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研究生:蔣欣妤
研究生(外文):Shin-Yu Chiang
論文名稱:探討培養基組成與IPTG誘導時間對基因轉殖菌與肌酸酵素生產之影響
論文名稱(外文):Different behaviors for the cell growth and creatinase productivity in various media and induction time of IPTG
指導教授:蔡少偉蔡少偉引用關係
指導教授(外文):Shau-Wei Tsai
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:66
中文關鍵詞:肌酸酵素大腸桿菌IPTG誘導時間
外文關鍵詞:creatinaseE-coliinduction time of IPTG
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中 文 摘 要

本基因重組菌為JM109大腸桿菌內殖入載體pQE-51-pSCR所構成,而此載體內含Pseudomonas putida NTU-8肌酸酶基因及前端插入一段來自於Aeromonas hydrophila幾丁質分解酵素的信號序列,此基因轉殖菌因質體具有幾丁質酵素的信號序列,所以可將肌酸酵素分泌至細胞間質。

本論文以LB培養基與將其中部分tryptone與yeast extract以3克、6克和9克葡萄糖置換的複合培養基進行醱酵培養,目的在於探討IPTG誘導時間對於個別培養基中菌體濃度與肌酸酵素活性變化之影響。實驗中發現不論在LB或其餘3個添加葡萄糖的複合培養基,在適當的時間誘導皆可促進菌體生長,推測原因可能為誘導後加速某特定胺基酸之利用效率所造成。為確定此構想,而嘗試分析與TCA cycle較接近的絲胺酸、丙胺酸、天門冬胺酸與麩胺酸在醱酵液中的濃度,以進一步瞭解誘導前後其濃度的變化。
ABSTRACT

The recombinant Escherichi coli was constructed by inserting the pQE-51-pSCR01 expression vector in Escherichia coli JM109. It can express the chitinase signal peptide – creatinase hybrid gene by way of the expression vector. Besides, the target protein can be excreted to the periplasmic space due to the fusing of the signal peptide.

We processed the fermentations by incubating the recombinant Escherichi coli in Luria-Bertani medium and the media with parts of tryptone and yeast extract replaced by glucose. Different behaviors for the cell growth and creatinase productivity in various media were found if the inducer of isopropyl-β-D-thiogalactopyranoside (IPTG) was introduced at different periods of time. The cell concentration and creatinase activity were enhanced when inducing IPTG at an appropriate time. Possible explanation for such enhancements was attributed to increasing usage of certain amino acid. For confirming this hypothesis, the analysis of the concentration of serine、aspartic acid、alanine and glutamic acid close to TCA cycle was performed.
目 錄

中文摘要 Ⅰ
英文摘要 Ⅱ
誌謝 Ⅲ
目錄 Ⅳ
表目錄 Ⅵ
圖目錄 Ⅶ

第一章 緒論 1
1-1 酵素 1
1-2 肌酸、肌酸酐與肌酸酵素簡介 3
1-3 基因選殖技術 6
1-4 基因調控 7
1-5 胺基酸代謝 10
1-6 研究動機 11

第二章 儀器與原理 13
2-1 離心 13
2-2 超音波粉碎細胞 14
2-3 BIO-RAD蛋白質分析試劑之定量原理 14
2-4 高效液相層析儀 15

第三章 實驗材料與方法 18
3-1 實驗材料 18
3-1-1 宿主-載體 18
3-1-2 藥品 21
3-1-3 儀器與裝置 22
3-2 實驗步驟 22
3-2-1 菌種保存 22
3-2-2 培養基 23
3-2-3 醱酵槽實驗 24
3-3 分析方法 25
3-3-1 菌體濃度 25
3-3-2 肌酸酵素活性 26
3-3-3 蛋白質濃度 28
3-3-4 胺基酸濃度 29

第四章 結果與討論 32
4-1 菌體濃度動力模式之參數計算 32
4-2 醱酵槽中IPTG誘導時間之影響 32
4-2-1 LB 培養基 33
4-2-2 培養基1 (葡萄糖添加濃度為每升3克) 37
4-2-3 培養基2 (葡萄糖添加濃度為每升6克) 43
4-2-4 培養基3 (葡萄糖添加濃度為每升9克) 43
4-2-5 綜合比較LB與等量增加葡萄糖濃度的影響 47
4-3 胺基酸濃度分析 53

第五章 結論與未來展望 60

參考文獻 62
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