Brevibacterium sp. TX4 與 Pseudomonas nitroreducens TX1 異化辛基苯酚聚氧乙基醇及其代謝物之生物分解途徑研究;Study of degradation pathway of octylphenol polyethoxylates by Brevibacterium sp. TX4 and octylphenol degradation by Pseudomonas nitroreducens TX1

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Title:	Brevibacterium sp. TX4 與 Pseudomonas nitroreducens TX1 異化辛基苯酚聚氧乙基醇及其代謝物之生物分解途徑研究;Study of degradation pathway of octylphenol polyethoxylates by Brevibacterium sp. TX4 and octylphenol degradation by Pseudomonas nitroreducens TX1
Authors:	林怡雯;Lin, Yi-Wen
Contributors:	生命科學系
Keywords:	辛基苯酚聚氧乙基醇;辛基苯酚;生物分解途徑;Brevibacterium sp. TX4;Pseudomonas nitroreducens TX1;octylphenol polyethoxylates;octylphenol;degradation pathway;Brevibacterium sp. TX4;Pseudomonas nitroreducens TX1
Date:	2017-02-03
Issue Date:	2017-05-05 17:13:11 (UTC+8)
Publisher:	國立中央大學
Abstract:	辛基苯酚聚氧乙基醇 (Octylphenol polyethoxylates, OPEOn) 屬於非離子性界面活性劑的一種，其一旦被排放至自然環境中，經常會生成為更不易分解且具環境賀爾蒙效力的代謝產物如辛基苯酚 (octylphenol) 與帶1-2單位氧乙基醇之辛基苯酚聚氧乙基醇，對於人體健康與環境生態具危害性。黃雪莉教授研究團隊先前從農藥工廠、稻田及中央大學排放水的表土及底泥中分離出43株可以辛基苯酚聚氧乙基醇為唯一碳源培養之菌株，其中Brevibacterium sp. TX4 為唯一的一株格蘭氏陽性菌。以高效能液相層析質譜儀 (HPLC-MS) 分析菌株TX4分解辛基苯酚聚氧乙基醇的代謝產物，推斷此菌株能在耗氧的情況下減短聚氧乙烯鏈，Brevibacterium sp. TX4為第一株被發現據此能力的格蘭氏陽性菌。另外，在43株具分解辛基苯酚聚氧乙基醇能力的菌株中，Pseudomonas nitroreducens TX1生長速率最快且對辛基苯酚聚氧乙基醇的耗氧活性最高，並能生長在0.05%到20%的辛基苯酚聚氧乙基醇濃度中，其生長速率在0.34到0.44 hr-1之間。以高效能液相層析質譜儀 (HPLC-MS) 分析菌株TX1分解辛基苯酚聚氧乙基醇的代謝產物，推斷此菌株能減短聚氧乙烯鏈並生成辛基苯酚。本研究發現菌株TX1可以進一步以支鏈型辛基苯酚 (4-t-octylphenol) 為唯一碳源生長。P. nitroreducens TX1為第一株被發現同時具有分解辛基苯酚聚氧乙基醇與支鏈型辛基苯酚能力的菌株。以高效能液相層析質譜儀 (LC-ESI-Q-TOF) 分析此菌株以支鏈型辛基苯酚為唯一碳源生長過程中的代謝產物，發現有中間代謝物辛基鄰苯二酚 (4-t-octylcatechol) 生成。其中偵測到兩個經間位裂解 (meta-cleavage) 途徑產生之代謝物: 5-formyl-2-hydroxy-6,6,8,8-tetramethylnona-2,4-dienoic acid 與 6,6,8,8-tetramethyl-2-oxonon-4-enoic acid，推測菌株TX1分解支鏈型辛基苯酚的過程首先藉由苯環羥化 (aromatic ring hydroxylation) 產生辛基鄰苯二酚，再經由間位裂解途徑以破壞辛基苯酚之結構進而降低環境賀爾蒙效力。除此之外，在菌株TX1分解支鏈型辛基苯酚的過程中發現有O-甲基化 (O-methylation) 的反應進行。生長實驗中測定菌株TX1對其他不同類型之烷基苯酚 (4-alkylphenols) 之利用特性，發現菌株TX1可利用廣泛不同類型之烷基苯酚，尤其是長鏈烷基苯酚。因此，菌株TX1具有應用於分解環境中之辛基苯酚聚氧乙基醇與不同類型之烷基苯酚污染物之潛力。 ;Octylphenol polyethoxylates (OPEOn) are surfactants and prone to be degraded into xenoestrogenic metabolites, such as octylphenol and OPEOn (n=1-2), are estrogenic-like compounds, and persistent to be degraded by bacteria in the environments. Due to the structural similarity to estradiol, nonylphenols/octylphenols influenced the growth, reproduction and sexual development of humans and animals, therefore, causing health and ecological concern. Forty three bacterial strains were isolated from soil and sediments of a pesticide factory, a rice field, and drainage of a dormitory by Huang’s group can utilize OPEOn as their sole carbon and energy source. Brevibacterium sp. TX4 was the only Gram-positive bacterium. Metabolites analysis by HPLC/MS revealed that Brevibacterium sp. TX4 could shorten the ethoxylate chain thereby degrading OPEOn, during which O2 was required. Brevibacterium sp. TX4 is the first Gram-positive bacterium which was demonstrated to shorten the ethoxylate chain of OPEOn. Among the isolates, Pseudomonas nitroreducens TX1 can grow on 0.05% to 20% of OPEOn with a specific growth rate of 0.34-0.44 hr-1. High-performance liquid chromatography–mass spectrometer analysis of OPEOn degraded metabolites revealed that strain TX1 was able to shorten the ethoxylate chain and produce octylphenol (OP). This study showed that P. nitroreducens TX1 was capable of utilizing 4-t-octylphenol, the endocrine disrupting compound, as a sole carbon source. This strain is the first bacterium which is able to degrade both octylphenol polyethoxylates (OPEOn) and 4-t-octylphenol to be reported. Along with the degradation of 4-t-octylphenol, 4-t-octylcatechol was identified as internal metabolite. Two key metabolites of the meta-cleavage pathway, 5-formyl-2-hydroxy-6,6,8,8-tetramethylnona-2,4-dienoic acid and 6,6,8,8-tetramethyl-2-oxonon-4-enoic acid, were detected by LC-ESI-Q-TOF. We concluded that degradation of 4-t-octylphenol by strain TX1 is initiated by aromatic ring hydroxylation to form 4-t-octylcatechol, followed by a meta-cleavage pathway thereby to disrupting the estrogenic activity of 4-t-octylphenol. In addition to the degradation reactions, O-methylation was observed during the 4-t-octylphenol degradation by strain TX1. Growth experiments with other 4-alkylphenols showed that strain TX1 could utilize a wide range of 4-alkylphenols, especially the long chain alkylphenols. Therefore, strain TX1 has potential be used in the bioremediation of environments polluted by OPEOn, 4-t-octylphenol and various other 4-alkylphenols.
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