Alginate lyases are commonly employed for producing alginate oligosaccharides (AOS), but their industrial application is often constrained by low thermal stability and catalytic efficiency. This study engineered mutants of alginate lyase Alyw203 from marine Vibrio based on B-factor values and negative ΔΔG values. The L172V mutant exhibited a 2.43-fold increase in half-life at 40 °C, reduced K_m (from 107 to 65 mg/mL), and enhanced k_cat/K_m (from 0.07 to 0.35 mL/mg/s), indicating improved thermal stability, substrate affinity, and catalytic efficiency. Molecular dynamics simulations revealed that these improvements originated from reconstructed hydrogen bond networks, which stabilized enzyme–substrate interactions and reduced conformational flexibility. These results demonstrate that rational design focused on strengthening hydrogen bonding can simultaneously improve both stability and activity, offering a promising strategy for industrial AOS production.

Proteolytic enzymes, which play a crucial role in peptide bond cleavage, are widely applied in various industries. In this study, protease-producing bacteria were isolated and characterized from marine sediments collected from the Yellow Sea, China. Comprehensive screening and 16S rDNA sequencing identified a promising G4 strain as Bacillus atrophaeus. Following meticulous optimization of fermentation conditions and medium composition via response surface methodology, protease production using strain G4 was significantly enhanced by 64%, achieving a yield of 3258 U/mL. The G4 protease exhibited optimal activity at 50 °C and pH 7.5, demonstrating moderate thermal stability with 52% residual activity after 30-min incubation at 50 °C—characteristics typical of an alkaline protease. Notably, the enzyme retained over 79% activity across a broad pH range (6–11) and exhibited excellent salt tolerance, maintaining over 50% activity in a saturated NaCl solution. Inhibition by phenylmethylsulfonyl fluoride, a serine protease inhibitor, confirmed its classification as a serine protease. The enzyme’s potential in generating bioactive peptides was further demonstrated through hydrolysis of sheep (Ovis aries) placenta, resulting in a hydrolysate with notable antioxidant properties. The hydrolysate exhibited a 64% superoxide anion scavenging activity, surpassing that of reduced glutathione. These findings expand the current understanding of Bacillus atrophaeus G4 proteases and provide a foundation for innovative sheep placenta utilization with potential industrial applications.