to be 0.23 +/- 0.04 nm for the AlgE4 and 0.10 +/- 0.02 nm for its A-module. The lack of interaction observed between the R-module and mannuronan suggest that the A-module contains the binding site for the polymer substrate. The ratio between the epimerase-mannuronan dissociation rate and the catalytic rate for epimerization of single hexose residues suggests a processive mode of action of the AlgE4 epimerase yielding the observed sequence pattern in the uronan associated with the A-module of this enzyme.

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.

Galactose moieties have been introduced on the uronic groups of alginates from different sources via an N-glycosidic bond, thus affecting the net charge on the polymer chain. The modified polymers have been analyzed by means of viscosity and of high-performance size-exclusion chromatography combined with refractive index multiple angle laser light scattering (HPSEC-RI-MALLS) measurements. The latter technique enabled us to determine the molecular weight of the modified polymers, proving that the synthetic procedure did not affect the chemical integrity of the chain. The intrinsic viscosity and the radius of gyration data showed that the hydrodynamic properties of the polymer chain varied with the degree and the pattern of substitution. In the presence of a relatively low galactose content (up to 19%), a decrease of the hydrodynamic dimensions of the coil was experienced, while on increasing the degree of substitution (especially on GG diads) a re-extension of the chain was discovered. Measurements of intrinsic viscosity at different values of the degree of dissociation have demonstrated that this effect cannot be solely explained by the reduction of the charge density of the polymer. Rather, it implies the occurrence of conformational changes of the chain that are specific to the chemical nature of the site of substitution. These data have been supported by the values of the persistence length of the natural and modified polymers obtained with the Doty-Benoit equation. The chiro-optical properties of the modified polymers studied by means of circular dichroism (CD) spectroscopy confirmed that conformational variations occurred to the polymeric chain upon introduction of galactose residues.

Coupling of alginate with 1-amino-1-deoxygalactose in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide results in a substituted polymer containing galactose side linked via an amide bond. To clarify the degree and pattern of substitution, a (1)H NMR study on the anomeric region of modified alginate, polymannuronate, alginate enriched in guluronic acid (G-enriched alginate), and polyalternating MG, was carried out (G, alpha-l-guluronic acid; M, beta-d-mannuronic acid). From the resonance of the proton at position 1 of galactosylamine, it was possible to determine the amount of galactose linked to mannuronic and to guluronic residues, respectively. Furthermore, (1)H NMR spectroscopy revealed a higher reactivity of guluronic residues for low degrees of conversion. Modified alginates with 7% and 19% of substitution are both able to form stable beads in the presence of calcium ions. The effect of galactose substitution on the dimensions, swelling, and stability of the beads has been studied and the cytotoxicity of the modified polymer evaluated in preliminary biological tests.

Konjak glucomannan (KGM) is a water-soluble linear copolymer of (1-->4) linked beta-D-mannopyranosyl and beta-D-glucopyranosyl units. It has been selectively C6-oxidized by a 2,2,6,6-tetramethylpiperidin-1-oxy mediated reaction to obtain the corresponding uronan. Oxidized KGM has been treated with three different C-5 epimerases, AlgE4, AlgE6, and AlgE1, to obtain uronans with a various content of alpha-L-gulopyranuronate residues, namely, KGME4, KGME6, and KGME1. By use of 1D selective and 2D NMR techniques, a full assignment of the high field (600 MHz) NMR spectra of the purified native KGM and of the oxidized and epimerized derivatives has been obtained. Since in the anomeric region of the (1)H NMR spectrum of native KGM, diads sensitivity is present, the glucose-glucose, glucose-mannose, mannose-mannose, and mannose-glucose distribution has been obtained. In the (13)C spectrum of oxidized KGM, due to the presence of triad sensitivity on the C-4 resonance of glucuronic and mannuronic units, a better sequential investigation has been possible. As a result the average length of mannuronic blocks, N(M) is obtained. When AlgE4, AlgE6, and AlgE1 enzymes are used for the epimerization of oxidized KGM, the reaction products differ significantly both in the proportion and in the distribution of the mannuronic and guluronic residues. In epimerized KGM derivatives, a careful deconvolution of (1)H spectra allows the measurement of the degree of epimerization. In the case of KGME1 and KGME6, the average blocks length, N(G), of the guluronic blocks introduced in the polysaccharidic chain with the epimerization has also been calculated. Due to the shortness of mannuronic blocks in the oxidized KGM before the epimerization, N(G) in the epimerized compounds is also very low.

Attention has been focused on two uronans, namely, mannuronan and galactose-depleted C-6 oxidized guar, the former of microbial origin and the latter of artificial nature, to provide original data on the extent of epimerization they can undergo in dilute aqueous solution using two C-5 mannuronic acid epimerizing enzymes, that is, AlgE-4 and AlgE-6, alone or in admixture. Original circular dichroism data coupled with 1H NMR spectra clearly point out that both uronans can be epimerized, depending on the enzyme or enzyme mixture used, to high levels yielding guluronic-rich alginate samples and guluronic-rich heteropolysaccharides, respectively. Mannuronan and its epimerization products can easily form clear, firm aqueous gels when an excess of HCl is added or when mixed with aqueous CaCl2, respectively. Depleted-guarox does not gel upon addition of excess HCl, while the heterouronan derived from it having a percent of epimerization nearly identical to that of epimerized mannuronan, that is, ca. 70%, can form gel in the presence of Ca(II) only at higher polymer and Ca(II) concentrations. With the latter, heterouronan alpha-D-galacturonic side groups exert hindrance to "junction zone" formation.

Different polysaccharides containing D-mannose residues have been C-6-oxidized by a selective TEMPO-mediated hypohalite oxidation to obtain the corresponding uronans. These have been treated with various recombinant mannuronan C-5-epimerases and the resulting products were analyzed by 1H NMR spectroscopy. Oxidized konjac mannan could be epimerized to obtain a uronan with a content of about 12% alpha-L-gulopyranuronate (G) residues. On prolonged epimerization, beta-elimination was observed. The oxidized galactomannan locust bean gum could only be scarcely epimerized, probably due to steric effects exerted by its 26% alpha-D-galacturopyranosyl side groups. Oxidized, galactose-depleted guar gum with a alpha-D-galactosyl content of 11% could be epimerized to a G content of about 15%. With oxidized cellulose as a substrate, mainly beta-elimination was observed. It thus seems that the mannuronan C-5-epimerases employed recognize glucuronate residues and abstract proton-5 but are unable to perform the second epimerization step and instead yield beta-eliminated products.