Pseudomonas nitroreducens TX1中二氫硫辛醯胺脫氫酶分解辛基苯酚聚氧乙基醇之研究

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孫玉齡(Yu-Ling Sun) 查詢紙本館藏

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Pseudomonas nitroreducens TX1中二氫硫辛醯胺脫氫酶分解辛基苯酚聚氧乙基醇之研究
(Degradation of octylphenol polyethoxylates by dihydrolipoamide dehydrogenase from Pseudomonas nitroreducens TX1)

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摘要(中)

烷基苯酚聚氧乙基醇 (alkylphenol polyethoxylates, APEOn)是一種非離子界面活性劑，結構為苯環的對位分別接上支鏈辛基及平均9.5個單位的聚氧乙基鏈之混合物，廣泛被應用於家庭日常生活中與工業上，也因之流放到環境中。其中間代謝產物烷基苯酚 (如壬基苯酚、辛基苯酚)具有雌激素活性，累積在環境中對環境生態與人類生殖造成影響，然而環境中之微生物及酵素分解烷基苯酚聚氧乙基醇與烷基苯酚的代謝機制卻尚未了解。先前本實驗是從一株能以0.05-20% (v/v) 辛基苯酚聚氧乙基醇 (octylphenol polyethoxylates,OPEOn)為唯一碳源生長的Pseudomonas nitroreducens TX1中，發現菌體之耗氧活性比不生長在此界面活性劑中高20倍，乃純化出一個具耗氧活性的酵素：二氫硫辛醯胺脫氫酶 (dihydrolipoamide dehydrogenase, LipDH)。同時在功能性蛋白質體學分析中，發現此酵素在以0.5 % OPEOn為唯一生長碳源，而增強表現4.6倍，以RT-PCR也驗證了在mRNA層次上亦有增加。本研究以重組DNA方法，構築dihydrolipoamide dehydrogenase之融合蛋白質表現在大腸桿菌中，經純化此酵素，以探討此脫氫酶與分解OPEOn的關係。以液相層析質譜儀分析發現dihydrolipoamide dehydrogenase能降解OPEOn之ethoxylate鏈，且ethoxylate鏈越長者，H2O2生成量與分解速率成正相關，被切短之速率也越快。其乃因dihydrolipoamide dehydrogenase能在NADH過量的情形下產生H2O2，同時此酵素可還原過量之鐵離子成亞鐵離子，當H2O2和亞鐵離子共存時發生Fenton reaction，因此產生hydroxyl radical，便攻擊OPEOn的ethoxylate chain，造成鏈的縮短而分解。Dihydrolipoamide dehydrogenase在大多數生物中皆存在，其生理功能為參與TCA循環中生成succinyl-CoA的反應，這是第一次發表有關於這個脫氫酶與細菌分解界面活性劑相關的報告。

摘要(英)

The alkylphenol polyethoxylates (APEOn) are commonly used non-ionic surfactants in industrial, agricultural and domestic applications. The release of APEOn in the environment has resulted in concern due to the accumulated metabolites, nonylphenol and octylphenol, for their estrogenic activity. However, little is known about the enzymes involved in the biodegradation of APEOn. Pseudomonas nitroreducens TX1, was isolated from the drainage on NCU campus, shown to grow on 0.05-20% of octylphenol polyethoxylates (OPEOn) as sole carbon source. An OPEOn-dependent oxygen consumption activity was induced in strain TX1 when grown on OPEOn as sole carbon source. Both gel-based proteomics and RT-PCR demonstrated that the expression such enzyme is up-regulated by OPEOn at both the protein and mRNA levels. Dihydrolipoaide dehydrogenase has been reported in most of the organisms involving in producing of succinyl-CoA from TCA cycle. An oxygen consumption enzyme was isolated and characterized as dihydrolipoamide dehydrogenase. Recombinant fusion protein constructed and expressed as soluble form in E. coli. In vitro, we detect the purified LipDH can produce H2O2 in the presence of excess NADH. This enzyme also catalyzes the reduction of Fe3+ to form Fe2+. In the presence of both H2O2 and Fe2+, Fenton reagent, the OPEOn were shown to be degraded by reducing the ethoxylate chain. The degradation mechanism by cleavage of ethoxylate chain on OPEOn is proposed to be carried by the hydroxyl radical produced from Fenton reaction. This is the first discovery on such as a novel function from bacterial dihydrolipoamide dehydrogenase.

關鍵字(中)

★ 二氫硫辛醯胺脫氫酶
★ 氫氧自由基
★ 烷基苯酚聚氧乙基醇

關鍵字(英)

★ dihydrolipoamide dehydrogenase
★ octylphenol polyethoxylates
★ hydroxyl radical

論文目次

目錄 I
表目錄 II
圖目錄 III
縮寫與全名對照表 IV
壹、緒論 1
一、界面活性劑的性質、種類與使用 1
二、烷基苯酚聚氧乙基醇類及其代謝產物之流佈及影響 2
三、烷基苯酚聚氧乙基醇代謝物之環境荷爾蒙效力 4
四、烷基苯酚聚氧乙基醇之分解 5
五、分解類似化合物之酵素 7
六、研究背景 8
七、本研究目的及大綱 10
貳、材料與方法 11
一、分解菌之培養 11
二、表現載體之建構 12
三、蛋白質純化 13
四、酵素定性 17
五、化學藥品與儀器設備 19
參、結果 21
一、序列分析 21
二、蛋白質表現 21
三、酵素純化 22
四、重組蛋白質定性 24
肆、討論 28
一、酵素定性 28
二、生物轉化OPEOn產物分析 30
三、經Fenton reaction降解OPEOn 31
四、dihydrolipoamide dehydrogenase分解OPEOn的氧乙基鏈之機制 32
伍、結論與建議 34
陸、參考文獻 36
表 44
圖 48
附錄 72

參考文獻

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2006-7-24

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