Chang Chang1, Siyi Gong1, Zhiping Liu1, Qiaojuan Yan2, Zhengqiang Jiang3. 1. 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. 2. Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Engineering, China Agricultural University, Beijing, 100083, China. 3. 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. zhqjiang@cau.edu.cn.
Abstract
BACKGROUND: 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. RESULTS: 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 (IC50 of 0.129 mg/mL). CONCLUSIONS: GsProS8 could be a potential candidate for industrial applications, especially the preparation of antihypertensive peptides.
BACKGROUND: 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. RESULTS: 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 (IC50 of 0.129 mg/mL). CONCLUSIONS: GsProS8 could be a potential candidate for industrial applications, especially the preparation of antihypertensive peptides.
Authors: Amaliawati Ahmad Latiffi; Abu Bakar Salleh; Raja Noor Zaliha Raja Abd Rahman; Siti Nurbaya Oslan; Mahiran Basri Journal: Genes Genet Syst Date: 2013 Impact factor: 1.517
Authors: Marta Pokora; Aleksandra Zambrowicz; Agnieszka Zabłocka; Anna Dąbrowska; Marek Szołtysik; Konrad Babij; Ewelina Eckert; Tadeusz Trziszka; Józefa Chrzanowska Journal: Acta Biochim Pol Date: 2017-04-07 Impact factor: 2.149