This study aimed to investigate the inhibitory effect and mechanism of alginate on xanthine oxidase (XO), and to evaluate its potential as a natural inhibitor or functional food supplement for alleviating hyperuricemia. Sodium alginate (SA) was used as a model compound. Enzymatic kinetics, fluorescence spectroscopy, time-resolved fluorescence spectroscopy (TRFS) and circular dichroism (CD) spectroscopy were employed for comprehensive analysis. The enzymatic kinetics results indicated that SA inhibited XO activity in a reversible mixed-type manner with an inhibition rate of 45.42% at a concentration of 10 mg/mL. Fluorescence spectroscopy analysis revealed that SA reduced the intrinsic fluorescence intensity of XO through a static quenching mechanism, with the interaction occurring at a single binding site. TRFS analysis further confirmed the static quenching process. CD spectroscopy demonstrated that SA induced secondary structural and conformational alterations in XO. The findings suggest that SA effectively inhibits XO activity by binding to its active site and inducing conformational changes in the enzyme, providing a theoretical foundation for the development of related functional foods or therapeutic agents.

This study aimed to improve the thermostability of κ-carrageenase from Pseudoalteromonas sp. JMUZ2 by rational design. A total of 10 single-site mutants were chosen using the PoPMuSiC program to analyze the sequence of the κ-carrageenase gene. A mutant gene was obtained by site-directed mutagenesis. The mutant enzyme was expressed under induced conditions, purified and identified. The mutant K155A could maintain the specific enzyme activity and had improved thermostability. After treatment at 50, 55, and 60 ℃ for 40 min, the thermal stability of the mutant K155A was 1.8, 2.7, and 4.5 times higher than that of the wild-type enzyme (WT), respectively. The structure and molecular dynamic simulation analysis showed that the increase in hydrophobic interactions and structural rigidity may be the cause of the improved thermal stability of K155A. This study is of great significance for improving the properties of κ-carrageenase, expanding its application scope, and studying its structural-functional relationship.

In order to clarify the effect of the non-catalytic carbohydrate-binding module (CBM) and F5/8 type C domains on the enzymatic properties of AlgL7, an alginate lyase from Microbulbifer sp. ALW1, the full-length enzyme AlgL7 and two truncated enzymes: CD1 (catalytic domain) and CD2 (containing F5/8 type C domain and catalytic domain) were constructed and characterized. The results showed that the truncated enzyme CD2 exhibited higher specific activity, thermostability, Michaelis constant (K_m), and maximum reaction velocity (V_max) compared to the full-length AlgL7, indicating that the CBM domain played an important role in maintaining the substrate affinity of the enzyme, but reduced the catalytic activity, thermostability, and V_max value the enzyme. Compared to the truncated enzyme CD1, CD2 exhibited higher specific activity, optimal reaction temperature, thermostability, V_max, and K_m, indicating that the F5/8 type C domain contributed to improve the enzymatic activity, optimum reaction temperature, thermostability, and V_max, but reduced the substrate affinity of the enzyme. Using sodium alginate as the substrate, the specific activity of CD2 was 183.9 U/mg. The optimal reaction temperature and pH were 40 ℃ and 7.0, respectively. The K_m and V_max were 39.80 mg/mL and 2000 U/mg, respectively. The major enzymatic hydrolysates were disaccharides and trisaccharides. This study promotes the understanding of the structureactivity relationship between the non-catalytic domains and the properties of alginate lyase, and lays a theoretical basis for using the non-catalytic domains to improve the catalytic properties of alginate lysate.