Pseudomonas nitroreducens TX1 異化辛基苯酚聚氧乙基醇之功能性蛋白質體學：以二維電泳法分析等電點4-8之蛋白質表現

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徐秉正(Ben-Cheng Hsu) 查詢紙本館藏

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論文名稱

Pseudomonas nitroreducens TX1 異化辛基苯酚聚氧乙基醇之功能性蛋白質體學：以二維電泳法分析等電點4-8之蛋白質表現
(Degradation of octylphenol polyethoxylate by Pseudomonas nitroreducens TX1: A functional proteomics study by two-dimensional gel electrophoresis (pI 4-8))

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摘要(中)

烷基苯酚聚氧乙基醇 (APEOs) 為非離子性界面活性劑界面活性劑，應用於家庭及工業用途極廣。其中Triton X-100屬於octylphenol polyethoxylate (OPEOn) 的，其平均值n為9.5，而其中間代謝物如OPEOn (n=1~3)　或是octylphenol，具環境賀爾蒙活性，已證實會累積在環境中並危害人體健康。本研究室先前自中央大學宿舍污水排放口之底泥中，篩選出可快速利用Triton X-100為唯一碳源而生長之分解菌株Pseudomonas nitroreducens TX1。本研究乃利用二維蛋白質電泳法 (pI範圍為4-8) 分析Pseudomonas nitroreducens TX1生長在0.5%或20% Triton X-100為唯一碳源之下所產生之蛋白質體，分析表現量增加強與抑制之一系列蛋白質並探討其代謝反應。研究結果所示，當Pseudomonas nitroreducens TX1生長於含0.5% succinate為唯一碳源的minimal salts basal (MSB) 培養基為對照組時，在等電點pI 4-8之間呈現的蛋白質點平均有600個；在以0.5%的Triton X-100為唯一碳源時，則可分離平均500蛋白質點：而在以20%的Triton X-100為唯一碳源時，可分辨到約600個蛋白質點。續以介質輔助雷射離子化飛行時間串聯四極質譜儀 (MALDI-Q-TOF) 鑑定Pseudomonas nitroreducens TX1的蛋白質體發現，於0.5% Triton X-100生長環境中計有17個蛋白質表現增加、11個蛋白質表現降低。其中，表現增加的蛋白質有6個屬於ABC (ATP binding cassette transporter) 運輸蛋白質，其中包括運輸支鏈胺基酸與運輸極性胺基酸兩大類，推測其中屬Leu/ Ile/ Val-binding protein family的ABC 運輸蛋白質可能與Triton X-100的輸入有關。在抗逆境反應方面，發現此菌增加表現了GroEL、ClpB、IbpA等維持蛋白質構型的抗逆境蛋白質。同時亦發現具有還原烷基過氧化物能力的AhpC蛋白質，此為第一次在界面活性壓力中被發現。加上抗鍗蛋白TerD、TerE表現量的增加，推測Triton X-100對菌體所造成的逆境與過氧化物造成的逆境相近。在滲透壓調節方面，本研究發現鉀離子的運輸蛋白KdpD與KdpE表現增加，而與滲透壓有關的調節蛋白OmpR (藉由OmpC、OmpF調整滲透壓) 則被抑制，推測此菌可能存在與Triton X-100相關的滲透壓調節系統。在能量的生成方面，dihydrolipoamide dehydrogenase在Triton X-100逆境下表現增加，此酵素參與pyruvate形成acetyl-CoA的反應，所生成acetyl-CoA進入三羧酸循環並產生能量。另一方面，其他如酯肪酸生合成蛋白質、DNA的合成蛋白質、胺基酸合成相關蛋白質則表現被抑制。本研究進一步探討此菌生長於20% Triton X-100環境中的蛋白質體，相對於0.5% Triton X-100的蛋白質體發現，ATP生合成酶、鞭毛蛋白、與酯肪酸的生合成相關蛋白等細胞周邊蛋白質的表現有增加的現象。而關於Triton X-100的代謝方面，由於乙醛酸循環中的重要酵素malate synthetase被抑制，則推測此菌切斷Triton X-100聚氧乙基醇鏈之代謝產物，以乙醛酸進入三羧酸循環的假設之可能性較低，然而已有研究發現乙醛會抑制PhoP蛋白質的表現，而本實驗亦另外觀察到PhoP蛋白質的表現被抑制，故聚氧乙基醇鏈形成乙醛而進一步被利用的可能性則較高。

摘要(英)

Alkylphenol polyethoxylates (APEOs) are non-ionic surfactants that have been used in the household and as industrial detergents for more than 40 years. Triton X-100 belongs to the octylphenol polyethoxylates, and their metabolism intermediates such as octylphenol and octyl phenol monoethoxylate have been proved the environmental hormone. A bacterial strain, Pseudomonas nitroreducens TX1 was previously isolated from the sediments of drainage near the dormitory of National Central University in Taiwan. The bacterial strain is able to use Triton X-100 as the sole carbon and energy source to grow. This study was aimed to use 2D gel electrophoresis-based proteomic approach to identify proteins up- and down-regulated by 0.5% and 20% Triton X-100 in P. nitroreducens TX1, respectively. Results showed that there are about 500 protein spots from the pI 4-7 gels for P. nitroreducens TX1 grown under minimal salts basal medium (MSB) containing 0.5% Triton X-100 (experiment group) and about 600 spots for cells grown under 0.5% succinate as the sole carbon source (control group). 600 spots for cells grown under 20% Triton X-100 as the sole carbon source (experiment group). Moreover, the matrix-assisted laser desorption ionization-quadruples-time of fly mass spectrometer (MALDI-Q-TOF MS) was used to identify the proteins separated by two dimension gel electrophoresis (2DE). In the case of 0.5% Triton X-100, there are 17 up-regulated proteins and 11 down-regulated proteins were identified. Among the up-regulated proteins, several classes of proteins were found: (1) Six proteins were found putative ATP binding cassette (ABC) transporters, including two main classes of ABC transporters. One of the classes, the Leu/ Ile/ Val-binding protein family was suggested to involve in the transportation of Triton X-100; (2) Three were found the stress-responsive proteins (GroEL, ClpB, IbpA), which perform the function of chaperone meditating protein folding; (3) The alkyl hydroperoxide reductase (AhpC), which was first discovered in surfactant stress response; (4) Other stress proteins (TerD, TerE), which are presumed to relate to oxidative stress; (5) The K+ transporters KdpD and KdpE are highly expressed while the osmotic regulator OmpR was depressed, which suggest there may exist osmotic control system in the presence of Triton X-100. (6) One of the up-regulated enzyme is dihydrolipoamide dehydrogenase. It is related to the energy synthesis and catalyze the transformation of acetyl CoA from pyruvate. On the other hand, down-regulated enzymes are involved in the fatty acid synthesis, DNA biosynthesis and serine synthesis. The proteins up-regulated by 20% Triton X-100 were further studied in comparison to 0.5% Triton X-100. Up-regulated proteins included ATP synthetase, flagellin and fatty synthesis related proteins. Malate synthetase, one of key enzyme involved in the glyoxylate cycle, which catalyzes glyoxylate to malate was down-regulated, These suggested the degradation of polyethoxylate chain in Triton X-100 is less likely to generate glyoxylate. However, study founded acetaldehyde inhibit the expression of PhoP protein, which familiar to our experiment. It suggested the degradation of polyethoxylate side chain in Triton X-100 is more likely to from acetaldehyde.

關鍵字(中)

★ 蛋白質體學
★ 二維電泳
★ 假單胞菌屬
★ 辛基苯酚聚氧乙基醇
★ 界面活性壓力
★ 介質輔助雷射離子化飛行時間串聯四極質譜儀

關鍵字(英)

★ Proteomics
★ two dimensional electrophoresis
★ pseudomonas
★ Triton X-100
★ surfactant stress
★ MALDI-Q-TOF

論文目次

中文摘要-------------------------------------------------------------------------------- II
英文摘要-------------------------------------------------------------------------------- IV
目錄--------------------------------------------------------------------------------------- VI
圖目錄----------------------------------------------------------------------------------- X
表目錄----------------------------------------------------------------------------------- XI
縮寫與全名對照表------------------------------------------------------------------ XII
壹、緒論--------------------------------------------------------------------------------- 1
一、烷基苯酚聚氧乙基醇化合物之基本特性與應用--------------------------- 1
二、細菌於界面活性劑環境中的逆境反應---------------------------------------- 2
三、蛋白質體學應用於微生物分解有機污染物之文獻------------------------- 3
四、假單胞菌屬基因體之特色------------------------------------------------------- 6
五、非離子界面活性劑Triton X-100之生物分解------------------------------- 7
六、研究動機---------------------------------------------------------------------------- 9
貳、材料與方法---------------------------------------------------------------------- 11
研究大綱及實驗流程示意圖---------------------------------------------------- 11
一、菌種與培養基---------------------------------------------------------------------- 12
二、生長曲線與蛋白質粗萃液的製備---------------------------------------------- 13
1. 生長曲線的製作--------------------------------------------------------------- 13
2. 蛋白質粗萃液的製備--------------------------------------------------------- 13
3. 蛋白質定量分析--------------------------------------------------------------- 14
三、等電點焦集與聚丙烯醯胺膠體二維電泳------------------------------------- 15
1. 等電點膠條覆水作用--------------------------------------------------------- 15
2. 等電點焦集電泳--------------------------------------------------------------- 16
3. 等電點焦集第一維電泳分離電壓條件------------------------------------ 18
4. 聚丙烯醯胺膠體二維電泳--------------------------------------------------- 19
5. 膠體顯色------------------------------------------------------------------------ 21
四、蛋白質體分析及樣品前處理---------------------------------------------------- 21
1. 影像分析------------------------------------------------------------------------ 21
2. 膠體內消化法------------------------------------------------------------------ 22
五、蛋白質鑑定原理與方法---------------------------------------------------------- 23
1. 蛋白質鑑定原理說明--------------------------------------------------------- 23
2. 蛋白質功能搜尋--------------------------------------------------------------- 28
六、實驗儀器與藥品------------------------------------------------------------------- 29
1. 儀器與設備--------------------------------------------------------------------- 29
2. 藥品------------------------------------------------------------------------------ 30
參、結果-------------------------------------------------------------------------------- 32
一、Pseudomonas nitroreducens TX1蛋白質初萃液的製備-------------------- 32
二、二維電泳分離Pseudomonas nitroreducens TX1 蛋白質體條件--------- 32
三、二維電泳蛋白質圖譜統計分析------------------------------------------------ 33
1. 以0.5% Triton X-100培養之胞內蛋白質體---------------------------- 34
2. 以20% Triton X-100培養之胞內蛋白質體----------------------------- 35
3. 以0.5% Triton X-100培養之胞外蛋白質體---------------------------- 36
4. 經質譜分析但未被鑑定出的蛋白質-------------------------------------- 37
四、蛋白質身分鑑定----------------------------------------------------------------- 37
1. MS MS ion search使用NCBInr資料庫搜尋結果--------------------- 38
2. MS MS ion search使用OWL資料庫搜尋結果------------------------ 40
3. 使用胜肽指紋鑑定技術的搜尋結果------------------------------------- 41
五、蛋白質功能鑑定------------------------------------------------------------------ 43
肆、討論-------------------------------------------------------------------------------- 52
一、二維電泳圖譜分析--------------------------------------------------------------- 52
二、蛋白質功能之探討-------------------------------------------------------------- 52
1. Pseudomonas nitroreducens TX1於Triton X-100環境之代謝反應 52
1.1 代謝反應分析---------------------------------------------------------------- 52
1.2 運輸蛋白質功能----------------------------------------------------------------- 54
1.3 蛋白質之修復功能--------------------------------------------------------- 55
1.4 ATP合成酶與鞭毛之分析----------------------------------------------------- 57
1.5 滲透壓的調控-------------------------------------------------------------------- 59
1.6 鐵離子的運輸與利用----------------------------------------------------------- 59
1.7 氮的利用-------------------------------------------------------------------------- 60
2. Triton X-100可能的代謝途徑與相關酵素------------------------------ 61
3. 細胞膜與能量生合成的關係----------------------------------------------- 62
4. 胞外蛋白質-------------------------------------------------------------------- 63
三、二維電泳法之探討---------------------------------------------------------------- 64
1. 還原劑TBP (tributylphosphine)的影響------------------------------------ 64
2. 分子量和等電點値的判定--------------------------------------------------- 64
四、蛋白質鑑定----------------------------------------------------------------------- 65
1. 菌屬之間的關係--------------------------------------------------------------- 65
2. 胜肽指紋鑑定技術的探討--------------------------------------------------- 65
3. 蛋白質功能鑑定之資料庫--------------------------------------------------- 66
伍、結論與建議---------------------------------------------------------------------- 67
一、結論----------------------------------------------------------------------------------- 67
二、建議----------------------------------------------------------------------------------- 68
陸、參考文獻------------------------------------------------------------------------- 69
圖------------------------------------------------------------------------------------------ 78
表------------------------------------------------------------------------------------------ 91
附錄一二維電泳條件之最適化--------------------------------------------- 112
附錄二蛋白質鑑定結果------------------------------------------------------- 129
附錄三蛋白質家族 (domain) 查詢結果-------------------------------- 134
附錄四 KEGG資料庫搜尋結果--------------------------------------------- 140
附錄五蛋白質搜尋參數調較------------------------------------------------ 145
附錄六已鑑定之蛋白質原始資料----------------------------------------- 152
附錄七胜肽指紋鑑定之比對情形----------------------------------------- 250

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指導教授

黃雪莉(Shir-Ly Huang)

審核日期

2004-1-29

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