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There is an increased need for alginate materials with both enhanced and controllable mechanical properties in the fields of food, pharmaceutical and specialty applications. In the present work, well-characterized algal polymers and mannuronan were enzymatically modified using C-5 epimerases converting mannuronic acid residues to guluronic acid in the polymer chain. Composition and sequential structure of controls and epimerized alginates were analyzed by (1)H NMR spectroscopy. Mechanical properties of Ca-alginate gels were further examined giving Young's modulus, syneresis, rupture strength, and elasticity of the gels. Both mechanical strength and elasticity of hydrogels could be improved and manipulated by epimerization. In particular, alternating sequences were found to play an important role for the final mechanical properties of alginate gels, and interestingly, a pure polyalternating sample resulted in gels with extremely high syneresis and rupture strength. In conclusion, enzymatic modification was shown to be a valuable tool in modifying the mechanical properties of alginates in a highly specific manner.

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.

In the present experiments we have demonstrated increased protection against a pathogenic bacterium in juvenile turbot (Scophthalmus maximus L.), after feeding the juveniles the live food organism Artemia enriched with a suspension of alginate micro-spheres containing an immunostimulant. Most likely the effect was due to stimulation of the non-specific defence of the juveniles. Poor control of the microbial conditions in the intensive rearing facilities may adversely affect the health of fish larvae, and is most likely one of the main factors restricting production of marine juveniles. Methods which help to control the microflora in the system or improve the resistance of the larvae against bacterial infection may substantially improve larval viability. Newly hatched marine fish larvae have not developed a specific immune defence. Except for eventual maternally transferred immunity, as described for tilapias, the larvae therefore strongly depend on non-specific immunity against bacterial infections. Several studies have shown that the non-specific defence system of fish can be stimulated (Robertsen et al., 1991; Anderson, 1992). Alginate is reported to have a strong stimulatory effect on cytokine production by human monocytes, with the mannuronic acid as the major active component. Incubation of yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.) in water to which mannuronic acid rich alginate (FMI) was added, improved the survival rate (Vadstein et al., 1993). This suggests that FMI may stimulate the non-specific system of marine fish larvae.

Through enzymatic modification, we are now able to manipulate the composition and sequential nanostructures of alginate, one of the most versatile gelling polymers found in nature. Here we report the application of a set of processive polymer-modifying epimerases for the preparation of novel alginates with highly improved functional properties essential for numerous applications as gel matrices. Gels of enzymatically engineered alginate were found to be more elastic and compact, less permeable, and extremely stable under physiological conditions, offering significant advantages over native alginates. As a result, this study shows that, by controlling alginate nanostructure, its macroscopic properties can be highly controlled. The ability to tailor alginate has a great impact on the wide use of this biomaterial in industry and medicine. More importantly, this adds more knowledge to the link between polymer nanostructure and macroscopic properties and may serve as a model system for other polymer-based materials.

Microcapsules of alginate cross-linked with divalent ions are the most common system for cell immobilization. In this study, we wanted to characterize the effect of different alginates and cross-linking ions on important microcapsule properties. The dimensional stability and gel strength increased for high-G alginate gels when exchanging the traditional Ca2+ ions with Ba2+. The use of Ba2+ decreased the size of alginate beads and reduced the permeability to immunoglobulin G. Strontium gave gels with characteristics lying between calcium and barium. Interestingly, high-M alginate showed an opposite behavior in combination with barium and strontium as these beads were larger than beads of calcium−alginate and tended to swell more, also resulting in increased permeability. Binding studies revealed that different block structures in the alginate bind the ions to a different extent. More specifically, Ca2+ was found to bind to G- and MG-blocks, Ba2+ to G- and M-blocks, and Sr2+ to G-blocks solely.

The physical properties of alginate gels correlate with alginate composition. Blocks of guluronic acid (G) strongly contribute to gel formation. Recently, the role of alternating sequences in calcium-alginate gels has been elucidated. The present contribution aimed at extending the analysis already reported (Donati, I.; Holtan, S.; Mørch, Y. A.; Borgogna, M.; Dentini, M.; Skjåk-Braek, G. Biomacromolecules 2005, 6, 1031) and at explaining some apparent mismatch of experimental data. In the present work, calcium hydrogels from different alginate samples have been analyzed by means of uniaxial compression and puncture tests to evaluate their Young's modulus and work at break. The role of long MG blocks in mechanical deformations (small and large domains) as well as in swelling experiments was investigated with natural and MG-enriched (AlgE4 epimerized) alginate samples. Alginates with elongated alternating sequences displayed, upon treatment with saline solution, a notable increase in swelling behavior, which was not paralleled by increased mechanical properties (Young's modulus). This behavior was traced back to the disentanglement of MG/MG junctions, which increased the local charge density, reducing the osmotic contribution to hydrogel swelling. The analyses of the large deformation curves for natural and epimerized alginates revealed an increase in the energy to breakage in the latter case caused by the dissipation effect of "sliding" MG/MG junctions.

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.

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.

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.