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Student Number 92224002
Author Min-Chung Ko(柯旻君)
Author's Email Address No Public.
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Department Life Science
Year 2004
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Functional proteomic study on the metabolism and stress response of Pseudomonas putida TX2 grown on octylphenol polyethoxylates and octylphenol
Date of Defense 2005-07-21
Page Count 315
Keyword
  • octylphenol
  • octylphenol polyethoxylates
  • Abstract Octylphenol polyethoxylates (OPEOn) are nonionic surfactants that have been used for more than 40 years in household and industrial detergents, crop protection agents, chemical, plastics and textiles manufacturing. The degradation metabolites of OPEOn and octylphenol (OP) have become ubiquitous in the aquatic environment and can serve as environmental hormones. The ultimate fate of OPEOn and their metabolites is not adequately understood. A bacterial strain, Pseudomonas putida TX2, was previously isolated in the microcosm of farm soil with addition of OPEOn followed by 2 month of adaptation. It can grow effectively on 0.05%~20% OPEOn as the sole carbon source. This study was aimed to use functional proteomic approach to identify the TX2 proteins up- and down-regulated under 0.5% OPEOn, 0.02% OPEOn or 0.02% OP and the same concentration of succinate as control. 1D-SDS-PAGE and 2D-SDS-PAGE were used for protein separation, and those protein changing by more than 4-fold were identified by MALDI-Q-TOF or ESI-MS/MS. There are 64 up-regulated proteins and 46 down-regulated proteins in the proteome of P. putida TX2 grown in 0.5% OPEOn; 25 up-regulated proteins and 16 down-regulated proteins in 0.02% OPEOn, and 47 up-regulated proteins and 9 down-regulated proteins in 0.02% OP. The physiological responses of P. putida TX2 to environmental stress under 0.5% or 0.02% OPEOn and 0.02% OP are very similar. Take enzymes responsible for alcohol hydrolysis under OPEOn stress as example, the up-regulated responses in 0.5% OPEOn is alcohol dehydrogenase while in 0.02% OPEOn are methanol dehydrogenase and benzoate 1,2- dioxygenase. There is another strain named P. nitroreducens TX1 studied and cultured as previously described. P. nitroreducens TX1 also degrades OPEOn but is not able to further degrade OP, a product with estrogen activity. However, the small molecular transportation protein in 0.02% OPEOn is ABC transporter while transportation protein in 0.02% OP is outer membrane protein and branched-chain amino acid ABC transporter. Increases of protein expression under 0.5% OPEOn of both P. putida TX2 or P. nitroreducens TX1 are heat-shock proteins, protective proteins for antioxidants and structural protection as well as small transportation proteins, metabolic enzymes for protein, peptides and nitrogen. However, the main metabolic enzymes for amino acids in P. putida TX2 are glutamate、histidine and methionine but in P. nitroreducens TX1 is aspartate. The breakthrough of biodegradation in OPEOn or OP is that P. putida TX2 is able to cleave ethoxylate chain and to oxidize the alcohol terminal into aldehyde and then to carboxyl group. Also, the existence of enzyme for digesting benzene ring structure equally contributes to biodegradation in OPEOn or OP. In conclusion, the biotransformation performed by P. putida TX2 to OPEOn or OP is through the oxidation of alcohol into aldehyde. Intriguingly, enzyme responsible for benzene ring cleavage seemed to be necessary in 0.5% OPEOn, but is not detected in 0.02% OP, and which is the key for further study.
    Table of Content 目錄
    中文摘要 I
    英文摘要III
    目錄V
    表目錄VIII
    圖目錄X
    名詞縮寫對照表XIV
    壹、前言1
    一、蛋白質體學簡介1
    二、微生物逆境中之蛋白質體學4
    三、微生物分解環境污染物之蛋白質體學應用9
    四、烷基苯酚聚氧乙基醇及其環境宿命14
    五、烷基苯酚聚氧乙基醇及烷基苯酚之微生物分解23
    六、本研究之目的33
    貳、材料方法37
    一、培養基與菌種品系37
    二、辛基苯酚代謝產物分析38
    三、蛋白質體學之聚丙烯醯胺膠與二維電泳40
    四、軟體分析電泳膠體52
    五、膠體內消化52
    六、質譜儀分析與資料庫搜尋54
    七、實驗儀器、實驗藥品56
    參、結果59
    一、辛基苯酚對P. putida TX2 之影響59
    二、P. putida TX2 之聚丙烯醯胺膠體圖及二維電泳圖(10%
    1D-SDS-PAGE;12~20% 2D-SDS-PAGE,pI 4-7)61
    三、P. putida TX2 於0.5%辛基苯酚聚氧乙基醇逆境下表現量
    有改變之蛋白質64
    四、P. putida TX2 於0.02%辛基苯酚聚氧乙基醇逆境下表現
    量有改變之蛋白質73
    五、P. putida TX2 於0.02%辛基苯酚逆境下表現量有改變之
    蛋白質77
    六、P. putida TX2 生長於於0.02%辛基苯酚或0.02%辛基苯
    酚聚氧乙基醇逆境下蛋白質體之比較82
    七、P. putida TX2 生長於0.5%或0.02%辛基苯酚聚氧乙基醇
    逆境下蛋白質體之比較83
    八、P. putida TX2 或P. nitroreducens TX1 生長於於0.5%辛基
    苯酚聚氧乙基醇逆境下蛋白質體之比較85
    肆、討論87
    一、P. putida TX2 攝入辛基苯酚聚氧乙基醇與辛基苯酚87
    二、P. putida TX2 代謝辛基苯酚聚氧乙基醇與辛基苯酚89
    伍、結論與建議94
    陸、參考文獻97
    表117
    圖177
    附錄209
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