Pseudomonas putida SH1異化

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題名:	Pseudomonas putida SH1異化
作者:	梁福翥;Fu-Zhu Liang
貢獻者:	生命科學研究所
日期:	2001-07-09
上傳時間:	2009-09-22 10:16:24 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	I 蛋白質體為細胞或組織所表現的全部蛋白質圖譜，可以分析出同ㄧ物種在不同環境下所表現蛋白質的差異，其方法為先將細胞中總蛋白質利用二維膠體電泳分析，再以質譜儀配合資料庫比對做蛋白質的鑑定。Pseudomonas putida SH1 為一株能分解各種苯環化合物的革蘭氏陰性桿菌。先前研究得知P. putida SH1 可以、酚或鄰-甲酚為唯一碳源生長，且其在minimal salts basal (MSB) 培養基之最適碳源濃度皆為0.05%，以P. putida SH 在其中鄰苯二酚加氧酵素比活性最高時的菌體製成蛋白質粗萃液，進行二維膠體電泳，分析此菌在利用不同苯環化合物為唯一碳源生長時所表達蛋白質圖譜的差異。結果顯示 P. putida SH1 分別生長在0.05% 、酚或鄰-甲酚的MSB 培養基與與控制組(即含0.3% succinate 為碳源的MSB培養基) 中所誘發的蛋白質有明顯的不同。將這些被誘發出來的蛋白質經過介質輔助雷射揮離離子化飛行時間(MALDI-TOF) 質譜儀的偵測與質量指紋做比對，結果分別有10 個蛋白質是參與異化與5 個蛋白質是參與異化酚或鄰-甲酚。由二維電泳圖譜與蛋白質鑑定結果推測此菌在異化酚或鄰-甲酚時可能利用相同的代謝途徑，而分解時則走另一途徑。另外發現P. putida SH1 在異化苯環化合物時都會誘發烷基過氧化還原蛋白(AhpC)，這是第一次有關細菌在異化、酚或鄰-甲酚的環境下有逆境蛋白質AhpC 的表現的發現，表示此菌利用苯環化合物的同時還有逆境蛋白的表現，但未知其相互關聯。此外，從P. putida mt-2 純化出C23O，以兔子製備的抗血清對二維電泳膠做西方墨點分析，結果顯示P. putida SH1 在異化酚或鄰-甲酚時表現出來的鄰苯二酚加氧酵素是相同的，而在異化時則表現出另一種鄰苯二酚加氧酵素，與本實驗室之前的生化結果相符。 Proteomics is a systematic analysis of the proteins expressed by a cell or tissue. The combination of two-dimensional electrophoresis for protein separation with mass spectrometry for protein identification is the essential analytical tools by proteomics approach. Pseudomonas putida SH1 was capable to catabolize naphthalene, phenol or o-cresol as sole source of carbon and energy. The protein spots in 2-D gel showed that different profile induced in P. putida SH1 during growth in naphthalene, phenol or o-cresol as sole carbon source compared to using succinate as a control. Protein spots were digested by trypsin and the peptide mass fingerprints were analyzed by matrix assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. Ten and five enzymes were identified to be involved in the degradation pathways of naphthalene and phenol/o-cresol, respectively. The degradation pathway of individual aromatic compound can then be constructed. The results indicated that the degradation of phenol and o-cresol may shared the same catalytic pathway in this bacterium. In addition, a general bacterial stress protein, alkyl hydroperoxide reductase (AhpC), was induced by P. putida SH1 in the medium containing individual aromatic carbon source. This is the first observation of the involvement of an AhpC protein in the stress response to aromatic compound degradation. In the western blot analysis of 2D-gel, an aromatic ring cleavage enzyme, catechol 2,3-dioxygenase, was detected by using a polyclonal antibody against this protein. The enzyme also showed different in both molecular weight and pI in the proteome of naphthalene-grown strain SH1 and that from phenol (or o-cresol).
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