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研究生:黃俊諺
研究生(外文):Chun-Yen Huang
論文名稱:細菌血紅素對本土菌株PseudomonasaeruginosaJ16生產鼠李醣酯之探討
論文名稱(外文):A study on the production of rhamnolipids using Vitreoscalla hemoglobin with an indigenous isolate Pseudomonas aeruginosa J16
指導教授:魏毓宏
指導教授(外文):Yu-Hong Wei
學位類別:碩士
校院名稱:元智大學
系所名稱:生物科技與工程研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:59
中文關鍵詞:生物界面活性劑鼠李醣酯P. aeruginosa J16細菌血紅素比攝氧速率
外文關鍵詞:BiosurfactantRhamnolipidP. aeruginosa J16Vitreoscilla hemoglobinOxygen uptake rate.
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文獻指出當好氧性細菌Vitreoscilla 處於極低的溶氧環境下時,會合成出能增強微生物氧化代謝能力,加快細胞生長速率,增加細胞密度的細菌性血紅素Vitreoscilla hemoglobin(VHb)。本研究以構築帶有Vitreoscilla hemoglobin gene(vgb)之質體(pUV5G),對石化廢水篩選所獲得之台灣本土菌株Pseudomonas aeruginosa J16進行轉型作用,並進行一系列菌體生長、醱酵策略以及代謝產物鼠李醣酯的討論。其次經由調控攪拌式發酵槽醱酵期間之通氧量,並偵測培養液的氧傳遞係數與不同培養時間內菌體的比氧吸收速率,發現轉型株的比氧吸收速率是野生株的1.9倍,證實轉型株具有較高的氧氣擷取能力。以最適化半合成培養基醱酵培養,鼠李醣酯最佳產量可達16 g/L。更進一步修正轉速及通氣醱酵培養的調控方式,成功將菌體生長及鼠李醣酯代謝生成的時間有效提前將近24小時,並將鼠李醣酯
II
代謝產率由0.09 g/L/h提升至0.112 g/L/h,此結果顯示同步調控轉速及氧氣的確能有效幫助菌生長以及代謝鼠李醣酯。
Rhamnolipid, a glycolipid-type biosurfactant primarily produced by Pseudomonas aeruginosa, is among the most effective biosurfactants and has been applied in various industries and bioremediation. Literature point out that elevation of dissolved oxygen level seemed to have a positive effect on both cell growth and rhamnolipid production. This study aims to construct a hemoglobin gene to obtain a mutant stain for the Pseudomonas aeruginosa. Hemoglobin (VHb) produced by bacterial Vitreoscilla could enhance cell growth and metabolism at hypoxic environmental condition. The gene (vgb) encoding Vitreoscilla hemoglobin was constructed in plasmid pUV5G and electroporated into Pseudomonas aeruginosa J16 which was indigenous isolated bacteria. In this work, oxygen uptake rate and cell respiration intensity could be controlled via air flow using stirred fermenter. We found that the cell respiration of recombinant was 1.9 fold of obtained from wild type,
IV
which indicated recombinant has higher ability to uptake oxygen. Moreover, P. aeruginosa J16 pUV5G could produce 16 g/L of rhamnolipid in 120 hours by modify semi-synthetic medium. Amending the way of ferment in agitation and oxygen flow, we advanced the time of cell growth and rhamnolipid synthesis for 24 hours. We also raised the yield of rhamnolipid concentration from 0.09 g/L/h to 0.112 g/L/h. This result indicates simultaneously control agitation and oxygen flow could advance P. aeruginosa cell growth and rhamnolipid synthesis.
目錄
中文摘要.....................................................................................................I
英文摘要...................................................................................................III
致謝............................................................................................................V
目錄..........................................................................................................VI
表目錄........................................................................................................X
圖目錄......................................................................................................XI
第一章 文獻回顧......................................................................................1
1.1 生物界面活性劑(Biosurfatants)......................................................1
1.2 生物界面活性劑之生產菌株篩選..................................................2
1.3 醣酯類生物界面活性劑–鼠李醣酯................................................3
1.4 細菌血紅素Vitreoscilla hemoglobin(VHb).....................................5
1.5 鼠李醣酯之醱酵生產......................................................................6
1.6 鼠李醣酯之應用..............................................................................7
1.7 研究動機與目的..............................................................................8
第二章 材料與方法................................................................................12
2.1 實驗菌株........................................................................................12
VII
2.2 材料與試劑....................................................................................12
2.2.1 培養基......................................................................................12
2.2.2 實驗使用之質體......................................................................14
2.2.3 實驗使用之引子......................................................................14
2.2.4 限制酶酵素..............................................................................15
2.2.5 實驗藥品..................................................................................15
2.2.6 實驗儀器..................................................................................16
2.3 實驗方法........................................................................................17
2.3.1 菌株之保存..............................................................................17
2.3.2 目標基因純化與選殖.............................................................17
2.3.3 質體建構..................................................................................18
2.3.4 P. aeruginosa J16勝任細胞製備.............................................18
2.3.5 細胞轉型作用..........................................................................19
2.3.6 細胞篩選..................................................................................19
2.3.7 蛋白質電泳..............................................................................19
2.3.8 菌株之培養..............................................................................20
2.4 分析方法........................................................................................21
2.4.1 微生物生長分析......................................................................21
2.4.2 鼠李醣酯分析..........................................................................21
VIII
2.4.3 甘油定量分析..........................................................................22
第三章 結果與討論................................................................................23
3.1 論文架構........................................................................................23
3.2 菌種改殖........................................................................................23
3.2.1 選殖細菌血紅蛋白與抗生素基因片段.................................23
3.2.2 構築表現質體pUV5G.............................................................24
3.3 VHb蛋白質的表現.........................................................................28
3.4 野生株與轉型株之比較................................................................30
3.4.1 野生株與轉型株於LB之生長差異........................................30
3.4.2 野生株及轉型株於MMS代謝鼠李醣酯能力之比較............32
3.4.3 無機鹽培養基之液相體積氧傳遞係數測試.........................34
3.4.4 野生型及轉型株於MS菌體呼吸強度比較............................35
3.4.5 野生株及轉型株於MSS代謝鼠李醣酯能力比較.................38
3.4.6野生株與轉型株於二階段批次醱酵代謝鼠李醣酯能力之比較........................................................................................................40
3.5 醱酵策略........................................................................................42
3.5.1轉速對野生株及轉型株代謝鼠李醣酯能力之影響..............42
3.5.2 轉速控制溶氧對轉型株代謝鼠李醣酯之影響.....................45
3.5.3轉速與純氧控制溶氧對轉型株代謝鼠李醣酯之影響..........47
IX
3.5.4 添加不同濃度硫酸鐵對轉型株代謝鼠李醣酯之影響.........50
第四章 結論與未來展望........................................................................52
4.1 結論................................................................................................52
4.2 未來展望........................................................................................55
參考文獻...................................................................................................56
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