短小芽孢杆菌胞外蛋白质组双向电泳分析及碱性胁迫下胞外差异蛋白鉴定

Proteome analysis of Bacillus pumilus extracellular proteins by two dimensional electrophoresis and identifiation of differential proteins under alkaline stress

短小芽孢杆菌Bacillus pumillus SCU11是本实验室从富含蛋白质的生活垃圾中分离并诱变得到的具有高碱性蛋白酶活性的菌株,其分泌的碱性蛋白酶具有很好的生皮脱毛效果,在生物制革工业具有良好的应用前景.短小芽孢杆菌胞外蛋白质多达数百种,为了解其组成情况,采用LB培养基培养短小芽孢杆菌SCU11,以三氯乙酸/丙酮法沉淀发酵上清液中的蛋白质,通过双向电泳分析,建立了具有较高分辨率的短小芽孢杆菌胞外蛋白的双向电泳图谱.在此基础上,分析了碱胁迫条件下短小芽孢杆菌胞外蛋白质的双向电泳图谱,利用液相色谱-串联质谱(LC-MS/MS)鉴定了10个差异蛋白点,其中有5种为胞外分泌蛋白,包含2种胞外蛋白酶.本研究结果表明与呼吸作用及运动相关的蛋白可能参与了短小芽孢杆菌碱胁迫的应答反应.

A novel Bacillus pumilus strain SCUll was screened and mutagenized for its ability to produce extracellular alkaline protease with prominent leather dehairing function in our previous studies. B. pumilus secrets hundreds of extracellular proteins involved in many important biotechnological fields. However, there is so far no study about the extracelluar proteome of B. pumilus. To analyze the extracellular proteins of B. pumilus, the strain SCUll was cultured in LB medium and the supernatant was collected. Extracellular proteins were used for two-dimensional electrophoresis （2-DE） and mass spectrometry analysis. A high-resolution 2-DE map of extracellular proteins of B. pumilus SCUll was established. Furthermore, the extracellular proteomes of B. pumilus under alkaline stress were analyzed and ten differentially expressed protein spots were identified by liquid chromatography-mass spectrometry/mass spectrometry （LC-MS/MS）. The result showed five kinds of secreted proteins, including two kinds of extracellular protease. Data of this study indicated that proteins involved in respiration and movement may response to alkaline stress in B. pumilus.