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 (12248.16 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 200–1000 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.

In this study, a novel cellulase gene, CelL7, was cloned from the marine bacterium Zobellia sp. B2. Furthermore, a fusion gene, CelL7-CBM3, was constructed by fusing a carbohydrate-binding module family 3 (CBM3) to CelL7 and heterologously expressed in Escherichia coli BL21. The expressed fusion protein was purified by affinity column chromatography. The full length of the CelL7 gene was 1077 bp, encoding 358 amino acid residues, and the theoretical molecular mass of the encoded protein was 40.39 kDa. The specific enzyme activities of CelL7 and CelL7-CBM3 were 2249.81 and 2915.75 U/mg, respectively. The optimal reaction temperatures for both enzymes were 50 ℃, and the optimal pHs were 5.0 and 5.5, respectively. Mn²⁺ and Fe²⁺ activated the activity of CelL7, while Cu²⁺ inhibited it. CelL7 was capable of degrading carboxymethyl cellulose sodium, cellobiose, and xylan. When sodium carboxymethyl cellulose was used as a substrate, the Michaelis-Menten constant (K_m) of CelL7-CBM3 was 11.70 mg/mL, which was lower than that of CelL7 (K_m = 13.23 mg/mL), indicating that the fusion enzyme, with an added binding domain, exhibited enhanced affinity for carboxymethyl cellulose sodium. The maximum reaction rate (V_max) was 175.44 mg/(mL·min), the catalytic constant (K_cat) was 2.78 s^-1, and the K_cat/K_m was 0.24 mL/(mg·s), which were comparable to those of CelL7. Biofilm clearance experiments showed that concentrations of CelL7 ranging from 10.0 to 60.0 μg/mL and those of CelL7-CBM3 ranging from 30.0 to 60.0 μg/mL were effective in dispersing biofilm and reducing the amount of biofilm.