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.

中文翻译：

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酱油发酵过程中黑曲霉脯氨酰内肽酶催化大豆蛋白B3亚基的降解机理

_{由于B 3}亚基形成的酱油二次沉淀严重影响酱油的外观品质。在这项研究中，分离和鉴定了一种来自黑曲霉的脯氨酰内肽酶 (APE) ，它可以降解大约 50% 的 B ₃亚基，并使酱油中的脯氨酸含量增加 24%。结果表明，APE 是一种耐酸性盐的丝氨酸蛋白酶 (62 kDa)，在 40 °C 和 pH 4.0 时具有最佳活性，在 3 M NaCl 溶液中 10 天后仍保持 69% 以上的活性。作为 APE 的潜在底物，B ₃亚基含有 10 个脯氨酸残基。高盐度不会破坏APE的氢键、盐桥和内部疏水性；因此，APE 在 3 M NaCl 溶液中的空间结构和活性在 3 天内稳定，之后下降。高盐度使B ₃亚基更加坚硬，降低了APE对B ₃亚基的催化活性，阻碍了B 3 亚基的完全_水解。_{这是APE能够降解B 3}亚基并减少酱油二次沉淀的首次报道，为解决酱油二次沉淀提供了新的可能。