Analysis of culture phase based on proteome analysis and control of extracellular enzyme production

本研究的目的是根据细胞内和细胞外蛋白质组分析所获得的信息来分析培养时相，并控制细胞外酶的产生。以淀粉芽孢杆菌(ATCC 23350)为模板进行蛋白质组分析。从分批培养的不同生理状态，包括生长期和a-淀粉酶产生阶段，采集胞内和胞外蛋白质样品。用一种新型的双向凝胶图像分析仪分析硝酸银染色的双向凝胶图谱的动态过程。利用这种新型的图像分析仪，可以将2张DE图谱上重叠的蛋白质斑点识别为单独的斑点。与传统的图像分析方法相比，新型图像分析仪检测到的蛋白质斑点数目有所增加。利用肽质量指纹图谱分析枯草芽孢杆菌蛋白质数据库对检测到的蛋白质斑点进行鉴定。在生长阶段，与糖酵解和TCA循环有关的蛋白质被清楚地识别出来，而没有观察到胞外蛋白质。然而，在a-淀粉酶的产生阶段，许多胞外蛋白被鉴定出来。因此，利用双向凝胶电泳法进行蛋白质组分析，可以在蛋白质水平上有效地分析培养相。

The aim of this study was to analyze culture phase based on informations obtained by intracelluar and extracelluar proteome analysis using a two dimensional electrophoresis (2 DE) and to control extracellular enzyme productions. Bacillus amyloliqucfaciens (ATCC 23350) was employed for the proteome analysis. Intracellular and extracelluar protein samples were taken from batch cultures at different physiological states including growth phase and a-amylase production phase. A novel 2 DE image analyzer was employed to analyze dynamic processes of 2 DE maps stained with silver nitrate. Using the novel image analyzer, overlapped protein spots on the 2 DE maps could be recognized as individual spots. The number of protein spots detected by the novel image analyzer was increased as compared with the conventional image analysis method. The detected protein spots were identified by use of the database of Bacillus subtilis proteins analyzed by a peptide mass fingerprinting. At the growth phase, proteins concerning glycolysis and TCA cycle were clearly identified, while extracellular proteins were not observed. However, at a-amylase production phase, many extracellular proteins were identified. Therefore, the culture phase was effectively analyzed at a protein level by proteome analysis using 2 DE.