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研究生:賁煜
研究生(外文):Yu Pen
論文名稱:混合碳源對大腸桿菌生產重組蛋白之影響
論文名稱(外文):Effect of Mixed Carbon Sources on the Production of Recombinant Protein by Escherichia coli
指導教授:陳特良
指導教授(外文):Teh-Liang Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:63
中文關鍵詞:大腸桿菌醱酵白細胞介素-20混合碳源
外文關鍵詞:Escherichia colimixed carbon sourcesIL-20fermentation
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以葡萄糖為碳源的批次醱酵,因大腸桿菌快速生長,及葡萄糖對乙醛酸鹽分流的抑制,而造成過多的醋酸分泌,20 g/L 是此操作下葡萄糖濃度的上限。菌體二階段生長的現象,暗示了混合碳源培養也許是個解決之道。選擇以甘油、果糖、蔗糖為碳源與葡萄糖等比例混合,甘油(10 g/L) + 葡萄糖(10 g/L) 混合醱酵及果糖(10 g/L) +葡萄糖(10 g/L) 混合醱酵,相較於葡萄糖(20 g/L) 醱酵,菌體濃度分別提高46%及18%。混合碳源醱酵亦使菌體將系統中之醋酸當作碳源來代謝,有效地減少醋酸的累積量。在指數生長期終點,同時饋入新培養基200 mL 和IPTG 1 mM 來誘導菌體生產IL-20。以葡萄糖(20 g/L) 醱酵的IL-20 產量,遠不及甘油(20 g/L) 及果糖(20 g/L)醱酵之量,但卻跟蔗糖(20 g/L) 醱酵所得之IL-20 產量相近。用甘油(10 g/L) + 葡萄糖(10 g/L),果糖(10 g/L) + 葡萄糖(10 g/L) 及蔗糖(10 g/L) + 葡萄糖(10 g/L)雙碳源醱酵所得IL-20 產量,較單一碳源的甘油(20 g/L),果糖(20 g/L) 及蔗糖(20g/L) 醱酵高出0.4,0.4 及3.3 倍。
Fermentation using glucose as a carbon source results in excess acetate secretion due to the rapid growth of Escherichia coli and the repression of glyoxylate shunt by glucose. Glucose concentration of 20 g/L is the critical value in batch system for E. coli. Diauxic growth of the organism implies that cultivation with mixed nutrients is probably a resolution. Glycerol, fructose, and sucrose were chosen to blend with glucose in equal ratio. Biomass rose 46% and 18% in the fermentations with glycerol (10 g/L) + glucose (10 g/L) and fructose (10 g/L) + glucose (10 g/L) media, respectively compared to that in glucose (20 g/L) one. Fermentation with mixed-substrate led the acetate in system to be metabolized as a carbon source thus reduced the acetate accumulation effectively. Host cells were induced to produce IL-20 by pulse addition of fresh media 200 mL as well as IPTG 1 mM at the termination of exponential phase. IL-20 yield in glucose (20 g/L) culture was less than that in glycerol (20 g/L) and fructose (20 , but approximated to that in sucrose (20 g/L) one. IL-20 yields in dual-carbon-source fermentations with glycerol (10 g/L) + glucose (10 g/L), fructose (10 g/L) + glucose (10 g/L), and sucrose (10 g/L) + glucose (10 g/L) mixtures were 0.4, 0.4, and 4.3-fold more than single carbon source operations with glycerol (20 g/L), fructose (20 g/L), and sucrose (20 g/L), respectively.
中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅱ
誌謝……………………………………………………………………Ⅲ
目錄……………………………………………………………………Ⅳ
表目錄…………………………………………………………………Ⅶ
圖目錄…………………………………………………………………Ⅷ

第一章 緒論…………………………………………………………1
  1.1 前言…………………………………………………………1
  1.2 包含體的形成………………………………………………1
  1.3 牛皮癬………………………………………………………3
  1.4 白細胞介素…………………………………………………3
  1.5 研究動機及目的……………………………………………5
第二章 實驗材料及方法……………………………………………7
  2.1 菌株…………………………………………………………7
  2.2 藥品…………………………………………………………7
  2.3 儀器裝置……………………………………………………10
  2.4 實驗方法……………………………………………………11
    2.4.1 固態培養基之製作…………………………………11
    2.4.2 菌株之保存…………………………………………12
    2.4.3 前培養………………………………………………12
    2.4.4 菌體濃度檢量線……………………………………12
    2.4.5 溶氧電極之校正……………………………………13
    2.4.6 酸鹼電極之校正……………………………………13
    2.4.7 主培養………………………………………………13
  2.5 醋酸濃度之分析……………………………………………16
    2.5.1 醋酸濃度檢量線……………………………………16
    2.5.2 樣品的醋酸濃度分析………………………………19
  2.6 葡萄糖濃度之分析…………………………………………19
  2.7 電泳原理……………………………………………………19
    2.7.1 蛋白質的帶電性……………………………………19
    2.7.2 聚丙烯醯胺膠體電泳………………………………21
  2.8 蛋白質之分析及定量………………………………………23
    2.8.1 鑄膠…………………………………………………23
    2.8.2 電泳操作……………………………………………23
    2.8.3 蛋白質之定量………………………………………26
  2.9 包含體中IL-20 之濃度分析………………………………26
第三章 結果與討論…………………………………………………30
  3.1 菌體濃度與醋酸累積量……………………………………30
    3.1.1 葡萄糖濃度之限制…………………………………30
    3.1.2 甘油—葡萄糖培養…………………………………33
    3.1.3 果糖—葡萄糖培養…………………………………36
    3.1.4 蔗糖—葡萄糖培養…………………………………39
  3.2 蛋白質的生產………………………………………………42
    3.2.1 甘油—葡萄糖醱酵…………………………………42
    3.2.2 果糖—葡萄糖醱酵…………………………………44
    3.2.3 蔗糖—葡萄糖醱酵…………………………………47
第四章 結論…………………………………………………………51
第五章 未來與展望…………………………………………………53
參考文獻………………………………………………………………54
附錄……………………………………………………………………59
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