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研究生:李權峰
研究生(外文):Chuan-Feng Li
論文名稱:柴油分解菌其功能蛋白之研究
論文名稱(外文):Study on functional protein of diesel-degrading bacteria
指導教授:方天熹方天熹引用關係陳俊德陳俊德引用關係
指導教授(外文):Tien-Shi FangChun-Te Chen
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:海洋環境資訊學系
學門:工程學門
學類:環境工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:88
中文關鍵詞:柴油降解菌功能性蛋白一維電泳二維電泳柴油污染Acinetobacter sp.
外文關鍵詞:diesel-degrading bacteriafunctional proteinsone-dimensional electrophoresistwo-dimentional electrophoresisdiesel oil pollationAcinetobacter sp
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摘要

為探討微生物降解海水中柴油之機制,本研究自高雄前鎮漁港內海水樣品分離與培養Acinetobacter sp.,並使用4階段連續萃取方法,於實驗室進行Acinetobacter sp.降解柴油時所產生的功能蛋白質之研究。4個連續萃取方法所得產物分別為細胞外產物(Extracellular product, ECP),細胞外套產物(Cell envelope product, CEP),細胞膜蛋白(Cytoplasmic membrane protein, CMP)與外膜蛋白(Outer membrane protein, OMP),之後再利用膠體電泳分離(SDS-PAGE)與不同膠體比例與濃度變化,尋找各萃取產物之相關功能蛋白。分析結果顯示膠體電泳分離出之外膜蛋白其分子量出現在59KDa與48KDa,其餘三種萃取產物並無相關功能蛋白出現。之後再以二維電泳進一步分離外膜蛋白之蛋白質,分析結果發現外膜蛋白有5個蛋白質出現,他們分別為:Spot A分子量為47KDa及PI6.2;Spot B分子量為39KDa與PI6.5;Spot C分子量為38.5KDa與PI6.65;Spot D分子量為26.8KDa與PI6.4;Spot E分子量為27.2KDa與PI6.85。
本研究期望利用柴油降解菌Acinetobacter sp.之功能蛋白研發,建立簡易之免疫分析方法,監測海洋環境有無柴油污染之依據,此外也期望此技術可繼續深入發展,成為海域柴油污染之生物復育技術。

關鍵字:柴油污染、柴油降解菌、Acinetobacter sp.、功能性蛋白、
一維電泳、二維電泳
Abstract

The purpose of this study is to analyse the mechanism of degrading the diesel oil by marine bacteria. Sea water samples were collected from the Chien-chen Fishing Harbour in Kaohsiung to separate and culture Acinetobacter sp.. A four-step sequential extraction method was applied to separate peoteins of extracellular product (ECP), Cytoplasmic envelope protein (CEP), Cytoplasmic membrane protein (CMP), and Outer membrane protein (OMP), in Acinetobacter sp.. This work aims to seek the functional proteins from the four kinds of proteins for degrading the diesel oil. Combining the four-step sequential extraction method with SDS-PAGE separation technique, it is found that only the Outer membrane protein (OMP) has two functional proteins and their molecular weights were do not possess any of the functional proteins. Further analysis by two-dimentional electrophoresis on OMP, it was found that there are five kinds of functional proteins. They are: Spot A of which molecular weight is 47KDa and PI6.2; Spot B of which molecular weight is 39KDa and PI6.5; Spot C of which molecular weight is 38.5KDa and PI6.65; Spot D of which molecular weight is 26.8KDa and PI6.4; and Spot E of which molecular weight is 27.2KDa and PI6.85.
Finally, this study is expecting to establish a fast and easy immune analytical method to quickly determine whether the seawater is contaminated by diesel oil or not. In addition, the knowledge of functional proteins of Acinetobacter sp. can be developed to bioremediate the diesel oil pollution in marine environments.

Key word: diesel oil pollution, diesel-degrading bacteria, Acinetobacter sp., functional proteins, one-dimensional electrophoresis, two-dimentional electrophoresis.
目錄


中文摘要………………………………………………………... Ⅰ
Abstract.…………………………………………………...….. Ⅱ
謝辭……………………………………………………………... Ⅲ
目錄………………………………………...…………………… Ⅳ
圖目錄…………...……………………………………………… Ⅵ
表目錄………………...………………………………………… Ⅶ
附錄目錄………………...……………………………………… Ⅶ
第壹章 前言……………………………………….…………. 1
一、研究動機………...….………………………………. . 1
二、文獻回顧………...….………………………………. . 3
(一) 海上洩油後之油污變化………………………. ...... 3
(二) 柴油簡介與微生物分解途徑…………………. .. .... 4
(三) 蛋白質體學……………………………………. ...... 5
(四) 微生物蛋白質體與環境壓力…………………. ...... 6
(五) Acinetobacter sp.的簡介…………………... ..... 7
第貳章 材料與方法………….………………………………. 11
一、材料………………...….…………………………………. 11
(一) 菌種來源………………………………………. 11
(二) 培養基…………………………………………. 11
(三) 萃取菌蛋白所需緩衝液………………………. 12
(四) 電泳常用之膠體比例與藥品…………………. 12
(五) 染劑與退染劑…………………………………. 15
(六) 二維電泳之膠體與藥品……………......... 15
(七) 其他……………………………………………. 18
(八) 儀器與設備……………………………………. 18
二、方法………………...….………………………………. 19
(一) 嗜油菌細胞之培養……………………………. 19
(二) 嗜油菌細胞蛋白質萃取………………………. 20
(三) 蛋白質濃度測定………………………………. 21
(四) 硫酸十二酯鈉聚丙烯醯胺膠體電泳分析……. 23
(五) 軟體分析………………………………………. 25
(六) 嗜油菌細胞蛋白質萃取……………………. 25
(七) 等電位聚焦分析 IEF…………………………. 25
(八) SYPRO Ruby 螢光染色……………………….. 28
(九) 軟體分析………………………………………. 28
第參章 結果與討論………….………………………………. 29
一、膠體電泳分離(SDS-PAGE)…….......………………. 29
(一) 不同培養基培養後細胞粹取之ECP、CEP、CMP、OMP各部分蛋白質移動性與強度之比較……….................. 29
(二) 利用不同膠體比例Acrylamide:N,N’-methylene-bis-acrylamide 29:1、59:1、69:1、79:1,分離CMP與OMP蛋白質,進行移動性與強度之比較 ………………............……... 30
(三) 利用不同膠體濃度6%、8%,分離CMP與OMP蛋白質,進行移動性與強度之比較……………….................... 33
(四) 利用膠體比例69:1,膠體濃度8%,分離CMP與OMP蛋白質,進行移動性與強度之比較……….…................. 35
二、二維電泳………………….……………………………. 37
第肆章 結論………………….………………………………. 41
參考文獻……………………………………...………………… 42
圖…………...…………………………………………………… 46
表………………...……………………………………………… 61
附錄………………...…………………………………………… 64

圖目錄


圖1各種工業所產生之主要污染物…………….....…....... 46
圖2高雄港圖……………………………………............... 46
圖3油在海中之動態與變化…………………………........…. 47
圖4烷類的氧化方式…………………………………......…... 47
圖5微生物氧化長鏈正烷類之方式……………..…........... 47
圖6好氧性細菌代謝苯環類化合物之途徑……..…........... 48
圖7有機物及碳氫化合物之生物分解機構及分解性............48
圖8蛋白質體學流程圖…………………………………......... 49
圖9蛋白質濃度測定檢量線……………….………............ 49
圖10LB與柴油培養基培養細胞後萃取之ECP、CEP電泳圖.......50
圖11LB與柴油培養基培養細胞後萃取之CMP、OMP電泳圖….....50
圖12LB與柴油培養基培養細胞後萃取之CMP、OMP在膠體A.29:1、B.59:1、C. 69:1、D. 79:1之電泳圖……….………......... 51
圖13柴油培養基培養後萃取細胞之CMP,在各膠體比例29:1、59:1、69:1、79:1下之移動性與強度…...........................52
圖14柴油培養基培養後萃取細胞之OMP,在各膠體比例29:1、59:1、69:1、79:1下之移動性與強度……………...….. .........53

圖15LB與柴油培養基培養細胞後萃取之CMP、OMP在膠體比例69:1濃度為6%與8%之電泳圖…………………............... 54
圖16柴油培養基培養後萃取細胞之CMP與OMP,在膠體濃度為6%、8%下之移動性與強度…………………..….........……...…. 54
圖17柴油培養基培養細胞後萃取之OMP與CMP稀釋成不同濃度之電泳圖…………………………………………...........……..…. 55
圖18LB與柴油培養基培養後萃取細胞之OMP與CMP,膠體比例69比1 ;濃度8% 下之蛋白質片段強度………….….................. 56
圖192D 標準品…………………………………………………… 56
圖20柴油培養基下之總蛋白質 2D gel 電泳圖……....…..… 57
圖21LB培養基下之總蛋白質 2D gel 電泳圖……….…..….. 57
圖22柴油培養基下之OMP 2D gel 電泳圖……….……...… 58
圖23LB培養基下之OMP 2D gel 電泳圖….…..…...…....... 59
圖24LB與柴油培養基2D gel Spot A-E的放大圖………..…... 60

表目錄

表1美國環保署所公佈之12項免疫分析………………………. 61
表2不同膠體比例下標準品之移動性…………………………... 61
表3不同膠體比例下標準品之Ban強度………………………... 62
表4不同膠體濃度下標準品之移動性與強度…………………... 62
表5LB與柴油培養基OMP 2D gel Spot A-E的強度……………. 63
表64次實驗中OMP spot A-E之強度平均值…………………… 63





附錄目錄

附錄1附錄1. ECP、CEP之Band強度與移動性…………….… 64
附錄2CMP、OMP之Band強度與移動性…………………...… 65
附錄3CMP LB在各比例下之Band強度與移動性…………...… 67
附錄4CMP OIL在各比例下之Band強度與移動性………...… 69
附錄5OMP LB在各比例下之Band強度與移動性………….… 71
附錄6OMP OIL在各比例下之Band強度與移動性…………… 73
附錄7CMP LB在6%與8%濃度下之Band強度與移動性…...… 75
附錄8CMP OIL在6%與8%濃度下之Band強度與移動性...… 76
附錄9OMP LB在6%與8%濃度下之Band強度與移動性…...… 77
附錄10OMP OIL在6%與8%濃度下之Band強度與移動性….… 78
附錄11CMP在8%濃度下之Band強度與移動性…………….…. 80
附錄12OMP在8%濃度下之Band強度與移動性………….……. 83
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中國石油網頁
http://www.cpc.com.tw/big5/files/燃料類產品.doc
美國環保署網頁
1. http://www.epa.gov/SW-846/pdfs/kits.pdf
2. http://www.epa.gov/epaoswer/hazwaste/test/pdfs/4030.pdf
3. http://www.epa.gov/epaoswer/hazwaste/test/pdfs/4035.pdf
高雄港務局網頁
http://www.khb.gov.tw/index_m.aspx?Link=AutoHtml/10/135/1-2-2 .htm
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