Muthu Sakthivel, Gopal Venkatesh Babu, Soundararajan Selvakumar, Nattarayan Karthikeyan, S Narayan Karthik, Lakshmikanthan Mythileeswari, Malairaj Sathuvan, Perumal Palani
Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu, India.
Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600025, Tamilnadu, India.
Plant Physiol Biochem. 2017 Mar;112:161-172. doi: 10.1016/j.plaphy.2017.01.003. Epub 2017 Jan 3.
A serine protease was purified from the leaves of Wrightia tinctoria by sequential flow through method comprising screening, optimization, ammonium sulfate precipitation, gel filtration and ion exchange column chromatography. The yield and purification fold obtained were 11.58% and 9.56 respectively. A single band of serine protease was visualized on SDS-PAGE and 2-D gel electrophoretic analyses were revealed with the molecular mass of 38.5 kDa. Serine protease had an optimum pH of 8.0 and was stable at 45°C with high relative protease activity. The addition of metal ions such as Mg2+ and Mn2+ exhibits a high relative activity. Serine protease had a potent antibacterial activity against both Gram-positive and Gram-negative bacteria. A 10 μg/ml of serine protease was tested against S. aureus, M. luteus, P. aeruginosa and K. pneumoniae which had 21, 20, 18 and 17 mm of zone of inhibition respectively. Serine protease from W. tinctoria degrades the peptidoglycan layer of bacteria which was visualized by transmission electron microscopic analysis.
通过包括筛选、优化、硫酸铵沉淀、凝胶过滤和离子交换柱色谱的连续流通法，从黄冠马利筋叶片中纯化出一种丝氨酸蛋白酶。获得的产量和纯化倍数分别为11.58%和9.56。在SDS-PAGE上观察到一条丝氨酸蛋白酶条带，二维凝胶电泳分析显示其分子量为38.5 kDa。丝氨酸蛋白酶的最适pH为8.0，在45°C下稳定，具有较高的相对蛋白酶活性。添加Mg2+和Mn2+等金属离子表现出较高的相对活性。丝氨酸蛋白酶对革兰氏阳性菌和革兰氏阴性菌均具有强大的抗菌活性。测试了10μg/ml的丝氨酸蛋白酶对金黄色葡萄球菌、藤黄微球菌、铜绿假单胞菌和肺炎克雷伯菌的抗菌活性，其抑菌圈分别为21、20、18和17mm。通过透射电子显微镜分析可见，黄冠马利筋的丝氨酸蛋白酶可降解细菌的肽聚糖层。
