Abstract: This study employed site-directed mutagenesis to molecularly engineer the alginate lyase Alya1, significantly enhancing its enzymatic activity and stability. Furthermore, this study evaluated the bioactivity of its degradation products, alginate oligosaccharides (AOS). Through homology modeling using Swiss-Model, multiple sequence alignment, and in silico mutation analysis, 16 key sites were selected for single and combinatorial mutations. Three beneficial mutants, S189L, S244Q, and D408W, were successfully obtained, whose specific activities were 2.70-, 2.23-, and 1.29-fold higher, respectively, than that of the wild-type enzyme (12 248.16 U/mg). Kinetic parameters indicated that the mutants possessed superior substrate affinity and catalytic efficiency constants compared with the native enzyme, demonstrating a significant overall enhancement in catalytic performance. The S189L mutant retained high activity after incubation at 20–35 ℃ for 60 min, while the D408W mutant showed acid tolerance with its optimal pH shifted to 4.0. Cell-based assays revealed that AOS, guluronate oligosaccharides (GOS), and mannuronate oligosaccharides (MOS) at concentrations of 200–1 000 μg/mL exhibited no cytotoxicity. Furthermore, they significantly inhibited the generation of reactive oxygen species (ROS) and the expression of inflammatory cytokines in a murine macrophage cell line, RAW264.7 cells. Among them, GOS exhibited the most potent inhibitory effects on the gene expression of interleukin-1β (IL-1β) and interleukin-6 (IL-6). No significant adverse effects were observed in zebrafish embryo toxicity tests. In a zebrafish model of acute hyperuricemia, all three oligosaccharides alleviated oxidative damage and improved renal metabolic function. MOS reduced superoxide dismutase (SOD) activity from 14.17 to 10.38 U/mg. AOS restored uric acid levels to normal and concurrently up-regulated the gene expression of organic anion transporter 1 (OAT1) to 6.18-fold that of the model group. This research provides a theoretical foundation for the molecular engineering of alginate lyase and the application of its oligosaccharide products in hyperuricemia intervention.

Key words: alginate lyase; site-directed mutagenesis; alginate oligosaccharides; hyperuricemia