博碩士論文 90224010 詳細資訊




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姓名 賴建宏(Jeff Lai)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 Pseudomonas nitroreducens TX1之具耗氧活性之麩胺酸合成酶之單離
(Purification of oxygen consumptive glutamate synthase from Pseudomonas nitroreducens TX1)
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摘要(中) 烷基苯酚聚氧乙基醇為最現今最為廣用的非離子性界面活性劑。由烷基鏈、酚、及聚氧乙烯鏈(ethoxylate chain)之結構組成。本論文探討可有效生長於0.05-20% 辛基苯酚聚氧乙基醇(octylphenol polyethoxylate,簡稱OPEOn)之分解菌(Pseudomonas nitroreducens TX1)其分解環境污染物之產物的分析方法,並推測其可能之代謝產物。另一方面,本實驗室先前研究顯示P. nitroreducens TX1細胞在以OPEOn為唯一生長碳源,會誘發對OPEOn具專一性之耗氧活性,而由succinate為唯一碳源生長之菌體則無此現象。故推測耗氧活性與此化合物的代謝有關,便以耗氧活性作為追蹤OPEOn代謝酵素,而分離出glutamate synthase。
在OPEOn代謝產物分析上,高效能液相層析質譜儀(HPLC-MS) 分析P. nitroreducens TX1細胞粗萃液及續經三個純化步驟(DEAE-Sepharose、Phenyl-Sepharose及Mono Q管柱)之部分純化的酵素,其轉化OPEOn及dodecyl octylethoxylate (AEO8)之產物發現兩種受質皆有長鏈聚氧乙烯鏈減短之產物,如AEO7及OPEOn(n=3-12)生成,同時發現末端醇官能基被羧化之產物,如octylphenol polyethoxycarboxylates (OPECn,n=5-9)。而由氣相層析質譜儀(GC-MS)所建立的分析方法中,發現產物中有OPEO2及結構特徵含有聚氧乙烯醇鏈被羧化及末端為醇官能基的產物,目前尚無法確認此分子完整結構。綜合上述結果初步推測P. nitroreducens TX1轉化OPEOn和AEO8,會在聚氧乙烯鏈之終端醇行羧化與醚鏈斷裂,進而造成OPEOn上之聚氧乙烯鏈的減短。
在酵素純化上第一步採用陰離子交換樹脂管柱(DEAE-Sepharose),此步驟可分離出兩個可耗氧之活性區,一個不會與此管柱結合,另一個會在0.2-0.4 M KCl鹽梯度沖提出,而兩者各約占細胞粗萃液50%活性。後者續經Phenyl-Sepharose管柱、 Mono Q管柱、hydroxyapatite管柱和Mono Q管柱分離可得到純的酵素。利用電噴灑游離串聯式質譜儀得到以胰蛋白酶水解之多個胜肽序列,資料庫(Mascot Search)比對其序列皆與P. aeruginosa之麩胺酸合成酶(glutamate synthase)最相近。由膠體過濾管柱定其分子量為190±10 kDa,聚丙烯醯胺膠電泳(SDS-PAGE) 分析有兩個次分子,其分子量分別為144±6 kDa及54±4 kDa,故其組成為???1。經由200-650 nm波長掃描測得純化的酵素含有黃素(FMN 或 FAD)在其中,且為氧化態型式。以LC-MS分析純化的glutamate synthase轉化OPEOn 1及3天,並無EO鏈減短的產物。同時回顧文獻已知的酵素功能,OPEOn應不為此酵素之受質,但是本研究所得的glutamate synthase,在含0.05% OPEOn和3 mM 混合金屬的40 mM鉀磷酸緩衝液(pH 7.0)中,於37℃中,加入0.5 mM NADPH後會有205 nmole/min/mg之耗氧比活性,當單獨外加金屬二價錳或銅,則比活性相較於無外加金屬之對照組提高2-3倍,為首次報導此酵素具耗氧特性之研究,其與OPEOn代謝的關聯值得未來深入探討。而細胞粗萃液經由DEAE-Sepharose、Phenyl Sepharose及Mono Q 三種管柱部分純化自P. nitroreducens TX1之酵素具分解OPEOn能力,主要由7個蛋白質組成,但繼續純化後則失去分解活性。其中ornithine carbamoyltransferase為本實驗室以蛋白質體學方法發現可被OPEOn up-regulated蛋白質之一。Glutamate synthase 則未在up-regulated 的蛋白質中發現。
摘要(英) Alkylphenol polyetholates (APEOn) are nonionic surfactants that are widely used today. They are composed of a polyethoxy chain and an alkyl chain connected to a phenyl structure at para-position. In this study, an analysis method to detect microbial biodegradation products from APEOn was established. In our previous study, Pseudomonas nitroreducens TX1 can grow on octylphenol polyethoxylates (OPEOn). The strain showed OPEOn-dependent oxygen consumption activity induced when it was grown on OPEOn as sole carbon source. But the activity was not found when grown on 0.5% succinate, suggesting oxygen uptake activity is involved in the metabolism of OPEOn. By using the oxygen uptake assay, glutamate synthase was purified from this bacterial strain.
In the analysis of OPEOn metabolic products, the transformed products from two substrates (OPEOn and dodecyl octylethoxylate(AEO8)) by crude extract or partially purified enzyme were analyzed by HPLC-MS. The ethoxylate chain was shortened and AEO7 formed by crude extract, while OPEOn with shortened ethoxylate chain (n=3-12) and octylphenol polyethoxycarboxylate (OPECn, n=5-9) were the transformation products by partially purified enzyme. By gas chromatography-mass spectrometry (GC-MS), metabolites such as OPEO2, some products with the structure of polyethoxylate, and carboxylated polyethoxylate were formed by crude extract. P. nitroreducens TX1 may undergo carboxylation of terminal ethoxylate alcohol and ether chain cleavage to metabolize OPEOn and AEO8.
In the purification of enzyme related to the metabolism of OPEOn, two oxygen consumption activity areas from DEAE-Sepharose chromatography of the crude extract of strain TX1 were detected; one that could not bind to the column and the other that was eluted by 0.2-0.4 M KCl. About equal activity were recovered from this step. The second pool was further purified by Phenyl-Sepharose, Mono-Q, hydroxyapatite, and second Mono-Q chromatography. The purified enzyme was identified as glutamate synthase by peptide finger-printing from ESI-MS/MS. A molecular mass of native protein was estimated as 190 ±10 kDa by gel filtration with two subunits of 144 ±6 and 54 ±4 kDa MW respectively from SDS-PAGE. The result indicates that the enzyme was a heterodimer (?1?1). The absorption spectrum at 200-650 nm showed that the pure enzyme contains flavin (FAD or FMN) in oxidized state. However, the transformation products by the purified glutamate synthase revealed no ethoxylate chain shortage. Based on the studies by other groups, OPEOn is very unlikely to be the substrate of the enzyme. In our study, the enzyme showed oxygen uptake activity in 40 mM potassium phosphate buffer, pH 7.0, containing 0.05% OPEOn, 3 mM metal mixture, and 0.5 mM NADPH. The specific activity of oxygen uptake was 205 nmole/min/mg. When the metal mixture was replaced by either 3 mM Mn2+ or Cu2+ ion, the specific activity was increased 2-3 fold compared to that with no metal control. This is the first report of oxygen uptake activity for glutamate synthase. The role of the enzyme in OPEOn metabolism and oxygen uptake activity from OPEOn-grown bacterium needs to be further investigated.
The partial purified enzyme that showed degraded products from OPEOn (short chain OPEOn and OPECn) was purified from P. nitroreducens TX1 using DEAE-Sepharose, Phenyl-Sepharose, and Mono Q chromatography. They were composed of seven major proteins. However, after further purification, the OPEOn degrading activity was lost from the next chromatography. One of the seven proteins, ornithine carbamoyltransferase, was found to be up-regulated by OPEOn from our proteomic study. However, glutamate synthase was not found to be up-regulated.
關鍵字(中) ★ 氣相層析質譜儀
★ 麩氨酸合成酶
★ 烷基苯酚聚氧乙基醇
★ 辛基苯酚聚氧乙基醇
★ 純化
★ 液相層析質譜儀
關鍵字(英) ★ octylphenol polyethoxylate
★ purificaiton
★ glutamate synthase
★ alkylphenol polyethoxyalte
★ GC-MS
★ LC-MS
論文目次 中文摘要 I
英文摘要 III
目錄 VI
表目錄 X
圖目錄 XI
名詞縮寫對照表 XV
壹、緒論 1
一、非離子性界面活性劑 1
二、辛基苯酚聚氧乙基醇之簡介 2
三、已知的Alkylphenol polyethoxylate去除法 4
四、APEOn代謝之微生物性醚鍵斷裂可能途徑 4
五、APEOs及其代謝物之分析方法 7
六、研究背景與大綱 14
貳、材料與方法 18
一、細菌培養與培養基 18
二、微生物醱酵槽培養 18
三、酵素活性分析 20
四、酵素純化 21
五、聚丙烯醯胺膠之蛋白質分離法 25
六、Glutamate synthase 活性偵測方法 27
七、高效能液相層析質譜儀分析 27
八、氣相層析質譜儀分析法 28
九、化學藥品與儀器設備 31
參、結果 33
一、耗氧活性偵測之最適化 33
二、鯡魚精蛋白處理的TX1細胞粗萃液在4℃及-20℃的耗氧活性穩定性測試 33
三、TX1細胞粗萃液通過DEAE-Sepharose管柱分離之結果 34
四、具耗氧性酵素glutamate synthase之純化 34
五、具耗氧性的glutamate synthase酵素定性 37
六、輔以質譜儀分析TX1胞內酵素轉化AEO8之速率試圖再純化OPEOn相關於OPEOn分解酵素之純化結果 37
七、分別以0.5%的succinate及OPEOn為唯一碳源培養出來的TX1細胞對0.05%及1% OPEOn之耗氧活性 38
八、關於OPEOn可能之分解酵素 39
九、高效能液相層析質譜儀分析OPEOn/AEO8及其代謝產物 39
十、GC-MS分析OPEOn/AEO8及其代謝產物 40
肆、討論 42
一、酵素耗氧活性最適化 42
二、鯡魚精蛋白處理的P. nitroreducens TX1細胞粗萃液在4℃及-20℃的耗氧活性穩定性測試 43
三、P. nitroreducens TX1細胞粗萃液通過DEAE-Sepharose管柱分離之結果討論 44
四、利用耗氧活性追蹤而純化到的酵素_glutamate synthase 45
五、具耗氧性的glutamate synthase酵素定性 47
六、輔以質譜儀追蹤OPEOn分解速度而純化OPEOn相關分解酵素 48
七、分別以0.5%的succinate及OPEOn為唯一碳源培養出來的TX1細胞對0.05%及1% OPEOn之耗氧活性 49
八、關於OPEOn分解酵素 50
九、LC-MS分析OPEOn降解產物結果討論 52
十、GC-MS分析方法建立之討論 52
十一、關於OPEOn分解酵素可能之異化機制 55
伍、結論與建議 56
一、結論 56
二、建議 58
陸、參考文獻 62
表 67
圖 80
附錄 115
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指導教授 黃雪莉(Shir-Ly Huang) 審核日期 2004-9-29
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