Pseudomonas putida TX2中催化辛基苯酚聚氧乙基醇之醇類脫氫酶的初步定性

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鍾依靜(Yi-Ching Chung) 查詢紙本館藏

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生命科學系

論文名稱

Pseudomonas putida TX2中催化辛基苯酚聚氧乙基醇之醇類脫氫酶的初步定性
(Preliminary characterization of alcohol dehydrogenase interacting with octylphenol polyethoxylates in Pseudomonas putida TX2)

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摘要(中)

烷基苯酚聚氧乙基醇(Alkylphenol polyethoxylates, APEOn)為一常被用於工業、農業及ㄧ般家庭使用之非離子界面活性劑;它包含辛基苯酚聚氧乙基醇(Octylphenol polyethoxylates, OPEOn)及壬基苯酚聚氧乙基醇(Nonylphenol polyethoxylates, NPEOn)，其中以NPEOn的使用量佔全世界的80%。大量的界面活性劑被釋放到環境當中已知會在環境中生成短鏈(n≦3)之APEOn及alkylphenol (AP)對於水中之生物以及人類皆具類似雌激素之環境荷爾蒙效應。目前對於環境中微生物體中烷基苯酚聚氧乙基醇的降解途徑並非十分清楚。先前本實驗室篩選出一株可利用0.05~20%辛基苯酚聚氧乙基醇或0.02% octylphenol (OP)為唯一碳源生長之菌株，將之命名為Pseudomonas putida TX2。根據液相層析串聯式質譜儀的分析，P. putida TX2可逐步的切斷OPEOn的氧乙基鏈，生成octyl phenol (OP)，再生成octyl catechol (OC)，顯示此菌可對具雌激素活性OP進行轉化，特別的是，此菌亦可直接以OP為唯一碳源生長(同時會偵測到OC生成，但下游產物不明)，顯示P. putida TX2可成為一個研究AP分解機制之模式生物。本研究主要目的即在由P. putida TX2中純化在此代謝途徑中第一個利用OPEOn的酵素。在P. putida TX2中發現有一醇類脫氫酶 (alcohol dehydrogenase, ADH)，其在細胞粗萃取液中占總活性的48%，3%在周質間，16%的活性在高速離心分離後的細胞質及細胞膜中間的沉澱物中。發現此ADH在100 mM citric acid phosphate緩衝液, pH 7, 40 oC時活性最高，在pH 6及pH 8的100 mM citric acid phosphate緩衝液下酵素活性可穩定42小時。利用偵測不同輔因子的醇類脫氫酵素活性法，推估對OPEOn具活性之醇類脫氫酶應含有pyrroloquinoline quinine (PQQ)與heme c；在595 μg細胞粗萃取液中之ADH對於OPEOn、NPEOn及AEO8分別有6.5, 5.2及5.6 mU/mg的活性，並且也對於nonyl aldehyde具有19.8 mU/mg活性，而對polyethylene glycol (PEG)、methanol、butanol、benzyl alcohol、formaldehyde和benzaldehyde無活性。利用離子交換管柱層析及疏水性管柱層析得知P. putida TX2中至少有兩個PQQ-linked ADH，ㄧ個為pI＜6.5而另ㄧ為pI＞8；在酵素純化方面，第ㄧ步採用疏水性管柱層析，才可得到較高之回收率 (20%)以及純度 (1.1~2.5倍)。由P. putida TX2經離子交換管柱層析後之部分純化的ADH (1048 μg的蛋白質)對於OPEOn、NPEOn及AEO8分別有5.53 mU/mg, 5.22 mU/mg和5.07 mU/mg的活性，其中，此部份純化之ADH對於nonyl aldehyde也具7.63 mU/mg活性，而對PEG、methanol、butanol、benzyl alcohol、formaldehyde和benzaldehyde無活性，因此推測P. putida TX2 之ADH可先將OPEOn末端氫氧基氧化成醛基再將其氧化成羧基。未來將選殖此ADH，以進一步確認ADH在P. putida TX2中所扮演之角色。

摘要(英)

Alkylphenol polyethoxylates (APEOn) are non-ionic surfactants and extensively used in industrial, agricultural, and household activities. APEOn includes octylphenol polyethoxylates (OPEOn) and nonylphenol polyethoxylates (NPEOn). Large quantities of surfactants are released into environment and formed short chain APEOn (n≦3)and alkylphenol (AP). These metabolites mimic estrogenic activity, as environmental hormones, which are harmful to aquatic organisms and human. The biodegradation pathway and fate of these compounds in the environment and in organisms still remains unclear. Pseudomonas putida TX2 was isolated in Taiwan and was able to grow on 0.05~0.5% octylphenol polyethoxylates (OPEOn) or 0.02% octylphenol (OP) as sole carbon source. According to the Liquid Chromatograph-Mass Spectrometry analysis, P. putida TX2 was capable of degrading OPEOn to form OPEOn (n≦3). It also transformed OP to form octylcatechol (OC) through a sequential cleavage of ethoxylate chain. Since P. putida TX2 can grow on OP as sole carbon source, it can be a model organism to study the degradation mechanism of estrogenic-like AP. The purpose of this research is to characterize the first enzyme react on OPEOn in P. putida TX2. Using crude extract as our sample, an alcohol dehydrogenase (ADH) was found to be 48% of activity in the crude extract, 3% in periplasm and 16% of activity in the fraction between crude extract and membrane after an ultra-speed centrifugation. ADH from P. putida TX2 showed highest activity in 100 mM citric acid phosphate buffer, pH 7 at 40oC and is stable in pH 6 and 8 100 mM citric acid phosphate buffers for 42 hours. Using different enzyme assays to detect different cofactors involved in ADH, the ADH has activity toward OPEOn is a PQQ-linked and heme c containing ADH. 595 μg of protein in crude extract, ADH has 6.5, 5.2 and 5.6 mU/mg activity toward OPEOn, NPEOn, AEO8 and nonyl aldehyde, respectively and no activity to polyethylene glycol (PEG), methanol, butanol, benzyl alcohol, formaldehyde and benzaldehyde. By ionic exchange chromatography and hydrophobic interaction chromatography, there are at least two different PQQ-linked ADHs, pI＜6.5 and pI＞8. The first purification is a hydrophobic interaction chromatography because of the higher recovery and purification fold compared to anionic and cationic exchange chromatographies. The partially purified ADH (pI>6.5) by ionic exchange chromatographies has 5.53 mU/mg, 5.22 mU/mg and 5.07 mU/mg activity toward OPEOn, NPEOn and AEO8, respectively. It also has 7.63 mU/mg of activity toward nonyl aldehyde. No activity on PEG, methanol, butanol, benzyl alcohol, formaldehyde and benzaldehyde were detected .Therefore, we proposed that ADH (pI>6.5) in P. putida TX2 is the first to oxidize the terminal OH group of OPEOn to aldehyde then to carboxylic acid to form octylphenol polyethoxycarboxylate (OPECn). For the future work, we will clone this ADH to confirm the role in the OPEOn degradation pathway by P. putida TX2.

關鍵字(中)

★ 醇類脫氫酶
★ 辛基苯酚聚氧乙基醇

關鍵字(英)

★ alcohol dehydrogenase
★ octylpheonol 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
　ㄧ、界面活性劑之性質與使用及其代謝物的影響1
二、烷基苯酚聚氧乙基醇之分解4
三、研究動機9
貳、材料與方法11
一、菌株與培養11
二、蛋白質大量表現14
三、酵素活性測試20
四、酵素純化23
五、蛋白質偵測方式27
六、酵素定性28
七、化學藥品與儀器設備29
參、結果31
一、微生物培養31
二、蛋白質大量表現31
三、酵素活性測試32
四、酵素純化37
五、酵素定性39
肆、討論41
伍、結論與建議50
陸、參考文獻52
表58
圖72
附錄96

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2008-1-21

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