Key Laboratory of Food Bioengineering (China National Light Industry), College of Food Science and Nutritional Engineering, China Agricultural University, No. 17 Qinghua Donglu, Beijing, 100083, China.
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Engineering, China Agricultural University, Beijing, 100083, China.
BMC Biotechnol. 2021 Mar 11;21(1):21. doi: 10.1186/s12896-021-00678-7.
Proteases are important for hydrolysis of proteins to generate peptides with many bioactivities. Thus, the development of novel proteases with high activities is meaningful to discover bioactive peptides. Because natural isolation from animal, plant and microbial sources is impractical to produce large quantities of proteases, gene cloning and expression of target protease are preferred.
In this study, an alkaline serine protease gene (GsProS8) from Geobacillus stearothermophilus was successfully cloned and expressed in Bacillus subtilis. The recombinant GsProS8 was produced with high protease activity of 3807 U/mL after high cell density fermentation. GsProS8 was then purified through ammonium sulfate precipitation and a two-step chromatographic method to obtain the homogeneous protease. The molecular mass of GsProS8 was estimated to be 27.2 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 28.3 kDa by gel filtration. The optimal activity of GsProS8 was found to be pH 8.5 and 50 °C, respectively. The protease exhibited a broad substrate specificity and different kinetic parameters to casein and whey protein. Furthermore, the hydrolysis of whey protein using GsProS8 resulted in a large amount of peptides with high angiotensin-I-converting enzyme (ACE) inhibitory activity (IC of 0.129 mg/mL).
GsProS8 could be a potential candidate for industrial applications, especially the preparation of antihypertensive peptides.
蛋白酶对于蛋白质的水解作用非常重要，因为它可以生成具有多种生物活性的肽。因此，开发具有高活性的新型蛋白酶对于发现生物活性肽具有重要意义。由于从动物、植物和微生物来源中天然分离大量蛋白酶是不切实际的，因此基因克隆和目标蛋白酶的表达是首选方法。
本研究成功地从嗜热脂肪地芽孢杆菌中克隆并表达了一种碱性丝氨酸蛋白酶基因（GsProS8）。在高密度发酵后，重组 GsProS8 的蛋白酶活性达到 3807 U/mL。通过硫酸铵沉淀和两步层析法对 GsProS8 进行了纯化，得到了均一的蛋白酶。SDS-PAGE 电泳和凝胶过滤法估计 GsProS8 的分子量分别为 27.2 kDa 和 28.3 kDa。GsProS8 的最适活性分别为 pH 8.5 和 50°C。该蛋白酶表现出广泛的底物特异性和不同的动力学参数，对酪蛋白和乳清蛋白均有作用。此外，使用 GsProS8 水解乳清蛋白可产生大量具有高血管紧张素转化酶（ACE）抑制活性（IC 为 0.129 mg/mL）的肽。
GsProS8 可能是工业应用的潜在候选酶，特别是用于制备具有降压作用的肽。
