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

Abstract

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 (1)H 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.

Analysis of G-Block Distributions and Their Impact on Gel Properties of in Vitro Epimerized Mannuronan

“…We have previously suggested that such long G-blocks might function as reinforcement bars in the ionic gel network 5 . To test this hypothesis, we studied how addition of long G-blocks into in vitro epimerized alginates affected gel properties.…”

Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications

“…They can be classed into 2 groups due to their substrate specificities, 13 molecular weights 19 and action modes. Now alginate lyases, especially endolytic alginate lyases, have been widely used in the production of alginate oligosaccharides, 20 the elucidation of the fine structures of alginate [21][22][23] and the preparation of protoplast of red and brown algae. [24][25][26] Furthermore, alginate lyase also shows great potential application in treatment of cystic fibrosis by degrading the polysaccharide biofilm of bacterium.…”

Physical and Chemical Hyaluronic Acid Hydrogels and Their Biomedical Applications

“…An important improvement along this line has been recently accomplished and it is based on alginate sequencing using a combination of different analytical techniques such as 1 H-NMR, high-performance anion exchange chromatography-pulsed amperometric detection (HPAED-PAD) and size exclusion chromatographymulti-angle laser light scattering (SEC-MALLS), and alginate lyases ( Fig. 13.4) [2]. These enzymes are endolyases that have been isolated from many sources [160], including marine algae [88], marine mollusks [12,67,105], and various microorganisms like Pseudomonas aeruginosa [40] and Azotobacter vinelandii [74].…”

“…At variance, lyase from P. alginovora cleaves only MM diads [15]. Aarstad et al used four different lyases with appropriate specificity, to obtain small alginate fragments which were then characterized and the results interpreted in statistical terms [2]. This novel approach showed already its enormous potential allow- ing to unveil the processive character of the epimerization by C-5 epimerases [14,70].…”