細菌降解辛基苯酚聚氧乙基醇酵素活性分析： 醇類脫氫酶、醛類脫氫酶、乙醯輔酶A合成酶、 異檸檬酸裂解酶及蘋果酸合成酶;Enzyme activity involved in bacterial degradation of octylphenol polyethoxylate：alcohol dehydrogenase, aldehyde dehydrogenase, acetyl-CoA synthetase, isocitrate lyase and malate synthase

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Title:	細菌降解辛基苯酚聚氧乙基醇酵素活性分析：醇類脫氫酶、醛類脫氫酶、乙醯輔酶A合成酶、異檸檬酸裂解酶及蘋果酸合成酶;Enzyme activity involved in bacterial degradation of octylphenol polyethoxylate：alcohol dehydrogenase, aldehyde dehydrogenase, acetyl-CoA synthetase, isocitrate lyase and malate synthase
Authors:	葉辰瑋;Yeh,Chen-Wei
Contributors:	生命科學系
Keywords:	辛基苯酚聚氧乙基醇;醇類脫氫酶;醛類脫氫酶;乙醯輔酶A合成酶;異檸檬酸裂解酶;蘋果酸合成酶
Date:	2016-07-19
Issue Date:	2016-10-13 13:02:12 (UTC+8)
Publisher:	國立中央大學
Abstract:	烷基苯酚聚氧乙基醇 (Alkylphenol polyethoxylate, APEOn)，包含：辛基苯酚聚氧乙基醇 (Octylphenol polyethoxylate, OPEOn) 和壬基苯酚聚氧乙基醇 (Nonylphenol polyethoxylate, NPEOn)，屬於非離子界面活性劑，廣泛用於工業、農業與家庭用途。一般家庭、河川底泥與都市廢水系統中皆發現此類化合物之累積。APEOn在環境中會經由微生物降解其ethyleoxylate鏈，產生一些代謝產物，如壬基酚 (nonylphenol) 和辛基酚 (octylphenol)，這些代謝物將會累積在環境和生物體內，對生物帶來慢性毒性及內分泌干擾活性。Pseudomonas nitroreducens TX1為從放流水/灌溉渠道底泥中分離出的細菌，能以高濃度之APEOn為唯一碳源生長。在蛋白體學及transcriptome (RNA-seq) 分析中發現到P. nitroreducens TX1以0.5% OPEOn為唯一碳源生長時，其alcohol dehydrogenase、aldehyde dehydrogenase及acetyl-CoA synthetase的表現量，明顯高於以0.5% succinate為唯一碳源生長的菌體，因此使用以OPEOn為唯一碳源生長的P. nitroreducens TX1取其細胞粗萃取液，分別進行此三種酵素之活性分析，發現會比以succinate為唯一碳源生長的菌體細胞粗萃取液，每單位蛋白質量中酵素活性分別增加4.1倍、6.9倍及2.1倍，再經由基質特異性比較結果發現參與OPEOn生物轉化的alcohol dehydrogenase，發現只對於APEOn系列界面活性劑具有高度基質專一性的酵素，對於其他PEG系列界面活性劑與乙醇則無活性。P. nitroreducens TX1的基因體序列草圖已於2014年1月發表，為APEOn的降解基因定位與分析提供基本資訊，本研究使用轉位子突變法 (Transposon mutagenesis) 製作無法生長在此界面活性劑的突變株。目前自約30,000株突變菌株中篩選出145株以OPEOn為唯一碳源培養，而生長緩慢或無法生長的突變株；其中130株突變菌株已完成突變基因鑑定，發現6株突變株發生在isocitrate lyase基因上及8株於malate synthase基因，他們皆無法生長於以OPEOn為唯一碳源的培養基中。因此使用以OPEOn為唯一碳源生長的P.nitroreducens TX1細胞粗萃取液，分別進行isocitrate lyase和malate synthase之酵素活性分析，發現會比以succinate為唯一碳源生長的菌體細胞粗萃取液，每單位蛋白質量中酵素活性分別增加8.9倍及1.7倍。此外，我們成功將P. nitroreducens TX1的isocitrate lyase表達在E. coli中，也以相同酵素活性法進行基質專一性測試，顯示只對isocitrate具活性，對於OPEOn及結構類似物沒有任何活性，因之判斷isocitrate lyase不是直接參與降解OPEOn EO chain結構的生物降解酵素，而是發生在下游代謝中，將轉化後代謝產物進入glyoxylate cycle的關鍵酵素。最後根據蛋白質體、代謝產物分析、轉位子突變法突變菌株的突變庫及酵素活性測試結果，推測OPEOn可能經由P. nitroreducens TX1細胞外膜上的運輸蛋白質，進入periplasm中後經由ABC transporter進入細胞內，經alcohol dehydrogenase進行EO鏈末端醇基氧化為醛類，並由aldehyde dehydrogenase氧化為羧酸，再經由EO鏈cleavage酵素將EO鏈斷開產生acetate後，使用acetyl-CoA synthetase轉化成acetyl-coA進入glyoxylate cycle中，並產生succinate進入中央代謝途徑TCA cycle以轉化碳源供P. nitroreducens TX1生長，故此代謝步驟為本研究提出P. nitroreducens TX1使用OPEOn的推測代謝途徑。 ;Alkylphenol polyethoxylates (APEOn) that include octylphenol polyethoxylate (OPEOn) and nonylphenol polyethoxylate (NPEOn) are non-ionic surfactants and widly used in industy, agriculture and domestic purposes. APEOn are often found as pollutants in natural aquatic environments and in raw municipal wastewater. Ethyleoxylate chain of APEOn is degraded by microorganisms in environment to produce estrogenic metabolites, such as nonylphenol and octylphenol. They accumulate in environments and cause chronic ecotoxicity as endocrine disrupters to aquatic organisms, wildlife, and humans. Pseudomonas nitroreducens TX1 was found from sediment of irrigation farmland and was capable to use a wide range concentrations of APEOn, as a sole carbon source. By proteomics and transcriptome RNA seq analysis, alcohol dehydrogenases, aldehyde dehydrogenases and acetyl-CoA synthetase have higher expression level when growth in MSB medium containing 0.5% OPEOn as a sole carbon source than that in 0.5% succinate. Therefore, crude extract of P. nitroreducens TX1 growth in 0.5% OPEOn was used to optimize the activity assay for three enzyme. The enzyme activities of alcohol dehydrogenase, aldehyde dehydrogenase and acetyl-CoA synthetase have been shown 4.1, 6.9 and 2.1 fold increase than those in 0.5% succinate. The substract specificty of OPEOn-dependent alcohol dehydrogenases was demonstrated that APEOn related compounds are the enzyme substrates but not using polyethoxyl glycol (PEG) and ethanol. Due to the draft genome sequence of P. nitroreducens TX1 was already published and provides basal informations in investigating gene loci and sequence related to significant roles in alkylphenol polyethoxylate degradation. Transposon mutagenesis was used to create mutant library of P. nitroreducens TX1 for the screening of growth defected mutants on OPEOn. Over 30,000 mutants have been isolated after triparental mating between transposon DNA containg E.coli and P. nitroreducens TX1. 145 mutants grew slowly or did not grow on OPEOn medium. The mutated genes in 130 mutants have been identified. Among these mutants, six transposon insertion mutants and eight mutants were idendifed in isocitrate enzyme and malate synthase, respectively. Therefore, the two enzyme were selected for further enzymatical activity study. The two enzymes have 8.9 fold and 1.7 fold increased in their activities in crude extracts from growth in OPEOn MSB medium than in succinate MSB medium. Furthermore, isocitrate lyase was cloned and expressed in E.coli. The isocitrate lyase has high enzyme activity for its putative target, isocitrate, but has no activity for OPEOn and its derivatives. Therefore, according to these results, the isocitrate lyase is not directly involved in the biodegrading of OPEOn. The isocitrate lyase may paly an important role at the downstream metabolic step, converting to glyoxylate cycle. Based on current evidences by proteomics, metabolites, transposon mutagenesis mutants and enzyme activities in this study, we proposed the OPEOn metabolic pathway in P. nitroreducens TX1 that the OPEOn biotransformation follows the proposed polyethoxylate chain biodegrading mechanism that OPEOn processes the terminal end of polyethoxylatic alcohol oxidation by alcohol dehydrogenases to form terminal aldehyde and further oxidizes to form carboxylic acid, named OPEC, by aldehyde dehydrogenase. The two carbon source, acetate, was somehow produced from OPEC, and catalyzed by acetyl-CoA synthetase to form acetyl-CoA. It goes into glyoxylate cycle via isocitrate lyase and malate sythase to produce OAA and succinate in TCA cycle to support P. nitroreducens TX1 growth.
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