Preliminary note

Biosynthesis of alginate. Epimerisation of d-mannuronic to l-guluronic acid residues in the polymer chain

Alginates are valued in many industries, due to their versatile properties. These polysaccharides originate from brown algae (Phaeophyceae) and some bacteria of the Azotobacter and Pseudomonas genera, consisting of 1 → 4 linked β-d-mannuronic acid (M), and its C5-epimer α-l-guluronic acid (G). Several applications rely on a high G-content, which confers good gelling properties. Because of its high natural G-content (F_G = 0.60–0.75), the alginate from Laminaria hyperborea (LH) has sustained a thriving industry in Norway. Alginates from other sources can be upgraded with mannuronan C-5 epimerases that convert M to G, and this has been demonstrated in many studies, but not applied in the seaweed industry. The present study demonstrates epimerisation directly in the process of alginate extraction from cultivated Saccharina latissima (SL) and Alaria esculenta (AE), and the lamina of LH. Unlike conventional epimerisation, which comprises multiple steps, this in-process protocol can decrease the time and costs necessary for alginate upgrading. In-process epimerisation with AlgE1 enzyme enhanced G-content and hydrogel strength in all examined species, with the greatest effect on SL (F_G from 0.44 to 0.76, hydrogel Young's modulus from 22 to 34 kPa). As proof of concept, an upscaled in-process epimerisation of alginate from fresh SL was successfully demonstrated.

The mannuronan C-5 epimerases catalyze epimerization of β-d-mannuronic acid to α-l-guluronic acid in alginate polymers. The seven extracellular Azotobacter vinelandii epimerases (AvAlgE1–7) are calcium-dependent, and calcium is essential for the structural integrity of their carbohydrate binding R-modules. Ca²⁺ is also found in the crystal structures of the A-modules, where it is suggested to play a structural role. In this study, the structure of the catalytic A-module of the A. vinelandii mannuronan C-5 epimerase AvAlgE6 is used to investigate the role of this Ca²⁺. Molecular dynamics (MD) simulations with and without calcium reveal the possible importance of the bound Ca²⁺ in the hydrophobic packing of β-sheets. In addition, a putative calcium binding site is found in the active site, indicating a potential direct role of this calcium in the catalysis. According to the literature, two of the residues coordinating calcium in this site are essential for the activity. MD simulations of the interaction with bound substrate indicate that the presence of a calcium ion in this binding site increases the binding strength. Further, explicit calculations of the substrate dissociation pathways with umbrella sampling simulations show and energetically higher dissociation barrier when calcium is present. The present study eludes to a putative catalytic role of calcium in the charge neutralizing first step of the enzymatic reaction. In addition to the importance for understanding these enzymes’ molecular mechanisms, this could have implications for engineering strategies of the epimerases in industrial alginate processing.

Alginate is a linear polysaccharide composed of β-d-mannuronate (M) and α-l-guluronate (G). Alginate and its degradation products (alginate oligosaccharides) possess abundant biological activities such as antitumor activity and antimicrobial activity, which have great application value in food, pharmaceutical, and agricultural fields. The M/G ratio of alginate determine its biological activity and physicochemical properties. Mannuronan C5-epimerases can catalyze the conversion of β-d-mannuronate (M) to its C5 epimer α-l-guluronate (G) in vitro. With the development of technology, the number of mannuronan C5-epimerases characterized gradually increased. As the main mannuronan C5-epimerases, the properties, catalytic mechanisms and structures of AlgE-type and AlgG-type enzymes have been reported. The potential applications of mannuronan C5-epimerases on tailored alginate have also been explored. Modification engineering helps to improve the properties of mannuronan C5-epimerases, thus making it possible to tailor alginate in vitro. This comprehensive information should be helpful to expand the research and application of mannuronan C5-epimerases.

Carrageenan, agar, and alginate are seaweed-derived carbohydrate hydrocolloids which are used as thickening and gelling agents in foods, pharma and biotechnology applications due to their unique gelation properties. These hydrocolloids are extracted commercially from a set of high-yielding, cultivated seaweeds (marine macroalgae). The seaweed type and biological and environmental factors during seaweed growth determine the chemical and rheological hydrocolloid properties. New insight into biosynthesis and microbial degradation of these seaweed-derived hydrocolloids includes discovery of unique enzymes that catalyze specific structural changes in the carbohydrate polymers which affect the hydrocolloid properties.

In this review we describe the intricate chemical structures, gelation mechanisms and biosynthesis routes of seaweed hydrocolloids. We provide an overview of novel enzymes and enzyme reactions that catalyze changes in these hydrocolloids, and discuss how this new enzyme knowledge may enable further technological advancements in processing and applications of seaweed hydrocolloids.

The red seaweeds from the genera Kappaphycus and Eucheuma are key commercial sources of carrageenan, species from the genera Gracilaria and Gelidium are the main sources of agar, and brown seaweeds from the genera Laminaria and Macrocystis are currently the main sources of alginate. Recent progress has advanced our understanding of enzymatic reactions involved in the biosynthesis and microbial degradation of these hydrocolloids and especially uncovered unique epimerase, desulfatase, and alginate lyase processes. This new knowledge provides a basis for rethinking the sources and extraction protocols of these hydrocolloids because such enzymes can catalyze distinct molecular changes to improve the physical properties of the hydrocolloids.

The present study examined the growth of alginate-encapsulated marine macroalgal spores of a green alga (Ulva intestinalis) and brown algae (Undaria pinnatifida and Ecklonia cava). We compared the initial germination and growth of gametes and thalli using alginate-encapsulated and non-encapsulated spores. Spores of the three algal species germinated easily; there were no significant differences between alginate-encapsulated and non-encapsulated spores (p > 0.05). After 45 days of culture, the alginate-encapsulated and non-encapsulated U. intestinalis spores were 35.315 ± 0.252 mm and 33.616 ± 0.815 mm in size, respectively. Encapsulated and non-encapsulated U. pinnatifida thalli (including gametophytes) were 24.928 ± 0.956 mm and 12.771 ± 0.458 mm, respectively. Encapsulated and non-encapsulated E. cava gametophytes were 648.35 ± 15.715 μm and 148. 33 ± 1.616 μm, respectively. Encapsulated spores of these three algal species tended to grow faster than non-encapsulated spores. Given these results, the artificial encapsulation with alginate was an effective way to enhance the growth of all three tested macroalgal spores.

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. Here, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).