Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brazil.
Universidade Federal de São Paulo, São Paulo, Brazil.
Prep Biochem Biotechnol. 2020;50(3):226-233. doi: 10.1080/10826068.2019.1683861. Epub 2019 Oct 29.
Traditionally, chymosin has been used for milk-clotting, but this naturally occurring enzyme is in short supply and its use has raised religious and ethical concerns. Because milk-clotting peptidases are a promising substitute for chymosin in cheese preparation, there is a need to find and test the specificity of these enzymes. Here, we evaluated the milk-clotting properties of an aspartic peptidase secreted by . The molecular mass of this enzyme was estimated at 36 kDa and Pepstatin A was determined to be an inhibitor. Optimal activity occurred at a pH of 5.5 and a temperature range of 50-60 °C, but the peptidase was stable in the pH range of 4-7 and a temperature as low as 45 °C. Proteolytic activity was significantly reduced in the presence of Cu and Al. When enzyme substrates based on FRET were used, this peptidase exhibited the highest catalytic efficiency for Abz-KNRSSKQ-EDDnp (4,644 ± 155 mM.s), Abz-KLRSSNQ-EDDnp (3,514 ± 130 mM.s), and Abz-KLRQSKQ-EDDnp (3,068 ± 386 mM.s). This study presents a promising peptidase for use in cheese making, due to its high stability in the presence of Ca and broad pH range of 4-7, in addition to its ability to efficiently clot milk.
传统上，凝乳酶被用于牛奶凝结，但这种天然存在的酶供应短缺，其使用引起了宗教和伦理方面的关注。由于乳凝肽酶是奶酪制备中凝乳酶的有前途的替代品，因此需要寻找和测试这些酶的特异性。在这里，我们评估了由 分泌的一种天冬氨酸肽酶的牛奶凝结特性。该酶的分子量估计为 36 kDa，Pepstatin A 被确定为抑制剂。最佳活性发生在 pH 5.5 和 50-60°C 的温度范围内，但该肽酶在 pH 4-7 和低至 45°C 的温度下稳定。在存在 Cu 和 Al 的情况下，蛋白水解活性显著降低。当使用基于 FRET 的酶底物时，该肽酶对 Abz-KNRSSKQ-EDDnp（4,644±155 mM.s）、Abz-KLRSSNQ-EDDnp（3,514±130 mM.s）和 Abz-KLRQSKQ-EDDnp（3,068±386 mM.s）表现出最高的催化效率。由于该酶在存在 Ca 和广泛的 pH 范围为 4-7 时具有高稳定性，并且能够有效地凝结牛奶，因此本研究提出了一种有前途的用于奶酪制作的肽酶。
