Alginate, the main cell-wall polysaccharide of brown algae, is composed of two residues: mannuronic acid (M-residues) and, its C5-epimer, guluronic acid (G-residues). Alginate lyases define a class of enzymes that cleave the glycosidic bond of alginate by β-elimination. They are classified according to their ability to recognize the distribution of M- and G-residues and are named M-, G- or MG-lyases. In the CAZy database, alginate lyases have been grouped by sequence similarity into seven distinct polysaccharide lyase families. The polysaccharide lyase family PL6 is subdivided into three subfamilies. Subfamily PL6_1 includes three biochemically characterized enzymes (two alginate lyases and one dermatan sulfatase lyase). No characterized enzymes have been described in the two other subfamilies (PL6_2 and PL6_3). To improve the prediction of polysaccharide-lyase activity in the PL6 family, we re-examined the classification of the PL6 family and biochemically characterized a set of enzymes reflecting the diversity of the protein sequences. Our results show that subfamily PL6_1 includes two dermatan sulfates lyases and several alginate lyases that have various substrate specificities and modes of action. In contrast, subfamilies PL6_2 and PL6_3 were found to contain only endo-poly-MG-lyases.

Alginate oligosaccharides (AOS) with a weight average molecular weight of 5 kDa were efficiently amidated with amino acids and carbohydrates in aqueous media in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). Here, alanine, leucine, serine, as well as mannose and rhamnose, were amidated at high yields with a good control of the degree of substitution (DS). Amino acid- and carbohydrate-grafted AOS showed improved stability against degradation by alginate lyases having different specificities. This enzyme resistance was correlated with the DS: hydrolysis was reduced by 60-70% for low DS (0.1), whereas AOS with DS ranging from 0.4 to 0.6 remained unhydrolyzed. Competitive inhibition assays demonstrated multivalent binding of mannose-amidated AOS to concanavalin A lectin. A 178-fold affinity enhancement was observed for AOS (DS 0.38) over α-methyl-mannoside with an IC of 5.6 μM, lending further evidence for the promising potential of AOS as multivalent scaffolds.