Enzymatic Fucoidan Extraction and Processing

Marine macroalge are renewable sources of interesting polysaccharides, which show diversity in structure as well as bioactivity. The exploitation of macroalgal polysaccharides for various commercial applications is essential to generate new potential products. Based on the hypothesis that efficient and gentle extraction of fucoidans can be achieved by enzymatic processing, the primary objective of this PhD study was development of a new enzyme-assisted method to extract sulfated polysaccharides (fucoidans) from minimum three different types of brown macroalgae. Then, to prepare and purify fucoidan oligosaccharides by enzymatic hydrolysis to deliver those to various bioactivity assessments (done by others).

Moreover, since alginate lyase is used for enzymatic fucoidan preparation, the study of alginate lyases, and notably characterization of a new alginate lyase, able to degrade alginate from different brown macroalgae was also studied.

The classical extraction methods of fucoidans are based on the use of acid and alkaline solvents, which effect the yield and chemical features of fucoidans. In this study, the enzyme-assisted extraction of fucoidans from Fucus evanescens, Saccharina latissima and Sargassum mcclurei using combinations of glucanase and an alginate lyase was developed and compared to a chemical extraction method. The yield, monosaccharide composition, sulfate content and molecular weight of crude fucoidans was determined. The yield of fucoidans based on extracted fucose amount in enzyme-assisted and chemical extraction was not statistically significantly different. However, the monosaccharide composition of enzymatic and chemical crude fucoidans were quite different in fucose, glucose and alginate level. Since the enzyme-assisted method showed high selectivity in degrading the surrounding cell wall components without harming the fucoidans, the sulfate content and molecular weight of fucoidans were conserved compared to the chemical method. The structure of purified fucoidan fractions from enzymatic extracts of F. evanescens and from both enzymatic and chemical extracts from S. latissima was also confirmed by nuclear magnetic resonance (NMR) spectroscopy.

The fucoidan oligosaccharides from F. evanescens and S. latissima were prepared by using specific fucoidanase enzymes. Hydrolysis products were separated into medium molecular weight and low molecular weight products. The native fucoidans extracted by enzymatic method from F. evanescens and S. latissima and fucoidan oligosaccharides from F. evanescens showed interesting bioactivity. Hydrolysis products from S. latissima were further separated and the structures were investigated. This is the first time the branched oligosaccharides from S. latissima was reported. Especially, based on the NMR data of fucoidan oligosaccharides, the new branch point at C4 of fucose residues was found in fucoidans from cultivated S. latissima compared to reported C2 branch point in fucoidans from wild S. latissima.

The characterization of a new alginate lyase PALy1 from sea cucumber gut marine bacteria Pseudoalteromonas sp was studied. The enzyme was found active on three different substrate alginates, polyM and polyG, in which the activity on polyG was highest. The optimal conditions for the alginate lyase PALy1 were 40 °C, pH 7, 256 mM NaCl. The enzyme was inhibited by alginate, polyM and polyG at high substrate concentrations.

Furthermore, it was inhibited by ethanol extracts from brown macroalgae, indicating inhibition by polyphenolic compounds. The alginate lyase PALy1 was able to hydrolyze alginate from F. evanescens, S. latissima and S. mcclurei by an endohydrolytic mechanism, which was confirmed by Carbohydrate - Polyacrylamide gel electrophoresis (C-PAGE).

In conclusion, the obtained data showed that the enzyme-assisted extraction using a combination of glucanase and an alginate lyase can extract fucoidans from brown seaweeds without affecting the chemical features of fucoidans. The study furthermore provided evidence that these fucoidans as well as fucoidan oligosaccharides with low molecular weight prepared by fucoidanase enzymes are promising for biomedical applications and also useful for determination of the chemical structures of fucoidans. Beside this, the new alginate lyase has potential for production of alginate oligosaccharides.