Screening and Characterization of Marine Bacillus atrophaeus G4 Protease and Its Application in the Enzymatic Hydrolysis of Sheep (Ovis aries) Placenta for the Preparation of Antioxidant Peptides

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 <i>Bacillus atrophaeus</i>. 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 (<i>Ovis aries</i>) 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 <i>Bacillus atrophaeus</i> G4 proteases and provide a foundation for innovative sheep placenta utilization with potential industrial applications.