以功能性蛋白質體學研究Pseudomonas putida TX2生長於 辛基苯酚聚氧乙基醇與辛基苯酚之代謝與逆境反應

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以功能性蛋白質體學研究Pseudomonas putida TX2生長於辛基苯酚聚氧乙基醇與辛基苯酚之代謝與逆境反應
(Functional proteomic study on the metabolism and stress response of Pseudomonas putida TX2 grown on octylphenol polyethoxylates and octylphenol)

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摘要(中)

辛基苯酚聚氧乙基醇（octylphenol polyethoxylates、OPEOn）為一非離子性界面活性劑，使用於家庭與工業用清潔劑、保護作物之化學藥劑、化學、塑膠與紡織製業已逾四十年。OPEOn的代謝產物辛基苯酚（octylphenol、OP）普遍存在於水中且具有環境荷爾蒙之效應。而OPEOn之最終宿命以及其代謝產物還未被充分的證實。本實驗室於經常使用同類界面活性劑農田的土壤縮模，再外加OPEOn，經兩個月的馴化篩後選出Pseudomonas putida TX2，一株可有效分解利用OPEOn和OP為唯一碳源之微生物。本研究之主要目的為利用功能性蛋白質學的方法，包括一維和二維膠體電泳分離並以質譜儀鑑定出P. putida TX2分別生長於0.02%、0.5% OPEOn或、0.02% OP，再與相同濃度之succinate為對照組比較並鑑定所增生（up-regulation）或降低（down-regulation）之蛋白質，續以MALDI-Q-TOF以及ESI-MS/MS鑑定之。P. putida TX2生長於0.5% OPEOn之蛋白質體中共計有64個增生蛋白、46個降低蛋白；於0.02% OPEOn蛋白質體中共計有25個增生蛋白、16個降低蛋白；於0.02% OP蛋白質體中共計有47個增生蛋白、9個降低蛋白。P. putida TX2存在於含有0.5% OPEOn、0.02% OPEOn或0.02% OP的逆境反應之下的生理反應，大致相同，但是P. putida TX2在含有OPEOn環境下對於分解醇類化合物酵素量有增加的情況，像是在0.5% OPEOn偵測到alcohol dehydrogenase、於0.02% OPEOn 偵測到methanol dehydrogenase和benzoate 1,2 dioxygenase。本實驗室先前也利用相同的方法研究另一株可以快速代謝OPEOn卻無法利用其具有雌激素活性之代謝產物（OP）的菌株P. nitroreducens TX1。相較之下P. putida TX2或P. nitroreducens TX1存在於含有0.5% OPEOn的生理反應，其抗氧化蛋白質、保護蛋白質結構性蛋白質、熱休克蛋白、小分子運輸蛋白質、能量代謝蛋白質、胺基酸與酵素代謝蛋白質與氮代謝蛋白質的表現量均為增加表現的現象。但是，P. putida TX2在有關於胺基酸代謝機制有偵測到glutamate、histidine與methionine的代謝酵素均有被增加的現象，而P. nitroreducens TX1則為aspartate。總結來說，P. putida TX2對於OPEOn與OP的生物轉化的過程中指出，可將醇降解成醛，最有趣的是可能還有開環酵素的存在，但是，在0.02% OP並無偵測到，這是之後必須要再進一步研究的重點。

摘要(英)

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.

關鍵字(中)

★ 辛基苯酚聚氧乙基醇
★ 辛基苯酚

關鍵字(英)

★ octylphenol polyethoxylates
★ octylphenol

論文目次

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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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2005-7-28

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