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

Distribution and proportion of beta-D-mannuronic and alpha-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 H-1 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.