臺灣博碩士論文加值系統

將假單胞桿菌19-3於甲苯存在下繼代培養半年，可獲得甲苯耐性增強1000倍以上的超強耐性菌株。其中一些菌株在較高細胞密度(約109~1010cfu/ml)於無機鹽培養基中有較佳的甲苯耐性，因此具有潛力成為建構水不溶化合物，生物轉化用工業菌株的宿主之用。為了探討耐性增強的原因，針對不同耐性的菌株測定其細胞表面斥水性、脂多醣組成、抗生素耐性及電子傳遞鏈抑制劑的影響，結果發現排放幫浦與耐性有關，而細胞表面組成份及斥水性的改變更為重要。

After half a year-subculturing of Pseudomonas sp. 19-3 in the presence of toluene, super strains with more than 1000 folds of increase in toluene resistance were obtained. Some of them showed better toluene resistance in mineral medium with higher cell density(ca. 109~1010cfu/ml), so they might be capable of use as host in the construction of industrial strains for the bioconversion of water insoluble compounds. In order to explore the mechanism of increment in resistance, cell surface hydrophobicity, lipopolysaccharide composition, antibiotic resistance, and effect of electron transport chain inhibitor of strains with different level of resistance were examined. It was found that active efflux pump might be involved in toluene resistance, but changes in composition and hydrophobicity of cell envelope was more important.

謝誌 Ⅰ
中文摘要 Ⅱ
英文摘要 Ⅲ
目錄 Ⅳ
表目錄 Ⅹ
圖目錄 XI

第一章、前言 1
第一節 有機溶劑對菌株的傷害 1
第二節 耐性菌株的發現 3
第三節 耐性機制 5
1.3.1阻擋有機溶劑 5
1.3.1.1脂肪酸組成 5
1.3.1.2脂多醣 7
1.3.1.3細胞表面斥水性 9
1.3.2排除有機溶劑 9
1.3.2.1有機溶劑排除幫浦 9
1.3.2.2囊泡 11
第四節 耐性菌株的應用 11
第五節 實驗室前人研究 13
第六節 研究目的 14

第二章、材料與方法 15
第一節 菌株 15
第二節 培養基 16
第三節 菌液濃度測定 17
第四節 連續稀釋平板培養基活菌計數法 17
第五節 菌株保存 18
第六節 液體培養方式 18
2.6.1 種菌的製備 18
2.6.2 液體培養條件 19
第七節 萃取菌株DNA 19
第八節 聚合酵素連鎖反應(Polymerase Chain Reaction，PCR) 20
第九節 電泳 21
第十節 甲苯衝擊耐性測試 22
第十一節 馴化菌株 23
第十二節 營養缺乏測試 23
第十三節 馴化菌株在甲苯下之生長測試 24
第十四節 利用甲苯為碳源的測試法 24
第十五節 細胞表面斥水性 25
第十六節 蛋白質分析 25
2.16.1蛋白質定量分析 25
2.16.2 SDS-PAGE 26
2.16.2.1 SDS-PAGE的材料製備 26
2.16.2.2 SDS-PAGE的步驟 29
第十七節 脂多醣之萃取與分析 29
2.17.1脂多醣之萃取 29
2.17.2脂多醣之分析 30
2.17.3銀染 30
第十八節 菌株於抗生素中相對生長測試 31
第十九節 Carbonyl cyanide m-chlorophenyhydrazone對菌株
甲苯耐性影響 32

第三章、結果 33
第一節、菌株特性 33
3.1.1、菌株16S-rDNA定序 33
3.1.2、菌株利用甲苯生長 33
3.1.3、菌株19-3在甲苯衝擊及次培養後之生長情形 33
第二節、菌株對甲苯之耐性 34
3.2.1、甲苯適應處理對菌株甲苯耐性之影響 34
3.2.2、誘導處理對菌株甲苯耐性的影響 34
3.2.3、馴化菌株的甲苯耐受性 35
3.2.4、馴化菌株於甲苯存在下之生長情形 35
3.2.5、研究菌株在高菌數條件下對甲苯的耐性 36
第三節、耐性機制 37
3.3.1、細胞表面斥水性 37
3.3.2、脂多醣的分子量分佈 37
3.3.3、抗生素抗性 38
3.3.4、電子傳遞鏈抑制劑對菌株甲苯耐性影響 38
3.3.5、幫浦基因分析 39

第四章、討論 41
第一節、菌株特性 41
4.1.1、菌株16S-rDNA定序 41
4.1.2、菌株利用甲苯生長 41
第二節、菌株對甲苯之耐性 42
4.2.1、誘導影響菌株甲苯耐性 42
4.2.2、馴化菌株的甲苯耐受性 42
4.2.3、馴化菌株於甲苯存在下之生長情形 44
4.2.4、研究菌株在高菌數條件下對甲苯的耐性 45
第三節、耐性機制 46
4.3.1、細胞表面斥水性 46
4.3.2、脂多醣的分子量分佈 46
4.3.3、抗生素抗性 47
4.3.4、電子傳遞鏈抑制劑對菌株甲苯耐性影響 48
4.3.5、幫浦基因分析 49

第五章、結論 50

第六章、參考文獻 51

表目錄
表一、菌株19-1的16S-rDNA部分序列至NCBI資料庫比對結果 59
表二、菌株19-3經甲苯適應處理及馴化處理後之甲苯耐性 60
表三、甲苯誘導對菌株19-1及19-3之甲苯耐性影響 61
表四、各研究菌株細胞表面斥水性 62

圖目錄
圖一、菌株19-1之16S-rDNA部分序列定序結果 63
圖二、各種研究菌株利用甲苯為碳源之生長情形 64
圖三、菌株19-3在甲苯衝擊及次培養後之生長情形 65
圖四、甲苯存在下繼代培養菌株19-3所得分離菌株之甲苯耐受性 66
圖五、連續馴化所得分離菌株於甲苯存在下之生長曲線圖 67
圖六、甲苯對於高菌數的各種研究菌株的耐性影響 68
圖七、各種研究菌株脂多醣組成 69
圖八、連續馴化菌株於Ampicilline下之相對生長情形 70
圖九、連續馴化菌株於Tetracycline下之相對生長情形 71
圖十、連續馴化菌株於Chloramphenicol下之相對生長情形 72
圖十一、連續馴化菌株於Erythromycin下之相對生長情形 73
圖十二、連續馴化菌株於Streptomycine下之相對生長情形 74
圖十三、連續馴化菌株於Novobincin下之相對生長情形 75
圖十四、Carbonyl cyanide m-chlorophenyhydrazone對
菌株19-3和19S的甲苯耐性影響 76

附錄
附錄一、格蘭氏陽性與陰性細菌細胞外套的構造 77
附錄二、細菌排放幫浦的結構 78
附錄三、菌株於不同培養條件下的16碳與18碳飽和對不飽和
酯肪酸比例 79
附錄四、菌株於不同條件下的16碳與18碳反式對順式不飽和
酯肪酸比例 80
附錄五、目前對於P. putida以發表的排除幫浦所能排出的
各種抗生素種類 81
附錄六、本研究使用引子的核酸序列與來源 82
附錄七、Carbonyl cyanide m-chlorophenyhydrazone的結構 83

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