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

本研究的目的是利用双向电泳（2 DE）技术分析细胞内和细胞外蛋白质组信息，从而分析培养阶段，并控制胞外酶的产生。采用解淀粉芽孢杆菌（ATCC 23350）进行蛋白质组分析。在不同的生理状态下，包括生长期和α-淀粉酶产生期，从分批培养物中采集细胞内和细胞外蛋白样品。应用新型二维凝胶电泳图像分析仪，分析硝酸银染色二维凝胶电泳图谱的动态变化过程。利用新型图像分析仪，重叠的蛋白质点的二维凝胶电泳图谱可以识别为单独的点。与传统的图像分析方法相比，新的图像分析仪检测到的蛋白质点的数量增加。通过使用通过肽质量指纹分析的枯草芽孢杆菌蛋白质数据库来鉴定检测到的蛋白质点。在生长阶段，糖酵解和TCA循环相关蛋白被明确鉴定，而胞外蛋白未被观察到。然而，在α-淀粉酶生产阶段，许多胞外蛋白被鉴定。因此，通过使用2DE的蛋白质组分析，在蛋白质水平上有效地分析了培养相。

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.