Exploiting Mannuronan C-5 Epimerases in Commercial Alginate Production

Abstract

Alginates are one of the major polysaccharide constituents of marine brown algae in commercial manufacturing. However, the content and composition of alginates differ according to the distinct parts of these macroalgae and have a direct impact on the concentration of guluronate and subsequent commercial value of the final product. The Azotobacter vinelandii mannuronan C-5 epimerases AlgE1 and AlgE4 were used to determine their potential value in tailoring the production of high guluronate low-molecular-weight alginates from two sources of high mannuronic acid alginates, the naturally occurring harvested brown algae (Ascophyllum nodosum, Durvillea potatorum, Laminaria hyperborea and Lessonia nigrescens) and a pure mannuronic acid alginate derived from fermented production of the mutant strain of Pseudomonas fluorescens NCIMB 10,525. The mannuronan C-5 epimerases used in this study increased the content of guluronate from 32% up to 81% in both the harvested seaweed and bacterial fermented alginate sources. The guluronate-rich alginate oligomers subsequently derived from these two different sources showed structural identity as determined by proton nuclear magnetic resonance (¹H NMR), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and size-exclusion chromatography with online multi-angle static laser light scattering (SEC-MALS). Functional identity was determined by minimum inhibitory concentration (MIC) assays with selected bacteria and antibiotics using the previously documented low-molecular-weight guluronate enriched alginate OligoG CF-5/20 as a comparator. The alginates produced using either source showed similar antibiotic potentiation effects to the drug candidate OligoG CF-5/20 currently in development as a mucolytic and anti-biofilm agent. These findings clearly illustrate the value of using epimerases to provide an alternative production route for novel low-molecular-weight alginates.

Keywords: guluronate oligomers; mannuronan C-5 epimerases; potentiation of antibiotic effect; seaweed and bacterial alginate.

Figure 1

Content of guluronic acid (F_G determined by NMR analyses) in seaweed alginate from different species before (blue) and after epimerization with AlgE1 and AlgE6 alone (red and purple bars) and in combination with AlgE4 (green and turquoise bars).

Figure 2

Section of HPAEC-PAD chromatograms of alginate samples from L. hyperborea leaf before (black line) and after epimerization with AlgE1 (blue line) and AlgE6 (brown line) alone and in combination with AlgE4 (pink and green lines). The epimerized alginates were degraded with M-lyase prior to analysis, thus leaving only the G-blocks. The scale bar above the chromatograms indicates the degree of polymerization (DP) eluting at different time points, i.e., DP below 20, DP 20–50 and DP > 50.

Figure 3

Section of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) chromatograms of alginate samples from A. nodosum before (black line) and after epimerization with AlgE1 (blue line) and AlgE6 (brown line) alone and in combination with AlgE4 (pink and green lines). The epimerized alginates were degraded with M-lyase prior to analysis, thus leaving only the G-blocks. The scale bar above the chromatograms indicates the degree of polymerization (DP) eluting at different time points, i.e., DP below 20, DP 20–50 and DP > 50.

Figure 4

1H-NMR spectra showing alginate from Laminaria hyperborea leaf (A), after epimerization by AlgE4 and AlgE1 (B) and oligomers produced by hydrolysis of the epimerized alginate (C). The content of guluronic acid in the three materials are 0 (A), 79 (B) and 90% (C), respectively. AB-nC denotes proton n in uronic acid B with neighboring uronic acids A and C., e.g., MG-5 M is the resonance from H 5 in guluronic acid between two mannuronic acid units.

Figure 5

Overlayed HPEC-PAD chromatograms of oligomers produced by hydrolysis of bacterial mannuronan first epimerized with AlgE4 and AlgE1 (A), alginate from L. hyperborea leaf epimerized with AlgE4 and AlgE1 (B) and OligoG CF-5/20 (C). Numbers above the peaks indicate the oligomer length (degree of polymerization: DP).

Figure 6

NMR spectra showing mannuronan produced by P. fluorescens (A), mannuronan epimerized by AlgE4 and AlgE1 (B) and oligomers produced by hydrolysis of the epimerized mannuronan (C). The contents of guluronic acid in the three materials are 0 (A), 80 (B) and 93% (C), respectively. AB-nC denotes proton n in uronic acid B with neighboring uronic acids A and C., e.g., MG-5M is the resonance from H 5 in guluronic acid between two mannuronic acid units.