Bacterial alginate metabolism: an important pathway for bioconversion of brown algae

Brown macroalgae have attracted great attention as an alternative feedstock for biorefining. Although direct conversion of ethanol from alginates (major components of brown macroalgae cell walls) is not amenable for industrial production, significant progress has been made not only on enzymes involved in alginate degradation, but also on metabolic pathways for biorefining at the laboratory level. In this article, we summarise recent advances on four aspects: alginate, alginate lyases, different alginate-degrading systems, and application of alginate lyases and associated pathways. This knowledge will likely inspire sustainable solutions for further application of both alginate lyases and their associated pathways.

Degradation and utilization of Alginate by marine Pseudoalteromonas : a review

Alginate, which is mainly produced by brown algae and decomposed by heterotrophic bacteria, is an important marine organic carbon source. The genus Pseudoalteromonas contains diverse forms of heterotrophic bacteria that are widely distributed in marine environments and are an important group in alginate degradation. In this review, the diversity of alginate-degrading Pseudoalteromonas is introduced and the character of Pseudoalteromonas alginate lyases, including their sequences, enzymatic properties, structures and catalytic mechanisms, and the synergistic effect of Pseudoalteromonas alginate lyases on alginate degradation are introduced. The acquisition of the alginate-degradation capacity and the alginate utilization pathways of Pseudoalteromonas are also introduced. This paper provides a comprehensive overview of alginate degradation by Pseudoalteromonas, which will contribute to the understanding of the degradation and recycling of marine algae polysaccharides driven by marine bacteria.

Specificities and Synergistic Actions of Novel PL8 and PL7 Alginate Lyases from the Marine Fungus Paradendryphiella salina

Alginate is an anionic polysaccharide abundantly present in the cell walls of brown macroalgae. The enzymatic depolymerization is performed solely by alginate lyases (EC 4.2.2.x), categorized as polysaccharide lyases (PLs) belonging to 12 different PL families. Until now, the vast majority of the alginate lyases have been found in bacteria. We report here the first extensive characterization of four alginate lyases from a marine fungus, the ascomycete Paradendryphiella salina, a known saprophyte of seaweeds. We have identified four polysaccharide lyase encoding genes bioinformatically in P. salina, one PL8 (PsMan8A), and three PL7 alginate lyases (PsAlg7A, -B, and -C). PsMan8A was demonstrated to exert exo-action on polymannuronic acid, and no action on alginate, indicating that this enzyme is most likely an exo-acting polymannuronic acid specific lyase. This enzyme is the first alginate lyase assigned to PL8 and polymannuronic acid thus represents a new substrate specificity in this family. The PL7 lyases (PsAlg7A, -B, and -C) were found to be endo-acting alginate lyases with different activity optima, substrate affinities, and product profiles. PsAlg7A and PsMan8A showed a clear synergistic action for the complete depolymerization of polyM at pH 5. PsAlg7A depolymerized polyM to mainly DP5 and DP3 oligomers and PsMan8A to dimers and monosaccharides. PsAlg7B and PsAlg7C showed substrate affinities towards both polyM and polyG at pH 8, depolymerizing both substrates to DP9-DP2 oligomers. The findings elucidate how P. salina accomplishes alginate depolymerization and provide insight into an efficient synergistic cooperation that may provide a new foundation for enzyme selection for alginate degradation in seaweed bioprocessing.

Insights into alginate degradation through the characterization of a thermophilic exolytic alginate lyase

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to Polysaccharide Lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics dataset. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly β-d-mannuronate and poly α-l-guluronate within a broad range of pH, temperature and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as catalytic acid, indeed is essential for catalysis; whereas mutation of tyrosine Y446, previously proposed to act as catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l-erythro-5-hexulosuronate), which are formed after ring opening and, finally, two epimers of a 5-membered hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF) formed through intramolecular cyclisation of hydrated DEH. The detection and NMR assignment of these hemiketals refine our current understanding of alginate degradation. Importance The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme-cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme-cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at industrially relevant higher temperatures of 60 °C, this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation reveal previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.

Structural and molecular basis for the substrate positioning mechanism of a new PL7 subfamily alginate lyase from the arctic

Alginate lyases play important roles in alginate degradation in the ocean. Although a large number of alginate lyases have been characterized, little is yet known about those in extremely cold polar environments, which may have unique mechanisms for environmental adaptation and for alginate degradation. Here, we report the characterization of a novel PL7 alginate lyase AlyC3 from Psychromonas sp. C-3 isolated from the Arctic brown alga Laminaria, including its phylogenetic classification, catalytic properties, and structure. We propose the establishment of a new PM-specific subfamily of PL7 (subfamily 6) represented by AlyC3 based on phylogenetic analysis and enzymatic properties. Structural and biochemical analyses showed that AlyC3 is a dimer, representing the first dimeric endo-alginate lyase structure. AlyC3 is activated by NaCl and adopts a novel salt-activated mechanism; that is, salinity adjusts the enzymatic activity by affecting its aggregation states. We further solved the structure of an inactive mutant H127A/Y244A in complex with a dimannuronate molecule and proposed the catalytic process of AlyC3 based on structural and biochemical analyses. We show that Arg82 and Tyr190 at the two ends of the catalytic canyon help the positioning of the repeated units of the substrate and that His127, Tyr244, Arg78, and Gln125 mediate the catalytic reaction. Our study uncovers, for the first time, the amino acid residues for alginate positioning in an alginate lyase and demonstrates that such residues involved in alginate positioning are conserved in other alginate lyases. This study provides a better understanding of the mechanisms of alginate degradation by alginate lyases.

Separation and quantification of 2-Keto-3-deoxy-gluconate (KDG) a major metabolite in pectin and alginate degradation pathways

A rapid and sensitive High Performance Liquid Chromatography (HPLC) method with photometric and fluorescence detection is developed for routine analysis of 2-Keto-3-deoxy-gluconate (KDG), a catabolite product of pectin and alginate. These polysaccharides are primary-based compounds for biofuel production and for generation of high-value-added products. HPLC is performed, after derivatization of the 2-oxo-acid groups of the metabolite with o-phenylenediamine (oPD), using a linear gradient of trifluoroacetic acid and acetonitrile. Quantification is accomplished with an internal standard method. The gradient is optimized to distinguish KDG from its close structural analogues such as 5-keto-4-deoxyuronate (DKI) and 2,5-diketo-3-deoxygluconate (DKII). The proposed method is simple, highly sensitive and accurate for time course analysis of pectin or alginate degradation.HighlightsA fluorescent based-HPLC method report the quantification of KDG, a metabolite originating from alginate and from pectin degradation pathways, using derivatization with o-phenylenediamine (oPD)

Advances in Research on the Bioactivity of Alginate Oligosaccharides

Alginate is a natural polysaccharide present in various marine brown seaweeds. Alginate oligosaccharide (AOS) is a degradation product of alginate, which has received increasing attention due to its low molecular weight and promising biological activity. The wide-ranging biological activity of AOS is closely related to the diversity of their structures. AOS with a specific structure and distinct applications can be obtained by different methods of alginate degradation. This review focuses on recent advances in the biological activity of alginate and its derivatives, including their anti-tumor, anti-oxidative, immunoregulatory, anti-inflammatory, neuroprotective, antibacterial, hypolipidemic, antihypertensive, and hypoglycemic properties, as well as the ability to suppress obesity and promote cell proliferation and regulate plant growth. We hope that this review will provide theoretical basis and inspiration for the high-value research developments and utilization of AOS-related products.