Elsevier

Carbohydrate Polymers

Volume 67, Issue 4, 19 February 2007, Pages 465-473
Carbohydrate Polymers

Ionic gel formation of a (pseudo)alginate characterised by an alternating MG sequence produced by epimerising mannuronan with AlgE4

https://doi.org/10.1016/j.carbpol.2006.06.020Get rights and content

Abstract

The scope of this paper is the characterization, in terms of viscoelastic and mechanical properties, of ionic gels obtained from solutions of pseudoalginates characterized by a strictly alternating MG sequence (ALG(47%, 0%GG)) in the presence of calcium ions. ALG(47%, 0%GG) was obtained using a recombinantly produced mannuronan C-5 epimerase, named AlgE4 which catalyses the conversion of mannuronic residues into guluronic–mannuronic (GM) blocks. It was established that the kinetics of gelation as well as the mechanical properties and degree of syneresis of the ensuing gels are markedly dependent on both polymer concentration and Ca2+ content.

The molecular dynamic investigation was carried out on a comparative basis between poly(MG) vs. poly(G) and demonstrated that in the case of poly(MG) the structural unit composed by two calcium paired MG oligomer enjoy a higher degree of flexibility in comparison to the equivalent structure based on GG sequence. Furthermore, the electrostatic interaction between Ca2+ ions and carboxylate groups of M and G units is the main driving force to gel formation.

Introduction

Alginate is a binary co-polymer of β-d-mannuronate (M) and α-l-guluronate (G) arranged in a block-wise pattern along the linear chain. This polymer has wide industrial applications due to its viscosifying, gelling, suspending and ion-binding abilities. (Smidsrød & Draget, 1996).

The physical properties of alginates depend not only upon the uronate composition, i.e. the M/G ratio, but also upon the monomer sequence and distribution in the copolymer. The proportion and sequential arrangement of the uronic acid in alginates depends upon the species of algae and the kind of algae tissue from which the polysaccharide is extracted, and vary widely from species to species. This compositional heterogeneity represents a limitation in advanced technological applications in which is of vital importance the availability of alginates with a specific and constant primary structure. The lack of these requisites makes it difficult to obtain materials with the desired and reproducible characteristics. For instance, alginates beads from Laminaria hyperborea and Macrocystis pyrifera characterized by a different content of guluronate, were used as an immobilization matrix of hepatocytes (Murtas et al., 2005). The two kinds of beads exhibited a different permeability with respect to high molecular weight blue-dextrans, used as the probe macromolecules mimicking proteins and antibodies. Furthermore, alginate composition seems to affect also the metabolic activity of the entrapped hepatocytes: the secretion of albumin was better in the case of high mannuronic alginates than high guluronic alginates (Khalil et al., 2001). Thus, the primary structure of the alginates employed affects both the beads porous structure and the physiological response of the entrapped hepatocytes. Also the investigation of the relationship between the properties of alginates both in solution and in the gel state and their M/G ratio and residues sequential arrangement suffers of the variability and lack of control on the primary structure of natural occurring alginates. This drawback was well known to the scientific community involved in alginate research and efforts have been directed towards biosynthetic pathways capable of achieving control over the proportion and distribution of M and G in alginates. This work originated from the discovery that alginate-producing bacterium Azotobacter vinelandii encodes at least seven different mannuronan C-5 epimerases. These genes have been sequenced and cloned and expressed in Escherichia coli; the enzymes thus produced have been designed AlgE1–AlgE7 (Ertesvåg et al., 1994, Svanem et al., 1999). These enzymes are capable of converting M residues into G residues in the polymer chain with different patterns of epimerisation (Draget, Skjåk-Bræk, & Smidsrød, 1997), and can therefore be used to modify alginates in vitro to obtain new alginates with the desired content and distribution of G residues (Ertesvåg et al., 1995). For instance, the C-5 epimerase AlgE4 forms alginates with long strictly alternating sequences (Hartmann, Holm, Johansen, Skjåk-Bræk, & Stokke, 2002), while AlgE1 introduces stretches of G sequences. Thus, an alginate of whatever residue composition and sequence can be converted to an alginate with a composition close to the desired one by employing the appropriate single or blend of C-5 epimerases. Nevertheless, the compositional heterogeneity in natural alginates substrates still represents a limitation as far as the degree of control on the primary structure of the final product is involved. In this respect, a further improvement was represented by the microbial production of a homopolymeric mannuronan, using a C-5 epimerase negative mutant of recombinant Pseudomonas fluorescens (Gimmestad et al., 2003).

By using this polymer as substrates for a single or a blend of recombinant epimerises we are able to generate alginates with specific composition and sequential structures. This has opened the possibility to study the effect of increasing the content of G blocks and/or MG blocks on the mechanical properties of epimerised mannuronan both in solution and in the gel phase (Dentini et al., 2006, Dentini et al., 2005) and to make the important discovery of the ability of alternating MG alginate to undergo gelation in the presence of Ca2+ ions (Donati, Holtan, Mørch, Dentini, & Skjåk-Bræk, 2005) a behavior which was attributed, before the advent of the C-5 epimerases, to alginates rich in G stretches only.

In the present article, we focus on the rheological study of both the gelling kinetics and the viscoelastic properties of the ionic gels obtained from mannuronan epimerised with AlgE4 in the presence of calcium ions. The involvement of MG sequence in the formation of the junction zones induced by calcium ions was investigated by molecular dynamics and a new molecular model for poly(MG)–Ca2+ structure is proposed.

Section snippets

Materials

High molecular weight mannuronan was isolated from the fermentation of fructose in the presence of an epimerase-negative AlgG strain of P. fluorescens (Gimmestad et al., 2003). Purification and deacetylation were carried out as described earlier (Ertesvåg & Skjåk-Bræk, 1999). Pure mannuronan was epimerized by AlgE4 epimerase as described earlier. (Ertesvåg & Skjåk-Bræk, 1999).

The mole fraction of guluronate (G) residues, FG, of GM (MG) diad sequences, FMG, as determined by 1H-NMR (Grasdalen,

Study of the interaction ALG(47%G/0%GG)–Ca2+ ions in dilute aqueous solution by means of circular dichroism (CD) spectra

CD is a particularly valuable and sensitive technique able to reveal the existence and the extent of the interaction between a polymer and a potential ligand. The presence of molecules or ions able to induce conformational changes in the polymeric chain under appropriate physical conditions, leads to a more or less pronounced change in the specific ellipticity trace vs. wavelength.

It is well known that concentrated solutions of alginates are able to form solid-like structures (gels) in the

Conclusions

The availability of pseudoalginates with a strictly alternating sequence rendered possible a detailed characterization of its gelling behaviour. Both the gelling behaviour as well as the mechanical properties and the degree of syneresis strongly depend on the value of R. At relatively low value of R, gels of ALG(47%G/0%GG) even though endowed with characteristics typical of strong gels are relatively elastic. The increase in temperature enhances such feature. The degree of syneresis for these

Acknowledgements

This work has been carried out with the financial support of the European Union (contract QLK3-CT-1999-00034) and “Ateneo Funds” of “La Sapienza” University. C.A. would like to thank Loredana Vaccaro (University of Oxford, UK) for the fundamental support in electron density calculations.

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    Present address: SISSA/ISAS, Via Beirut 2-4, 34014 Trieste, Italy.

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