Sulfated fucans are matrix polysaccharides from marine brown algae, consisting of an alpha-L-fucose backbone substituted by sulfate-ester groups, masked with ramifications, and containing other monosaccharide residues. We here report on the characterization of a novel glycoside hydrolase (FcnA) specific for the degradation of sulfated fucans. This glycoside hydrolase was purified to electrophoretic homogeneity from a Flavobacteriaceae referred to as SW5. The gene fcnA was cloned and sequenced (3021 nucleotides), and the protein (1007 amino acids) was produced in Escherichia coli. FcnA exhibited a modular architecture consisting of a 400-residue-long N-terminal domain followed by three repeated domains predicted to adopt an immunoglobulin fold and by an 80-amino acid-long C-terminal domain. A truncated recombinant protein encompassing the N-terminal domain and the immunoglobulin-like repeats was shown to retain the enzyme activity. The N-terminal catalytic domain shared approximately 25% of sequence identity with two patented fucanase genes, and these three fucanases delineate a new family of glycoside hydrolases. As shown by size-exclusion chromatography (SEC) and 1H-NMR analyses, the fucanase FcnA proceeds according to an endolytic mode of action and cleaves the alpha-(1-->4) glycosidic linkages within the blocks of repeating motifs [-->4)-alpha-L-fucopyranosyl-2,3-disulfate-(1-->3)-alpha-L-fucopyranosyl-2-sulfate-(1-->]n.

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The structure and organization of Palmaria palmata cell walls, which are largely involved in biological and physiological functions as well as in biotechnological and food applications of this red marine alga, are principally assumed by the interactions and linkages of major mix-linked beta-(1-->3)/beta-(1-->4)-D-xylans. These partly acidic polysaccharides are essentially held in the cell wall by H-bonds. The location of the acid groups and the distribution of 1-->3-linkage were studied following the endo-beta-(1,4)-xylanase hydrolysis of sequentially extracted xylans, and fine analysis of the oligosaccharides produced by anion exchange chromatography, high performance anion exchange chromatography (HPAEC)-PAD, nuclear magnetic resonance (NMR) and electrospray ion trap mass spectrometry (ESI-MS) techniques. The results indicate that the acidity of the xylans was related to potential linkages to sulfated and/or phosphorylated xylogalactoprotein complexes. H-bonding of the mix-linked xylans involved a regular 1,3-linkages distribution idealized in a pentameric repeating structure (one 1,3-linkage and four 1,4-linkages). Furthermore, MS analysis of the xylo-oligosaccharides revealed a substitution of the mix-linked xylans by a non-osidic component of 175 g mol(-1). The presence of this substituent and of the proposed covalent linkage between the mix-linked xylans and charged glycoproteins are discussed with regard to the polysaccharides interactions in P. palmata cell walls.

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