关键词: 褐藻胶裂解酶；异源表达；酶学性质；聚古罗糖醛酸偏好性；耐有机试剂；抗氧化活性

Abstract: When Alteromonas sp. A1-6 was cultivated with sodium alginate as the sole carbon source, quantitative real-time PCR (qPCR) revealed a significant upregulation of the alg2579 gene, suggesting that it encodes an alginate-degrading enzyme. The alg2579 gene was successfully cloned from the genomic DNA of Alteromonas sp. A1-6. Heterologous expression and subsequent protein purification yielded the recombinant enzyme Alg2579, whose enzymatic characteristics and hydrolysis products were systematically studied. Bioinformatic analysis showed that Alg2579 contained a canonical polysaccharide lyase family 6 (PL6) domain and lacked transmembrane domains. Alg2579 exhibited maximal activity at 30 ℃ and pH 9.0 and remained stable under alkaline conditions. Its activity toward polyguluronate acid (Poly G) was higher than that toward alginate and polymannuronate acid (Poly M), with a specific activity of 1.383 U/mg, indicating a clear preference for Poly G. Ca2+ enhanced the enzymatic activity, consistent with the known Ca2+ dependence of many PL6 alginate lyases. Dithiothreitol (DTT) also stimulated its activity, increasing it by approximately 95% at high concentrations. Alg2579 demonstrated good tolerance to various organic solvents at low concentrations, retaining over 90% of its activity. The kinetic parameters Km and Vmax of Alg2579 were determined as 2.521 mg/ml and 0.019 μmol/min, respectively, with kcat = 0.579 s–1 and kcat/Km = 0.230 mL/(mg·s). Degradation of sodium alginate by Alg2579 produced substantial amounts of unsaturated alginate monosaccharides (DEH), unsaturated alginate disaccharides, and saturated monosaccharides. After an overnight reaction, a significant increase in oligosaccharide diversity and disaccharide concentration was observed, confirming that Alg2579 is a bifunctional alginate lyase with both endo- and exo-lytic activities. The ferric reducing antioxidant power (FRAP) assay indicated the hydrolysis products of Alg2579 had strong antioxidant activity. Furthermore, degradation products of Saccharina japonica by Alg2579 promoted wheat seed germination and root growth. In summary, the recombinant enzyme Alg2579 is a Poly G-preferring alginate lyase with good alkaline stability. These unique properties suggest considerable application potential in the preparation of brown algal oligosaccharides and the development of functional foods.

Key words: alginate lyase; heterologous expression; enzymatic properties; polyguluronic acid preference; resistance to organic reagents; antioxidant activity