臺灣博碩士論文加值系統

烷基苯酚聚乙氧基醇(alkylphenol polyethoxlates)為一廣泛使用於工業、農業與家用活動之非離子性界面活性劑，其一旦被排放至自然環境中，經常會生成更不易分解且具環境賀爾蒙效力的代謝產物如烷基苯酚，與帶1-2單位之烷基苯酚聚乙氧基醇，對於人體健康與環境生態具危害性。鑑於目前關於烷基苯酚聚乙氧基醇與烷基苯酚之分解菌株的研究仍非常有限，其生物分解途徑的探討更缺乏，因此，本研究之目的為以烷基苯酚及烷基鄰苯二酚為唯一生長碳源，藉由強化培養法篩選對於烷基苯酚聚乙氧基醇與烷基苯酚同時具有分解能力的台灣源菌株，並就利用液相層析質譜儀鑑定其代謝產物，同時推測其可能的代謝途徑。
本研究共篩選出28株能以烷基苯酚或烷基鄰苯二酚為唯一生長碳源之革蘭氏陰性菌，經BioLog碳源利用指紋法、脂肪酸圖譜及16S rDNA序列分析等三種鑑定方法發現，在所篩選的菌種中，屬革蘭氏陰性菌之Pseudomonas屬佔20株，其餘菌株包括Alcaligenes defragrans、Bordetella brochiseptica、Zoogloea ramigera及Inquilinus limosus。其中，菌株Pseudomonas putida TX2不僅能以寬廣濃度範圍的烷基苯酚聚氧乙基醇(0.05-20%)為唯一碳源生長，在最適濃度下之比生長率為0.5 h-1，亦能分解烷基苯酚，其培養濃度在0.02%以下時的菌株比生長率為0.47 d-1，故本研究續針對此一具有特殊活性之菌株進行探討。研究顯示，當菌株TX2以0.5 %辛基苯酚聚氧乙基醇為唯一碳源培養後，分別以0.05%辛基苯酚聚氧乙基醇、辛基苯酚及辛基鄰苯二酚為催化基質，在以菌液濃度OD600=0.3、溫度30˚C中，菌株TX2分別會表現192.6、4.2 及4.2 nmole/min之耗氧率，此表示菌株P. putida TX2具有特殊的耗氧酵素，對上述化合物具不同耗氧活性，故推測於好氧的環境中，此菌株不僅可分解辛基苯酚聚氧乙基醇，對於可能產生具雌激素活性之代謝產物如辛基苯酚，亦具有轉化成辛基鄰苯二酚並進一步予以開環的能力。此外，烷基苯酚與烷基鄰苯二酚之烷基鏈碳數的大小對於菌株TX2所產生的耗氧率會隨烷基鏈碳數減少而提高，然而若是苯環上完全不具烷基鏈時，則產生的耗氧率會大幅降低。本研究另利用西方墨點法發現，當菌株TX2分別以辛基苯酚聚氧乙基醇、辛基苯酚及辛基鄰苯二酚為唯一碳源培養時，均能以P. putida mt-2之鄰苯二酚2, 3-加氧酵素所製備之抗體辨識。而在初步酵素純化方面，可在尚未完全純化之蛋白質中，發現一分子量約為37 kDa之蛋白質，與西方墨點法所偵測到之開環酵素分子量接近，推測為TX2菌株之中對辛基鄰苯二酚開環的酵素。因此綜合上述實驗及液相層析質譜儀分析代謝產物之結果顯示，P. putida TX2會先經由逐一切斷聚氧乙基鏈的作用，將辛基苯酚聚氧乙基醇分解為辛基苯酚，同時累積許多烷基苯酚聚氧乙基酸等副產物。辛基苯酚再進一步藉由加氧作用轉化為辛基鄰苯二酚，然後可能先分解辛基鄰苯二酚之烷基鏈後，再經由meta-cleavage途徑予以開環，而破壞雌激素活性。

Alkylphenol polyethoxylates (APEOn), one kind of non-ionic surfactants, was extensively used in the industrial, agricultural and household activities. These compounds were frequently discharged into natural environment and transformed to alkylphenol (AP) and APEOn (n=1-2), which was more recalcitrant and has been demonstrated as the environmental hormone with estrogenic-like activity to aquatic organisms, wildlife and humans. Only a few of microorganism that can degrade alkylphenol polyethoxylates or alkylphenol were isolated. The biodegradation pathway of these compounds still remains unproven in previous study. The objectives of this study were aimed to isolate and characterize novel bacterial isolates able to grow on both APEOn and AP in order to elucidate the mechanism of disruption of estrogenic-like metabolites.
In this study, 28 bacterial strains are found to be able to degrade alkylphenol and/or from different topsoil samples. All of these isolates were Gram-negative bacteria. They were identified by three methods, Biolog breathprinting, fatty acid fingerprinting and 16S rDNA sequence analysis. 64% of them was belonged to Pseudomonas genus. Other species included Alcaligenes defragrans, Bordetella brochiseptica, Zoogloea ramigera and Inquilinus limosus. Among them, a strain Pseudomonas putida TX2 was shown to have a novel activity to grow on alkylphenol polyethoxylates (0.05~20%) or alkyphenol (<0.02%) as the sole carbon source, which is the first pure culture showing the feature. The strain TX2 reveals an oxygen uptake activity of 192.6, 4.2 and 4.2 nmole/min for a 5 ml cell suspension at OD600 = 0.3 using octylphenol polyethoxylates, octylphenol and octylcatechol as substrates, respectively. Moreover, the oxygen uptake rate of strain TX2 decreased with the increase of carbon numbers in alkyl-chain in bulk alkylphenol and alkylcatechol. In addition, Western blotting further demonstrated an catechol 2, 3-dioxygenase-like aromatic ring-cleavage enzyme that was inducible when strain TX2 was grown on octylphenol polyethoxylates, octylphenol, or octylcatechol as the sole carbon source. Furthermore, LC/MS analysis of the TX2 transformed metabolites from OPEOn and AEO8 showed that octylphenol polyethoxylates could be degraded as a sequential cleavage of the polyethoxylate chain and then produced octylphenol, which was further transformed to form octylcatechol followed by generating a metabolite with a molecular weight of 206. It was suggested as a aromatic ring-cleavage product. These results suggested that strain TX2 was able to shorten the alky chain followed by the cleavage of aromatic ring in the degradation of octylcatechol.

目錄…………………………………………………………………………….3
表目錄………………………………………………………………………….5
圖目錄………………………………………………………………………….6
名詞縮寫對照表……………………………………………………………….8
摘要…………………………………………………………………………….9
英文摘要……………………………………………………………………...11
第一章、前言…………………………………………………………………13
第二章、材料與方法…………………………………………………………25
2-1分解菌之篩選、分離與鑑定………….……………………………25
2-2微生物生長特性……………………………….……………………32
2-3菌體之耗氧活性………………………………….…………………32
2-4十二烷硫酸鈉-聚丙烯醯胺膠體電泳………………………………33
2-5西方墨點法………………….………………………………………34
2-6代謝產物鑑定………………………………………….……………35
2-7酵素純化……………………………………………….……………36
2-8蛋白質濃度測定……………………………………….……………40
2-9化學藥品……………..……………………………………………...40
第三章、結果…………………………………………………………………40
3-1分解菌之篩選、分離與鑑定………………………………………..40
3-2分解菌生長特性…………………………………..………………...45
3-3菌體之耗氧活性…………………………………………………….47
3-4 P. putida TX2開環酵素之純化與定性……………………………..50
3-5代謝產物鑑定……………………………………………………….51
3-6酵素純化…………………………………………………………….54
第四章、討論…………………………………………………………………56
4-1分解菌之篩選、分離與鑑定………………………………………..56
4-2分解菌之碳源利用特性…………………………………………….60
4-3菌體之耗氧活性…………………………………………………….61
4-4代謝產物鑑定……………………………………………………….63
4-5酵素純化…………………………………………………………….65
4-6 Pseudomonas putida TX2 降解OPEOn之可能機制………………67
第五章、結論與建議…………………………………………………………69
第六章、參考文獻……………………………………………………………71
第七章、表……………………………………………………………………80
第八章、圖……………………………………………………………………94
第九章、附錄………………………………………………………………...123

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