Alginate Degradation: Insights Obtained through Characterization of a Thermophilic Exolytic Alginate Lyase

Abstract

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 data set. 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 pHs, temperatures, and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as a catalytic acid, indeed is essential for catalysis, whereas mutation of tyrosine Y446, previously proposed to act as a 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-member hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF), formed through intramolecular cyclization of hydrated DEH. The detection and nuclear magnetic resonance (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 a high industrially relevant temperature (≥60°C), this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation revealed 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.

Keywords: DEH; DHF; NMR; Saccharina latissima; alginate lyase; biorefining; exo-activity; nuclear magnetic resonance; thermal stability.