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研究生:蔡青宴
研究生(外文):Ching Yen Tsai
論文名稱:以體學方法研究去硝化菌對固醇類物質的無氧代謝
論文名稱(外文):OMIC studies on the anoxic steroid metabolism by Steroidobacter denitrificans
指導教授:江殷儒
指導教授(外文):Y. R. Chiang
學位類別:碩士
校院名稱:長庚大學
系所名稱:中醫學系天然藥物
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
論文頁數:114
中文關鍵詞:基因體學蛋白質體學代謝體學基因定序去硝化菌環裂解固醇類物質厭氧代謝
外文關鍵詞:genomicsproteomicsmetabolomicsDNA sequencingSteroidobacter denitrificansring cleavagesteroidanoxic degradation
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自然界中存在許多豐富之固醇類物質。此類物質及其衍生物主要為真核生物所合成,調控許多重要之生理活動。許多微生物對固醇類物質具有生物轉換甚至完全降解的能力,因此本研究具有環境生物技術以及製藥方面之潛在應用價值。微生物在有氧環境下,參與固醇類物質降解之相關酵素已經被純化且定性。其反應之關鍵酵素為氧化酶,此酵素之特性為絕對需要氧氣的參與。然而,微生物在厭氧環境下,參與雌二醇以及睪固酮降解之酵素資訊卻付之闕如。本研究之模式生物為去硝化菌 Steroidobacter denitrificans。其可在厭氧環境下,以硝酸作為電子接受者,並利用雌二醇或睪固酮作為單一碳源及能量來源。本研究亦用碳-13標定之睪固酮進行胞內試驗,隨後利用代謝質體學之相關分析技術,分離並鑑定出一系列經過碳-13標定的睪固酮降解之相關衍生物。進一步利用核磁共振儀及質譜儀鑑定純化後之固醇類相關代謝物。此外,本篇論文在蛋白質定序初步分析結果中,發現數個可能和固醇類代謝相關之蛋白質。本論文進一步結合基因體、蛋白質體以及代謝體學之研究,針對微生物在厭氧環境下降解固醇類物質做更深入之探討,使其代謝路徑更為完整。
Steroids are ubiquitous and abundant compounds in nature. Steroids are produced by eukaryotes where they have a variety of chemical structures and play important physiological roles. Many bacteria are capable of transforming and completely degrading steroids under oxic conditions. The microbial metabolism of steroids has gained considerable interest due to its potential applications in industrial and environmental biotechnology. The oxic degradation pathways of steroids by aerobic bacteria were established, and some of the involved enzymes were well characterized. The key players in these pathways are oxygenases which utilize dioxygen as a co-substrate. Steroidobacter denitrificans able to grow anaerobically on testosterone or estradiol was adopted as the model organism in this study. Our investigations revealed unique and interesting biochemical reactions and enzymes. We added [2,3,4-13C3]testosterone as the tracer in in vivo assays to explore the testosterone-derived intermediates. Steroid products purified from the in vivo assays were then identified using NMR spectroscopy and UPLC-Mass spectrometry. In this investigation, we applied OMICs approachs to identify the genes、enzymes and intermediates involved in the anoxic testosterone catabolism. According to our present data, a novel testosterone catabolic pathway is proposed.
指導教授推薦書..................................................................................................................I
論文口試委員會審定書.....................................................................................................II
長庚大學授權書...............................................................................................................III
國家圖書館授權書............................................................................................................IV
誌謝................................................................................................................................... V
摘要....................................................................................................................................VI
Abstract ..........................................................................................................................VII
總目錄.............................................................................................................................VIII
附圖、表目錄.................................................................................................................... XII
專有名詞縮寫對照表................................................................................................... XVII

一、 緒論....................................................................................................................P. 1
1.1 固醇類物質之概述.....................................................................................P. 1
1.2 固醇類物質在動物體內之生合成途徑.....................................................P. 4
1.3 微生物對固醇類物質之有氧代謝.............................................................P. 6
1.4 微生物之無氧代謝.....................................................................................P. 8
1.5 去硝化菌 Steroidobacter denitrificans FST (=DSMZ18526T=JCM14622T)
之介紹......................................................................................................P. 12
1.6 固醇類物質對於環境的影響...................................................................P. 13
1.7 固醇類物質之生物轉換研究的應用價值...............................................P. 15
1.8 體學 (OMICs)之概念以及研究目標.....................................................P. 16
1.9 DNA定序 (sequencing)..........................................................................P. 18

二、 材料與方法......................................................................................................P. 21
2.1 實驗材料...................................................................................................P. 21
2.1.1 菌株..................................................................................................P. 21
2.1.2 培養 Steroidobacter denitrificans (DSMZ 18526) 之培養基
..........................................................................................................P. 21
2.1.3 本研究所使用之有機碳源..............................................................P. 24
2.1.4 其它重要藥品..................................................................................P. 25
2.1.5 有機溶劑..........................................................................................P. 26
2.1.6 重要儀器..........................................................................................P. 26
2.1.7 重要分析材料..................................................................................P. 26
2.2 實驗方法...................................................................................................P. 27
2.2.1 去硝化菌 Steroidobacter denitrificans 之厭氧培養....................P. 27
2.2.2 去硝化菌 Steroidobacter denitrificans 之生長測定....................P. 27
2.2.3 總蛋白質沉澱之測定......................................................................P. 28
2.2.4 固醇類物質之萃取及定量分析......................................................P. 28
2.2.5 細菌蛋白質之製備..........................................................................P. 28
2.2.6 胞外生物轉換試驗 (in vitro biotransformation assays)..............P. 29
2.2.7 薄層層析法......................................................................................P. 29
2.2.8 二維膠體電泳分析..........................................................................P. 29
2.2.9 膠體之染色 (Coomassie Blue R250 染色法)...............................P. 33
2.2.10 去硝化菌 Steroidobacter denitrificans genomic DNA之製備.....P. 33
2.2.11 胞內睪固酮降解試驗......................................................................P. 34
2.2.12 胞外睪固酮降解試驗......................................................................P. 35
2.2.13 高效液態層析法..............................................................................P. 35
2.2.14質譜儀分析法....................................................................................P. 36
2.2.15 核磁共振儀分析法..........................................................................P. 36
2.2.16 紅外線光譜儀..................................................................................P. 37
三、 結果與討論..................................................................................................P. 38
3.1 Steroidobacter denitrificans (DSMZ18526) 菌在無氧條件下依序使用 同之固醇類受質之生長測定..................................................................P. 38
3.2 二維膠體電泳分析:S. denitrificans 生長於不同之固醇類受質所獲得之可溶性蛋白質的比較..............................................................................P. 40
3.3 胞外生物轉換試驗 (in vitro biotransformation assays) .....................P. 42
3.3.1細菌生長於睪固酮下所得到的可溶性蛋白質,進行雌二醇的生物轉
換試驗.................................................................................................P. 42
3.3.2以細菌生長於雌酮下所得到的可溶性蛋白質,進行四種雄激素的生物轉換試驗.............................................................................................P. 44
3.3.3胞外雌激素之生物轉換試驗一 (使用生長於雌酮的S. denitrificans細
胞) .......................................................................................................P. 48
3.3.3.1不同電子接受者...........................................................................P. 48
3.3.3.2不同 pH值...................................................................................P. 50
3.3.4 胞外雌激素之生物轉換試驗二 (使用生長於雌酮的S. denitrificans
細胞).................................................................................................P. 52
3.3.5 二維膠體電泳之初步分析..............................................................P. 54
3.3.6 胞內睪固酮降解試驗......................................................................P. 62
3.3.7 胞內試驗 (in vivo assay)產生之中間代謝物的結構分析............P. 66
3.3.8 胞外生物轉換試驗得到之環裂解代謝物 (Ring Cleavage).........P. 72
3.3.9 S. denitrificans於不同受質或環境下培養,進行雄激素的胞內和胞
外之生物轉換試驗 (in vivo and in vitro assay) .......................................P. 78
3.3.9.1 胞內生物轉換試驗.....................................................................P. 78
3.3.9.2 胞外生物轉換試驗.....................................................................P. 81
3.3.10 Steroidobacter denitrificans基因定序之結果.................................P. 83
3.4 討論與結論.............................................................................................P. 88

四、 參考文獻........................................................................................................P. 95

附圖、表目錄

圖 1. 膽固醇之化學結構.....................................................................................P. 1
圖 2. Mevalonate之形成過程 (Eisenreich et al., 2004)...................................P. 2
圖 3. Isopentenyl pyrophosphate和 dimethylallyl pyroph-osphate之間的相互
轉換 (Stryer et al., 2006)..........................................................................P. 2
圖 4. Squalene轉變成 lanosterol之過程 (Lamb et al., 2007).......................P. 3
圖 5. Lanosterol轉換形成膽固醇的簡化過程 (Stryer et al., 2006)...............P. 4
圖 6. 各類固醇類荷爾蒙之生合成途徑 (Bell et al., 2008) ..............................P. 5
圖 7. 雄激素 (androgens)和雌激素 (estrogens)之形成路徑
(Stryer et al., 2006) ...................................................................................P. 6
圖 8. 以細菌 (Comamonas testosterone)為模式生物對於睪固酮之有氧降解路徑 (Horinouchi et al., 2003) …................................................………….P. 7
圖9. S. denitrificans在厭氧環境下代謝膽固醇之初步反應 (Chiang et al.,
2007)...........................................................................................................P. 9
圖10. 睪固酮之化學結構 (testosterone)..........................................................P. 10
圖11. Steroidobacter denitrificans (DSMZ18526) 對睪固酮之無氧代謝預期的反應路徑 (Leu et a l., 2011)...................................................................P. 11
圖12. 環境中氮循環之簡圖 (Bauman, 2004) ................................................P. 13
圖13. (A)雌二醇 (Estradiol﹐E2)及 (B)雌酮 (Estrone﹐E1)的化學結構
..................................................................................................................P. 14
圖14. (A)經由一支真菌菌株 (Rhizupos nigricans)來催化孕酮的 (proges-
terone)碳-11位置之氫氧化作用 (Madigan et al., 2009)。 (B)利用放射
菌 (actinomycete)之 P450為主的一系列酵素將 vitamin D3轉換成 1α,25-dihydroxyvitamin D3 (Sakaki et al., 2011).................................P. 16
圖15. 基因體、蛋白質體和代謝體之連結 (Mochida et al., 2011)..................P. 17
圖16. 第一代 Sanger DNA定序和雙去氧核苷酸 ddNTP (dideoxyribonucleot-
idetriphosphate) (Sanger et al., 1977)...................................................P. 19
圖17. 次世代定序技術- Illumina定序之簡單流程說明(Mardis, 2008).........P. 20
圖18. 先加入 1 mM雌酮,再加入 1 mM睪固酮之細菌生長 (以菌液中的總蛋白質表示)及兩種固醇類受質的消耗形..................................................P. 39
圖19. 先加入 1 mM雌酮,再加入 1 mM睪固酮之細菌生長 (以總蛋白質濃度表示)及兩種固醇類受質的消耗情形……..............................................P. 39
圖20. 利用二維電泳分析觀察 S. denitrificans在無氧條件下,分別培養於 (A)睪固酮及 (B)雌酮之可溶性蛋白質的表現............................................P. 41
圖21. 由睪固酮經由無氧培養所得之酵素,胞外雌酮之無氧生物轉換試驗
..................................................................................................................P. 43
圖22. 經由雌酮無氧培養後,得到水溶性蛋白質,做胞外雄激素之生物轉換試驗...............................................................................................................P. 46
圖23. 預測雌二醇氧化成雌酮後,再經過一系列的未知反應後,轉換成雄激素之相關產物...............................................................................................P. 47
圖24. 以雌二醇和雌酮做為受質,分別以不同之電子接受者進行胞外生物轉換試驗,以 254 nm的紫外光以及硫酸呈色法偵測固醇類物質的存在位置...............................................................................................................P. 49
圖25. 以雌二醇和雌酮做為受質,不同 pH值之胞外生物轉換試驗.............P. 51
圖26. 以水溶性蛋白質作用,並加入 carbinate、CoA及 ATP參與反應之胞外生物轉換試驗.......................................................................................P. 53
圖27. 二維膠體電泳分析 S. denitrificans在無氧條件下,以睪固酮為碳源並選擇 87個未知蛋白質點做質譜儀分析.....................................................P. 54
圖28. 檸檬酸循環之初步反應,其作用酵素為 Aconitate hydratas…….......P. 55
圖29. 依據積分值以及蛋白質之功能所做成的分類統計圖.........................,.P. 61
圖30. 胞內試驗之粗萃取物經超高效液相層析儀-高解析質譜儀 (UPLC-
HRMS)分析所得之層析譜......................................................................P. 62
圖31. 胞內試驗之粗萃取物經超高效液相層析儀-質譜儀 (UPLC-Mass)分析所得之圖譜...................................................................................................P. 65
圖32. 經由胞內試驗得到之產物TCY1-6.........................................................P. 67
圖33. 經由胞內試驗得到之產物 TCY3...........................................................P. 69
圖34. 經由胞內試驗得到之產物 TCY4...........................................................P. 71
圖35. 環裂解代謝物 (RC)之結構分析結果....................................................P. 75
圖36. 經由胞內試驗發現之睪固酮的衍生物質..............................................P. 77
圖37. (A)穀安酸鹽 (glutamate)之化學結構 (B)庚酸 (heptanoic acid)之化學
結構。........................................................................................................P. 78
圖38. S. denitrificans於不同受質或環境下培養,續以睪固酮作為受質之厭氧
小規模試驗..............................................................................................P. 80
圖39. S. denitrificans於不同受質或環境下培養,續以睪固酮作為受質之胞外
生物轉換試驗..........................................................................................P. 82
圖40. 簡單說明組裝關係流程圖......................................................................P. 84
圖41. 將 S. denitrificans之 contig序列做簡單的功能分類..........................P. 87
圖42. β-sitosterol之後續代謝步驟,為解環化物 (RC)碳支鏈切除參考路徑(Szentirmai, 1990)...................................................................................................P. 89
圖43. 膽固醇與睪固酮之無氧降解路徑之比較..............................................P. 90

(附件一) ..................................................................................................................P. 91
(附件二) ..................................................................................................................P. 92
(附件三) ..................................................................................................................P. 93
(附件四) ..................................................................................................................P. 94
表1. 基礎培養基 (1 L)..........................................................................................P. 21
表2. 微量元素溶液 SL-10 (1 L).........................................................................P. 22
表3. 硒和鎢溶液 (1 L)..........................................................................................P. 22
表4. 綜合維他命溶液 (1 L)..................................................................................P. 23
表5. 其它溶液.........................................................................................................P. 23
表6. 水合溶液 (Rehydration buffer)成分 (50 ml).............................................P. 30
表7. 平衡溶液 (Equilibration buffer)成分 (最終體積 100ml)........................P. 31
表8. 12.5 % SDS-PAGE膠體溶液 (Separating gel: pH 8.8,可供製備 6片 18
cm × 18 cm膠體) .......................................................................................P.31
表9. 5 % SDS-PAGE膠體溶液 (Stacking gel: pH 6.8﹐可供製備 6片 18 cm ×
18 cm膠體)..................................................................................................P. 32
表10. 電泳緩衝溶液之配製 (Running buffer﹐ 10 L ).......................................P. 32
表11. 雌二醇之胞外生物轉換試驗標示...............................................................P. 43
表12. 胞外雄激素之生物轉換試驗標示...............................................................P. 46
表13. 各種電子接受者進行胞外生物轉換試驗標示.......................................... P. 49
表14. 不同 pH值之胞外生物轉換試驗標示.......................................................P. 51
表15. 添加 carbinate、 CoA及 ATP參與反應之胞外生物轉換試驗標示......P. 53
表16. 二維膠體電泳-蛋白質相關資訊資料庫.....................................................P. 57
表17. 環裂解代謝物 (Ring Cleavage)之碳譜訊號整理表.................................P. 76
表18. 續以睪固酮作為受質之胞外生物轉換試驗標示.......................................P. 82
表19. S. denitrificans與基因資料庫做固醇類相關蛋白質之功能比對..............P. 84




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