World Journal of Microbiology and Biotechnology ( IF 4.0 ) Pub Date : 2021-12-13 , DOI: 10.1007/s11274-021-03195-z Maryam Rouhani 1, 2 , Sara Molasalehi 1, 2 , Vahideh Valizadeh 2 , Atousa Aghai 2, 3 , Sogol Pourasghar 2, 4 , Reza Ahangari Cohan 2 , Dariush Norouzian 2
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Serratiopeptidase is a bacterial protease that has been used medicinally in variety of applications. Though, some drawbacks like sensitivity to environmental conditions and low penetration into cells limited its usage as a potent pharmaceutical agent. This study aimed to produce four novel truncated serratiopeptidase analogs with different lengths and possessing one disulfide bridge, in order to enhance protease activity and thermal stability of this enzyme. Mutagenesis and truncation were performed using specific primers by conventional and overlap PCR. The recombinant proteins were expressed in E. coli cells then purified and their protease activity and stability were checked at different pH and temperatures in comparison to the native form of the enzyme, Serra473. Enzyme activity assay showed that T306 [12–302 ss] was not further active which could be due to the large truncation. However, T344 [8–339 ss], T380 [8–339 ss] and T380 [12–302 ss] proteins showed higher proteolytic activity comparing to Serra473. These analogs were active at temperatures of 25–90 °C and pH 6–9.5. Interestingly, remaining enzyme activity of T344 [8–339 ss], T380 [8–339 ss] and T380 [12–302 ss] forms at 90 °C calculated as 87, 83 and 86 percent, respectively, comparing to the activity at room temperature. However, residual activity at the same conditions was 50% for the full length enzyme. Formation of disulfide bond in engineered serratiopeptidases could be the main reason for higher thermal stability compared to Serra473. Thermostability of T344 [8–339 ss], as the most thermostable designed serratiopeptidase, was additionally confirmed using differential scanning calorimetry.
Graphical abstract
中文翻译：
具有增强的蛋白酶活性和热稳定性的新型工程化锯齿肽酶类似物的设计、表达和功能评估
Serratiopeptidase 是一种细菌蛋白酶，已在医学上用于各种应用。但是，一些缺点，例如对环境条件的敏感性和对细胞的低渗透性，限制了其作为有效药剂的用途。本研究旨在生产四种不同长度且具有一个二硫键的新型截短锯齿肽酶类似物，以提高该酶的蛋白酶活性和热稳定性。通过常规和重叠 PCR 使用特定引物进行诱变和截断。重组蛋白在大肠杆菌中表达然后纯化细胞，并与酶的天然形式 Serra473 相比，在不同的 pH 值和温度下检查它们的蛋白酶活性和稳定性。酶活性测定表明 T306 [12-302 ss] 没有进一步的活性，这可能是由于大截断。然而，与 Serra473 相比，T344 [8-339 ss]、T380 [8-339 ss] 和 T380 [12-302 ss] 蛋白显示出更高的蛋白水解活性。这些类似物在 25–90 °C 的温度和 pH 值 6–9.5 下具有活性。有趣的是，T344 [8-339 ss]、T380 [8-339 ss] 和 T380 [12-302 ss] 在 90 °C 形成的剩余酶活性分别计算为 87%、83% 和 86%，与在室内温度。然而，全长酶在相同条件下的残留活性为 50%。与 Serra473 相比，工程化 serratiopeptidases 中二硫键的形成可能是具有更高热稳定性的主要原因。T344 [8-339 ss] 的热稳定性作为最热设计的锯齿肽酶，还使用差示扫描量热法进行了确认。
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