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

Abstract

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.

Keywords: Bacillus atrophaeus; antioxidation; characterization; enzymatic hydrolysis; protease; sheep (Ovis aries) placenta.

Figure 1

Phylogenetic tree of strain G4 constructed based on 16S rRNA gene sequences, illustrating its phylogenetic placement within the Bacillus genus. GenBank accession numbers are shown in parentheses.

Figure 2

Protease production and growth kinetics of strain G4. The relative activity (100%) corresponds to protease activity of 1700 U/mL.

Figure 3

Effect of temperature (a) and pH (b) on protease production (black squares represent protease activity values) during fermentation by strain G4. The relative activity (100%) corresponds to protease activity of 1700 U/mL. All cultures were grown for 24 h at 200 rpm, with (a) pH 6.0 and (b) temperature at 30 °C.

Figure 4

Effects of nitrogen sources (a,b) and carbon sources (c,d) on protease production. Nitrogen source screening: The 100% relative activity corresponds to 1700 U/mL (basal medium: 0.5% (w/v) yeast extract, 1% (w/v) NaCl); carbon source screening: The 100% relative activity corresponds to 2500 U/mL (basal medium: 2% (w/v) casein, 1% (w/v) NaCl).

Figure 5

SDS-PAGE analysis of the purification process of protease from strain G4. M: Pre-stained protein molecular weight marker; Lane 1: Crude extract of protease from strain G4; Lane 2: Protease fraction after 20–60% (NH₄)₂SO₄ precipitation; Lane 3: Protease fraction after DEAE FF ion-exchange chromatography; Lane 4: Final purified protease.

Figure 6

(a) Effect of temperature on protease activity (black squares); (b) Effect of temperature on thermal stability of protease activity.

Figure 7

(a) Effect of pH on protease activity (black squares); (b) Effect of pH on the pH stability of protease activity.

Figure 8

Effect of NaCl concentration on the activity and salt stability of protease from strain G4.

Figure 9

Effect of hydrolysis time on free amino acid content and DPPH radical scavenging activity.

Figure 10

(a) DPPH radical scavenging activity of the hydrolysate; (b) Superoxide anion radical scavenging activity of the hydrolysate.