Engineering new alginate structures

Alginates are used in a number of pharmaceutical products. Rolf Myrvold*, Olav Gaserod and Edvar Onsoyen, from FMC BioPolymer, give a brief review of the current application of biotechnology in engineering new alginate structures

Alginate occurs as a skeletal component in marine brown algae, providing the necessary strength and flexibility to the plant and as capsular polysaccharides in soil bacteria. Currently, all commercially available alginates are produced from natural marine brown algae sources. A careful selection of alginates extracted from specific seaweeds is the current procedure applied when commercial grades with desired properties are developed. Today, alginate functionality as a function of primary chemical structure is fairly well understood, and alginates can be tailored to specific chemical and physical performances by using biotechnology. The level of understanding in this field of research has increased tremendously during the last decade.^1-4

alginate structure

In the pharmaceutical, medical device and dental industries, alginate finds application as the raft forming agent in medications for the treatment of esophageal reflux symptoms, as the matrix retard agent in oral dosage sustained release formulations, in dressings for moist wound care and in dental impression materials. It is believed that current and future biotechnological developments in alginate technology will improve the performance of alginate in both existing and new applications.

Alginate is a linear copolymer with homopolymeric blocks of (1'4)-linked b-D-mannuronate (M) and its C-5 epimer a-L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. The monomers can appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M-blocks), alternating M and G-residues (MG-blocks) or randomly organised blocks. The relative amount of each block type varies both with the origin of the alginate (seaweed type) and from which part of the plant the alginate is extracted. The structure of the mannuronate and guluronate monomer residues and examples of alginate sequences are illustrated in figure 1.

Alternating blocks form the most flexible chains and are more soluble at lower pH than the other blocks. Alginate with a very high M content has been found to be strongly immunostimulating.⁵ C-5 epimer a-L-guluronate blocks form stiff chain elements, and two G-blocks of more than six residues each form stable cross-linked junctions with divalent cations (e.g. Ca²+, Ba²+ and Sr²+ among others) leading to a three-dimensional gel network.^6,7

At low pH, protonised alginates will form acidic gels. In these gels, it is mostly the homopolymeric blocks that form the junctions, where the stability of the gel is determined by the relative content of C-5 epimer a-L-guluronate blocks.8

Determination of the primary structure is possible using NMR and figure 2 shows a typical spectrum. Table 1 gives typical structural information obtained from high-resolution proton NMR for several types of alginate.

biosynthesis

Alginates are not confined to the algal world as is the case with other algal marine polysaccharides such as agar and carrageenan. They are also synthesised as exocellular material in some types of bacteria.^9-12 The biosynthetic pathway of alginate was first studied in brown algae,¹³ but the present knowledge of the enzymes and genes controlling the alginate synthesis originates from the studies of two bacterial species, Pseudomonas aeruginosa and Azotobacter vinelandii. The alginate made by P. aeruginosa participates in adherence to surfaces, protects against the host's immune system and against certain antibiotics and is almost exclusively associated with lung infection in patients suffering from cystic fibrosis.¹⁴ In A. vinelandii the alginate production is a mandatory requirement for cyst formation. Cysts are metabolically dormant cells, characterised by several layers of polysaccharide material surrounding the cell.¹⁵ The polysaccharide coating protects the cell from desiccation and mechanical stress, and cysts have been reported to survive in dry soil for several years.¹⁶ Under favorable conditions, including the presence of water, the coating will swell, and the cysts germinate, divide and regenerate to vegetative cells.¹⁵

The most important difference between algal and bacterial alginate is the presence of O-acetyl groups in the latter.¹⁷ Apart from the acetyl groups, A. vinelandii alginates resemble the seaweed material in that they are true block copolymers with homopolymeric regions as described above. This is not the case for alginates derived from P. aeruginosa, which lacks G-blocks and consequently always contains less than 50% guluronic acid residues. These variations reflect the differences in the systems synthesising the polymer in the two organisms.

The biosynthetic route for alginates is shown schematically including the enzymes and different substrates for the seven-step synthesis. The genes involved in the biosynthesis were first identified in P. aeruginosa.¹⁴ As illustrated in figure 4, AlgA, encoding the bi-functional enzyme phosphomannoisomerase-guanosine diphosphomannose pyrophosphorylase, and AlgD, encoding GDP-mannose dehydrogenase, are flanking a gene cluster encoding other genes involved in the biosynthesis. The third gene involved in the synthesis of precursors, AlgC, encoding phosphomannomutase, is found as a cluster with genes involved in the synthesis of lipopolysaccharides.¹⁴ The membrane proteins Alg8 and Alg44 are probably involved in the polymerisation reaction, and it has been suggested that Alg60 also participates in this process. The lyase, epimerase and acetylase, AlgL, AlgG and AlgF, respectively, are all enzymes that modify the alginate. The enzyme AlgE is believed to be involved in the alginate secretion process.

applied alginate biotechnology

C-5-epimerase enzymes from A. vinelandii have been used to modify alginates in vitro,^18-20 resulting in polymers with improved gelling properties. Today eight different enzymes in the mannuronan C-5-epimerase family of A. vinelandii are known. Table 2 lists the different enzymes, the functionality and the resulting alginate structure where this has been determined and confirmed.

The epimerases have also been used to reduce the compositional heterogeneity in an alginate population. As shown before, the average composition of alginate may vary from plant to plant and from one tissue to another within the same plant.

Commercial grades of alginate can therefore be considered to be mixes of sub-populations of molecules. Applying the enzyme activity on the long homopolymeric sequences of mannuronic acid, an endpoint occurs where the M-blocks become too short to support further enzyme activity.²¹

By treating a commercially available alginate with C-5-epimerase, materials are obtained that have both a higher average content of guluronic acid and also a more uniform distribution of

composition and block structure. Modi-fying gene expression to optimise the desired enzyme activity may extend this technology further.

From an industrial point of view this new technology opens new potentials. Developing new alginate primary structure elements where the mode of action is known, may increase the performance in current applications and also increase the applicability in non-traditional applications, i.e. new alginates with functionalities not seen today.

Being able to define the natural polymer in terms of structure and molecular weight distributions is beneficial with respect to documentation, especially for highly regulated industries such as the pharmaceutical industry. The alginate production process is both time-consuming and cost-intense. However, applying the new epimerase technology in full-scale industrial productions may open new markets for tailored alginates with improved characteristics.