Characterization of a New Biofunctional, Exolytic Alginate Lyase from Tamlana sp. s12 with High Catalytic Activity and Cold-Adapted Features.

Alginate, a major acidic polysaccharide in brown algae, has attracted great attention as a promising carbon source for biorefinery systems. Alginate lyases, especially exo-type alginate lyase, play a critical role in the biorefinery process. Although a large number of alginate lyases have been characterized, few can efficiently degrade alginate comprised of mannuronate (M) and guluronate (G) at low temperatures by means of an exolytic mode. In this study, the gene of a new exo-alginate lyase-Alys1-with high activity (1350 U/mg) was cloned from a marine strain, Tamlana sp. s12. When sodium alginate was used as a substrate, the recombinant enzyme showed optimal activity at 35 °C and pH 7.0-8.0. Noticeably, recombinant Alys1 was unstable at temperatures above 30 °C and had a low melting temperature of 56.0 °C. SDS and EDTA significantly inhibit its activity. These data indicate that Alys1 is a cold-adapted enzyme. Moreover, the enzyme can depolymerize alginates polyM and polyG, and produce a monosaccharide as the minimal alginate oligosaccharide. Primary substrate preference tests and identification of the final oligosaccharide products demonstrated that Alys1 is a bifunctional alginate lyase and prefers M to G. These properties make Alys1 a valuable candidate in both basic research and industrial applications.

Production of a novel dimeric 4-deoxy-L-erythro-5-hexoseulose uronic acid by a PL-17 exolytic alginate lyase from Hydrogenophaga sp. UMI-18.

Hydrogenopahaga sp. strain UMI-18 is an alginolytic bacterium that can produce poly(3-hydroxybutylate) (PHB) using alginate as its sole carbon source. Genome analysis indicated that this strain harbors both PHB-synthesizing and alginate-assimilating gene clusters. In the present study, we cloned HyAly-I gene that encodes a PL-17 exolytic alginate lyase and investigated its enzymatic properties using recombinant HyAly-I (recHyAly-I) that was produced by Escherichia coli. The recHyAly-I preferably depolymerized poly(ß-D-mannuronate) block of alginate in an exolytic manner at an optimal temperature and a pH at 40 °C and pH 6.0, respectively. It released 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from the non-reducing terminus of polymer and oligomer substrates. Interestingly, recHyAly-I was found to produce a novel unsaturated disaccharide, i.e., dimeric DEH (diDEH), along with monomeric DEH. Production of diDEH was prominent in the degradation of trisaccharides.

A bifunctional exolytic alginate lyase from Microbulbifer sp. ALW1 with salt activation and calcium-dependent catalysis.

Alginate lyases can depolymerize alginate to oligomers with potential applications in many fields. Here a new alginate lyase, namely AlgL6, was characterized from Microbulbifer sp. ALW1, phylogenetically classified into the polysaccharide lyase family 6 (PL6). The recombinant alginate lyase AlgL6 exerted enzymatic activities towards polymannuronate, polyguluronate, and sodium alginate in an exolytic manner. AlgL6 had an optimum temperature of 35 °C and good stability at 30 °C or below. Its optimum pH was 8.0, and it had good stability over the pH range of 5.0-9.0. AlgL6 exhibited excellent halo-stability against Na+, and its activity can be increased up to about 1.8 times by 0.5 M NaCl. AlgL6 also showed strong stability in the presence of some nonionic detergents such as Tween 20 and Tween 80. The degradation products of sodium alginate by AlgL6 exhibited more effective antioxidant activities than the undigested polysaccharides. Structure analysis illustrated the catalytic mechanism defined by the coordination of the acid/base residues Arg269 and Lys248 of AlgL6. The replacement of Ca2+-interacting amino acid residues in AlgL6 and depletion of Ca2+ suggested the involvement of Ca2+ in the enzyme's catalytic activity. These properties of AlgL6 supply support to its industrial application for development of alginate bioresource.