分解辛基苯酚聚氧乙基醇之耗氧酵素（二氫硫辛醯胺脫氫酶）的純化與定性

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洪國展(Guo-Chan Hung) 查詢紙本館藏

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生命科學系

論文名稱

分解辛基苯酚聚氧乙基醇之耗氧酵素（二氫硫辛醯胺脫氫酶）的純化與定性
(Purification and characterization of a bacterial oxygen consumption enzyme, dihydrolipoamide dehydrogenase, able to degrade octylphenol polyethoxylates)

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摘要(中)

辛基苯酚聚氧乙基醇的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，為常用於農業、工業以及一般家庭使用之非離子界面活性劑。除本身具有毒性外，由於其化學結構的特殊性，會改變土壤中有機污染物如農藥、石化類碳氫化合物的吸附與分佈；且當其被分解至剩0 ~ 3個氧乙基醇單位時，在結構上類似雌激素，具環境荷爾蒙效力，故其在環境中的累積與廣泛流佈將對環境及生物體造成嚴重影響。先前本實驗室由中央大學放流水污泥中篩選出多株可利用辛基苯酚聚氧乙基醇為唯一碳源生長之菌株，其中1株Pseudomonas sp. stain 82.10.6，經三種鑑定法（BioLog呼吸碳源利用圖譜、脂肪酸圖譜及16S rDNA序列分析）與不具明膠水解活性，將之命名為Pseudomonas nitroreducens TX1。在whole-cell實驗中，已證實此菌株可將辛基苯酚聚氧乙基醇之氧乙基逐一斷裂，並消耗氧氣；故在酵素純化時，乃利用耗氧活性，將未吸附陰離子交換樹脂管柱之具有活性之酵素收集液繼以硫酸銨沉澱、疏水作用力、膠體過濾及色層焦集純化步驟分離後，得到一由單體分子量52.9 ± 1.0 kDa構成同質二聚體（a2）之酵素（分子量98.1 ± 7.5 kDa），其等電點為6.65 ± 0.06；酵素吸光光譜在360及457 nm具吸收波峰，並證實其為一每莫耳酵素包含1.87莫耳FAD為輔因子之酵素。而利用電噴灑游離串聯式質譜儀及介質輔助雷射脫附游離串聯式質譜儀獲得之多個胜肽序列，經比對後確認此蛋白質為二氫硫辛醯胺脫氫酉每（dihydrolipoamide dehydrogenase），與由P. fluorescens中分離的此酵素序列最接近。此酵素為生物中遍存之酵素，已知最主要為2-oxo acid dehydrogenase complexs家族中的E3部分，功能為氧化acetyltransferase上的dihydrolipoamide形成lipoamide。本研究為由P. nitroreducens中首次純化出此酵素，而於體外實驗證實純化酵素須外加0.5 mM NADPH時才具耗氧氣活性，且此活性在多加入1 mM過渡金屬離子如Fe2+或Mn2+時可促進耗氧活性表現達2.8倍以上；進一步藉由液相層析質譜儀分析酵素反應產物，首次證實它確有縮短OPEOn之聚氧乙基鏈的能力。前人曾推論OPEOn中聚氧乙基鏈之切斷的一種機制，可能是藉由Fenton反應中所產生之氫氧自由基攻擊所造成；而我們認為氫氧自由基若由Fenton反應生成，而Fenton反應的Fenton reagents（還原態過渡金屬離子和H2O2）是藉由dihydrolipoamide dehydrogenase在過量NADPH存在下，還原過渡金屬及消耗氧氣所生成H2O2所供給的。因此dihydrolipoamide dehydrogenase藉由執行此功能而造成縮短聚氧乙基鏈的能力在本研究中首次證實，為此酵素之新功能。

摘要(英)

Alkylphenol polyethoxylates (APEOn), including octylphenol polyethoxylates and nonylphenol polyethoxylates, are nonionic surfactants and are used in numerous commercial and industrial products including detergents, dispersants and emulsifiers. Large quantities of surfactants are released into the environment and these influence the fate of organic compounds in soils. The degradation products of these compounds, APEOn (n = 0 ~ 3), have been demonstrated to act as environmental hormones with estrogen activity. Octylphenol polyethoxylates (OPEOn) are composed of an aromatic ring with a branched eight-carbon alkyl polyethoxylate chain (average n = 9.5 ethoxylate units) at the para position. Our previous results have indicated that among the thirty-seven isolates capable of degrading this compound, a Gram-negative rod Pseudomonas sp. stain 82.10.6, showed the fastest growth and highest oxygen consumption activity using OPEOn as a sole carbon source and a transformation substrate, respectively. In this study, three identification methods, BioLog breath printing, fatty acid methyl ester fingerprinting and 16S rDNA sequence analysis, plus a negative activity for gelatin hydrolysis, were used to identify Pseudomonas sp. stain 82.10.6 as a Pseudomonas nitroreducens and the isolate was designated stain TX1. The sequential cleavage of the ethoxylate unit of OPEOn was confirmed by oxygen consumption during whole-cell fermentation of stain TX1. Using oxygen consumption activity as an enzyme assay, a NADPH-dependent enzyme was purified from stain TX1 and shown to shorten the ethoxylate chain. The OPEOn-oxygen consumption enzyme was isolated to homogeneity by flow-through anion exchange chromatography followed by ammonium sulfate precipitation, hydrophobic-interaction chromatography, gel filtration chromatography and chromatofocusing. The purified enzyme showed a subunit and native molecular weights of 52.9 ± 1.0 kDa (by SDS-PAGE) and 98.1 ± 7.5 kDa (by gel filtration), respectively. The enzyme configuration was a2. The pI of the enzyme was shown to be 6.65 ± 0.06 and 7.27 by chromatofocusing and 2-D gel electrophoresis, respectively. The absorption spectrum of the purified enzyme shows maximal absorbance at 375 and 458 nm, and the profile showed it contains FAD in a molar ratio of 1.87, that is one mole of FAD per subunit. Several peptide sequences from the enzyme were analyzed by ESI-Q-TOF and by MALDI-Q-TOF and most closely matched the same dihydrolipoamide dehydrogenase from Pseudomonas fluorescens (with a highest coverage of 36% of the amino acid sequence of the enzyme). Using the peptide sequences, molecular weight, configuration and absorption spectrum, the purified enzyme was identified as a dihydrolipoamide dehydrogenase. This is the first report of the isolation and characterization of dihydrolipoamide dehydrogenase from P. nitroreducens. The known function of this enzyme in all species up to the present is as one of the components in 2-oxo acid dehydrogenase complex family, with the function of oxidizing dihydrolipoamide on acetyltransferase modification. However, a new and very unique function has been demonstrated by this study. It is a NADPH-dependent, oxygen consuming enzyme and when exogenous transition metal ions, like Fe or Mn, are added to the enzyme reaction, the oxygen consumption activity is enhanced. The enzyme catalyzed product of this enzyme has been shown to be shortened ethoxylate chains of OPEOn (n = 6 is dominant) by LC/MS analysis. The cleavage of the ethoxylate chain of OPEOn occurs by hydroxyl radical attack and this was previously proposed by other researchers as one possible ethoxylate chain degradation mechanism. The hydroxyl radical is one of the products from the Fenton reaction. Where Fenton reagents (reduced metals and H2O2) are produced, there is both a reduction of transition metal ions and consumption of oxygen (O2 → O2•- → HOO• → H2O2) by dihydrolipoamide dehydrogenase in the presence of excess NADPH. This novel discovery is the first time that such a function has been shown for a bacterial dihydrolipoamide dehydrogenase.

關鍵字(中)

★ 氫氧自由基
★ 二氫硫辛醯胺脫氫酶
★ 烷基苯酚聚氧乙基醇

關鍵字(英)

★ hydroxyl radical
★ dihydrolipoamide dehydrogenase
★ alkylphenol polyethoxylates

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類之分解………………………………………. 6
四、研究背景與大綱……………………………………..…..……………. 9
貳、材料與方法………………………………………………………………. 12
一、分解菌之培養…………………………………………………..........12
二、菌種鑑定………………………………………………………………. 14
三、耗氧酵素活性測試……………………………………………………. 18
四、酵素純化………………………………………………………………. 20
五、耗氧酵素定性……………………………………………………..... 23
六、化學藥品與儀器設備…………………………………………………. 25
參、結果………………………………………………………………………. 27
一、菌種鑑定………………………………………………………………. 27
二、微生物大量培養………………………………….…………………… 27
三、酵素純化………………………………………………………………. 28
四、蛋白質定性……………………………………………………………. 29
肆、討論………………………………………………………………………. 32
伍、結論與建議………………………………………………………………. 41
陸、參考文獻…………………………………………………………………. 43
表………………………………………………………………………………. 50
圖………………………………………………………………………………. 56
附錄……………………………………………………………………………. 76

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2004-7-29

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