Sakthivel Muthu, Palani Perumal
Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600 025, India.
Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600 025, India.
Int J Biol Macromol. 2016 May;86:390-401. doi: 10.1016/j.ijbiomac.2015.11.086. Epub 2016 Jan 15.
A novel antimicrobial protein was purified from the seedlings of Bauhinia purpurea by sequential procedures entailing ammonium sulfate precipitation, cation exchange chromatography, preparative native-PAGE and a yield of 2.7% was obtained from the crude extract. The purified antimicrobial protein appeared as a single protein band on SDS-PAGE with the molecular mass of 20.9 kDa. Purified antimicrobial protein exhibited a potent antimicrobial activity against both Gram-positive and Gram-negative bacteria. Analysis of the trypsin digested peptides of purified protein using the MALDI-TOF MS/MS resulted in the identification of 174 amino acids. The purified protein had an optimum of pH of 5.5 and was stable at 35 °C for exhibiting its maximal antibacterial activity. The addition of metal ions such as Mn(2+) and Ca(2+) to the purified protein enhanced the antimicrobial activity of purified protein. The MIC of purified protein against Bacillus cereus and Escherichia coli were 13 μg/ml and 15 μg/ml, respectively. The purified protein digested the peptidoglycan layer of bacteria which was visualized by TEM analysis.
通过硫酸铵沉淀、阳离子交换色谱、制备型天然聚丙烯酰胺凝胶电泳等一系列步骤，从紫羊蹄甲幼苗中纯化出一种新型抗菌蛋白，粗提物的得率为2.7%。纯化后的抗菌蛋白在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上呈现为单一蛋白条带，分子量为20.9 kDa。纯化后的抗菌蛋白对革兰氏阳性菌和革兰氏阴性菌均表现出强大的抗菌活性。使用基质辅助激光解吸电离飞行时间串联质谱（MALDI-TOF MS/MS）对纯化蛋白的胰蛋白酶消化肽段进行分析，鉴定出174个氨基酸。纯化后的蛋白最适pH为5.5，在35℃下稳定，以表现出其最大抗菌活性。向纯化蛋白中添加锰离子（Mn(2+)）和钙离子（Ca(2+)）等金属离子可增强纯化蛋白的抗菌活性。纯化蛋白对蜡样芽孢杆菌和大肠杆菌的最低抑菌浓度（MIC）分别为13 μg/ml和15 μg/ml。通过透射电子显微镜（TEM）分析可见，纯化后的蛋白可消化细菌的肽聚糖层。
