Abstract
Despite the progress in massive gene analysis of brown algal species, no alginate-degrading enzyme from brown alga has been identified, impeding the understanding of alginate metabolism in brown alga. In the current study, we identified and characterized alginate lyase from Saccharina japonica using a protein-based approach. First, cDNA library was prepared from the S. japonica sporophyte. Expression screening was then performed; the encoding gene was identified and cloned; and the recombinant enzyme was purified and characterized. Alginate lyase production in algal tissues was evaluated by western blotting. The identified alginate lyase, SjAly (359 amino acids, with a predicted N-terminal secretion signal of 27 residues), is encoded by an open reading frame comprising seven exons. Recombinant SjAly exhibited endolytic alginate lyase activity, specifically toward stretches of consecutive β-d-mannuronic acid units. The optimum temperature, pH, and NaCl concentration were 30 °C, pH 8.0, and 100 mM, respectively. SjAly exhibited pronounced activity below 20 °C, the S. japonica growth temperature. SjAly was highly expressed in the blade but not the stipe and rhizoid. The data indicate that S. japonica possesses at least one active alginate lyase. This is the first report of a functional alginate lyase from brown alga, the major natural alginate producer.
Highlights
Alginate is a major polysaccharide of the cell wall matrix in brown alga
In alginate-assimilating bacteria, it has been proposed that alginate is metabolized to pyruvate and this process was reproduced in vitro using three alginate lyases, 4-deoxy-l-erythro-5-hexoseulose uronate (DEH) reductase, 2-keto-3-deoxy-D-gluconate (KDG) kinase, and 2-keto-3-deoxy-6-phosphogluconic acid (KDPG) aldolase, from Flavobacterium sp. strain UMI-0129
DEH was the smallest product of alginate degradation by SjAly (Fig. 2f), which raises an interesting question about alginate metabolism in brown alga
Summary
Alginate is a major polysaccharide of the cell wall matrix in brown alga. It is a linear heteropolysaccharide composed of two uronic acids, β-d-mannuronic acid (M) and its C5 epimer α-l-guluronic acid (G). To date, considering alginate-producing organisms, functional alginate lyases have been identified in only bacteria, and not in brown algae. AlgL, an alginate lyase from Pseudomonas aeruginosa, has been characterized[20] and functions in the periplasmic space during alginate biosynthesis[21,22,23]. Candidate genes encoding alginate degradation enzymes have not yet been identified in brown algae[31,32]. 31 and 105 of such candidate genes were identified in the genomes of E. siliculosus[33,34] and Saccharina japonica[35], respectively These multiple MC5Es are thought to contribute to the production of alginate molecules with a variety of M/G sequences. This is the first such discovery made for an alginate-biosynthesizing eukaryote and advances the understanding of alginate metabolism in the major alginate producer, the brown alga
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