Degradation Mechanism of Soybean Protein B3 Subunit Catalyzed by Prolyl Endopeptidase from Aspergillus niger during Soy Sauce Fermentation

Soy sauce secondary precipitate formed due to the B₃ subunit seriously affects soy sauce's appearance quality. In this study, a prolyl endopeptidase (APE) from Aspergillus niger, which could degrade approximately 50% of the B₃ subunit and increase proline content by 24% in soy sauce, was isolated and identified. The results showed that APE was an acidic salt-tolerant serine protease (62 kDa), which was optimally active at 40 °C and pH 4.0, and retained more than 69% activity in 3 M NaCl solution over 10 days. As a potential substrate of APE, the B₃ subunit contains 10 proline residues. High salinity could not damage the hydrogen bonds, salt bridges, and interior hydrophobicity of APE; thus, the spatial structures and activity of APE in 3 M NaCl solution were stable within 3 days and decreased thereafter. High salinity made the B₃ subunit more rigid and lowered the catalytic activity of APE on the B₃ subunit, hindering complete hydrolysis of the B₃ subunit. This was the first report about the APE capable of degrading the B₃ subunit and reducing the secondary precipitate of soy sauce, providing a new possibility to solve the secondary precipitate of soy sauce.