Pseudomonas nitroreducens TX1中催化辛基苯酚聚氧乙基醇之醇類脫氫酶的初步純化

Preliminary purification of an alcohol dehydrogenase for transformation octylphenyl polyethoxylates by Pseudomonas nitroreducens TX1

王大成(Da-chen Wang)

指導教授 ： 黃雪莉

國立中央大學/理學院/生命科學系/碩士(2008年)

摘要

烷基苯酚聚氧乙基醇(Alkylphenol polyethoxylate, 簡稱APEOn)，包含：辛基苯酚聚氧乙基醇(Octylphenol polyethoxylate, 簡稱OPEOn)和壬基苯酚聚氧乙基醇(nonylphenol polyethoxylate, 簡稱NPEOn)，是一種聚乙二醇(PEG)類的非離子界面活性劑。其結構是由數個親水性的氧乙基鏈(EO chain)在對位位置接連結一個疏水性的烷基苯酚(Alkylphenol, 簡稱AP)。APEOn可應用在各種工業、農業和家用，環境中的主要來源為廢水處理場的排放廢水和農田殘留的殺蟲劑和除草劑；當被排放到環境中，經由微生物的連續切斷APEOn的EO chain，會形成APEOn (n≦3) 和AP，亦會形成含有羧基的短鏈烷基苯酚聚氧乙基酸(Alkylphenol polyethoxycarboxylate, APECn, n≦3)生成；這些代謝物會累積在環境和生物體內，對生物、鳥類和哺乳動物都具有似雌激素的作用，故被稱為環境荷爾蒙。Pseudomonas nitroreducens TX1是一株從台灣分離出，可以0.05~20% (v/v)之OPEOn為唯一生長碳源的菌株；從液相層析質譜儀分析顯示，菌株TX1可以依序地切斷OPEOn和dodecyl octaethoxylate (AEO8)的EO chain，並伴隨著產生OPECn和AEO8的羧基產物。先前二維電泳和質譜儀分析菌株TX1生長於0.5% OPEOn和0.5% succinate為唯一生長碳源的蛋白質體差異，發現一種含Pyrroloquinoline quinine (PQQ)為輔因子的醇類脫氫酶(alcohol dehydrogenase(PQQ), 簡稱ADH(PQQ))和醛類脫氫酶(aldehyde dehydrogenas, 簡稱ALDH)，分別增加表現為15和10倍，且其他團隊曾也提到這類ADH(PQQ)可分解PEG，因此我們推測ADH(PQQ)酵素可能參與OPEOn的降解。我們已將ADH(PQQ)和ALDH基因選殖與表達，但大腸桿菌表現之ADH(PQQ)大部分在細胞膜，而無法偵測出活性。故我們從P. nitroreducens TX1中分離可代謝OPEOn之alcohol dehydrogenase (ADH)，發現可催化OPEOn脫氫的活性，不是以NAD+或只含有PQQ為輔因子的酵素，本研究暫無法確定此酵素之輔因子，因此暫稱為ADH。ADH在TX1菌株生長於0.5% OPEOn (實驗組)和0.5% succinate (控制組)為唯一生長碳源時，活性增加為3倍，顯示OPEOn能誘發ADH的增強表現。同時其活性在細胞粗萃液和細胞膜的分佈分別為74和26%，顯示酵素大部分存在於細胞質中。ADH在細胞粗萃液中的基質專一性為：對AEO8、OPEOn、NPEOn有顯著的活性(9~11 mU/mg)，對於PEG卻沒有催化活性，因此推論此ADH與他人發表的聚乙二醇脫氫酶(PEG dehydrogenase)並不相同，且對APEOn有作用，但對單純之醇類如：methanol、ethanol、butanol和benzyl alcohol無活性，為第一個發現對此類界面活性劑有催化專一性的酵素；在測試醛類時，對壬醛(nonyl aldehyde)有活性(22 mU/mg)，但對formaldehyde和benzaldehyde沒有活性，因此推測此酵素可以將OPEOn之EO chain末端之氫氧基依序氧化成醛基，再進而氧化成羧基，如此可解釋APEC代謝物之形成機制。以管柱層析方法追蹤ADH活性時，從離子交換管柱結合之特性中，推測ADH的pI值可能為5；第一步陰離子交換管柱層析，其回收率為22%，第二步經由膠體管柱層析，回收率為3%，第三步經由等電焦集管柱，推測ADH的pI值可能為5.8，但以上步驟之回收率顯示酵素之穩定性差，需再解決此問題，才能更進步得到純化ADH。經由第二步膠體管柱層析後得到部份純化的ADH，其基質專一性與細胞粗萃液相同。未來將繼續選殖ADH並利用液相層析串聯式質譜儀分析其代謝產物，以進一步確認在P. nitroreducens TX1中ADH所扮演之角色。

關鍵字

醇類脫氫酶 ； 辛基苯酚聚氧乙基醇

並列摘要

Alkylphenol polyethoxylate (APEOn), including octylphenol polyethoxylate (OPEOn) and nonylphenol polyethoxylate (NPEOn), are typical polyethylene glycol (PEG) type non-ionic surfactants. The structures of APEOn are a hydrophilic ethylene oxide (EO) chain attached to a hydrophobic alkylphenol at para-position. They are very important surfactants with industrial, agricultural, and domestic applications. The majority of environmental APEOn comes from wastewater treatment, plant discharge, pesticides and herbicides on farms. When APEOn are released into environment, the EO chains of APEOn are cleaved to APEOn (n≦3), APECn (n≦3) and alkylphenol (AP) which are known to cause estrogenic effects on aquatic organisms, birds, and mammals as environmental hormoens. Pseudomonas nitroreducens TX1, isolated in Taiwan, is shown to be able to grow on 0.05~20％(v/v) of OPEOn as sole carbon source. Liquid chromatography-mass spectrometry analysis showed that the biodegradation mechanism of OPEOn and dodecyl octaethoxylate (AEO8) by strain TX1 was through a sequential cleavage of the EO chain and accompany with the formation of carboxylated OPEOn and AEO8. Previously, P. nitroreducens TX1 was grown on minimal salts basal (MSB) medium containing 0.5％ OPEOn as sole carbon source and analyzed by a subtractive proteome comparisons against a succinate-grown proteome. An alcohol dehydrogenase containing pyrroloquinoline quinine (PQQ) and aldehyde dehydrogenase (ALDH) were shown to be up-regulated for 15-and 10-folds, respectively. Moreover, an alcohol dehydrogenase (PQQ) was shown to degrade PEG proposed by other group. Therefore, we cloned ADH(PQQ) and ALDH for expression in Escherichia coli. However, the ADH expressed in E. coli is mostly in the membrane fraction and we can not detect the activity in this fraction. We then identified an OPEOn degrading enzyme (ADH) from strain TX1 and found that ADH react on OPEOn is neither NAD+ nor PQQ-dependent enzymes. We can not determine the cofactor of this enzyme and temporarily named ADH. The alcohol dehydrogenase activity was up-regulated for 3 folds in cell grown on 0.5% OPEOn compared to that on 0.5% succinate, indicating OPEOn induced a higher expression of ADH. The activities of ADH in crude extract and in membrane are 74 and 26 percents, respectively. ADH in crude extract, has activity to AEO8, OPEOn and NPEOn (9~11 mU/mg of crude extract) but has no activity to PEG. Therefore, the ADH from P. nitroreducens TX1 is different from PEG dehydrogenase from Sphingomonas. terrae published previously. In addition, ADH has no activity to methanol, ethanol, butanol, benzyl alcohol, but the ADH also is a dehydrogenase for nonyl-aldehyde (22 mU/mg), but not on formaldehyde and benzaldehyde, indicating its role in the formation of APEC. The ADH from strain TX1 is specifically to catalyze OPEOn to form OPECn. By ionic exchange chromatography, the pI of ADH is estimated to be around 5. The first purification step is by anionic exchange chromatography. The recovery is 22% from the first step. Second step is by gel filtration and the recovery is 3%. Chromatofocusing was used as the third step for ADH purification. The pI of ADH is 5.8. Substrates specificity of the partial purified ADH from second-step purification is the same as ADH in crude extract. In the future work, the ADH will be cloned to analyze its products analyzed by LC-MS to confirm the role of ADH in the OPEOn degradation pathway in P. nitroreducens TX1.

並列關鍵字

octylphenyl polyethoxylates ； alcohol dehydrogenase

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