权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis and optimization of lysine fermentation based on proteome analysis

基于蛋白质组分析的赖氨酸发酵分析与优化

基本信息

批准号：
14550764
负责人：
HORIUCHI Jun-ichi
金额：
$ 2.37万
依托单位：
Kitami Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

The aim of this study is to develop a methodology to analyze and optimize bioprocesses based on intracellular information obtained by proteome analysis using a two-dimensional electrophoresis (2-DE). Threonine-limited lysine fermentation using Brevibacterium flavum was employed for the proteome analysis. Samples were taken from batch cultures of lysine fermentation at different physiological states including an exponential growth phase, an initial lysine production phase and a final lysine production phase. The cell-free extracts were obtained by homogenization using glass beads. The use of phenylmethylsulfonyl fluoride (protease inhibitor) and acetone precipitation greatly improved the extraction efficiency of intracellular proteins from intact cells. Approximately 480 and 450 protein spots were detected by 2-DE in the samples of the exponential growth phase and the initial lysine production phase, respectively. However, the number of protein spots decreased to approximately 320 in the sample of the final lysine production phase, which suggested the decline of microbial activity at the end of fermentation. The image analysis was then performed to identify protein spots of 2-DE maps by use of the database of Conrynebacterium glutamicum proteins analyzed by a peptide mass fingerprinting. At the exponential growth phase, proteins concerning glycolysis and TCA cycle were identified, while the proteins related to lysine biosynthesis was not observed. However, in the sample of the initial and final lysine production phases, 2 proteins (DapA and Doh) concerning lysine biosynthesis was clearly identified as well as proteins concerning glycolysis and TCA cycle. This shows that the difference in physiological states of lysine fermentation could be recognized at a protein level using 2-DE maps. Thus, the proteome analysis using 2-DE was found to be effective to obtain the intracellular information for the better understanding of bioprocesses.

本研究的目的是开发一种方法来分析和优化生物过程的基础上获得的细胞内信息的蛋白质组分析，使用二维电泳（2-DE）。采用黄色短杆菌苏氨酸限制性赖氨酸发酵进行蛋白质组学分析。从处于不同生理状态的赖氨酸发酵的分批培养物中取样，所述不同生理状态包括指数生长期、初始赖氨酸生产期和最终赖氨酸生产期。使用玻璃珠通过均质化获得无细胞提取物。苯甲基磺酰氟（蛋白酶抑制剂）和丙酮沉淀的使用大大提高了从完整细胞中提取胞内蛋白质的效率。在对数生长期和初始赖氨酸产生期的样品中，2-DE分别检测到约480和450个蛋白质点。然而，在最终赖氨酸生产阶段的样品中，蛋白质斑点的数量减少到约320个，这表明发酵结束时微生物活性的下降。然后进行图像分析，通过使用肽质量指纹分析的谷氨酸棒杆菌蛋白质数据库来鉴定2-DE图谱的蛋白质点。在对数生长期，鉴定出与糖酵解和TCA循环相关的蛋白，而未观察到与赖氨酸生物合成相关的蛋白。然而，在初始和最终赖氨酸生产阶段的样品中，清楚地鉴定了2种与赖氨酸生物合成有关的蛋白质（达帕和Doh）以及与糖酵解和TCA循环有关的蛋白质。这表明，赖氨酸发酵的生理状态的差异，可以识别在蛋白质水平上使用2-DE图谱。因此，利用2-DE技术进行蛋白质组分析，可以有效地获得细胞内的信息，以便更好地了解生物过程。