Alginate Sequencing: An Analysis of Block Distribution in Alginates Using Specific Alginate Degrading Enzymes

Distribution and proportion of β-d-mannuronic and α-l-guluronic acid in alginates are important for understanding the chemical-physical properties of the polymer. The present state of art methods, which is based on NMR, provides a statistical description of alginates. In this work, a method was developed that also gives information of the distribution of block lengths of each of the three block types (M, G, and MG blocks). This was achieved using a combination of alginate lyases with different substrate specificities, including a novel lyase that specifically cleaves diguluronic acid linkages. Reaction products and isolated fragments of alginates degraded with these lyases were subsequently analyzed with 1H NMR, HPAEC-PAD, and SEC-MALLS. The method was applied on three seaweed alginates with large differences in sequence parameters (F G = 0.32 to 0.67). All samples contained considerable amounts of extremely long G blocks (DP > 100). The finding of long M blocks (DP ≥ 90) suggests that also algal epimerases act by a multiple attack mechanism. Alternating sequences (MG-blocks) were found to be much shorter than the other block types. In connection with method development, an oligomer library comprising both saturated and unsaturated oligomers of various composition and DP 2–15 was made.