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研究生:胡紹揚
研究生(外文):Shao-Yang Hu
論文名稱:利用重組大腸桿菌生產N-Carbamoylase-D-AminoAcidAmidohydrolase之研究
論文名稱(外文):Production of N-Carbamoyl-D-Amino Acid Amidohydrolase from Recombinant Escherichia coli
指導教授:蘇遠志蘇遠志引用關係黃健雄黃健雄引用關係
指導教授(外文):Yuan-Chi SuJan-Hsiung Huang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:108
中文關鍵詞:合成培養基
外文關鍵詞:N-Carbamoylase-D-Amino Acid AmidohydrolaseEscherichia coliHigh cell density culture
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N-Carbamoyl-D-amino acid amidohydrolase(Nca)在工業上具有相當高之應用價值,可用來生產D型胺基酸以做為許多半合成抗生素、賀爾蒙製劑及殺蟲劑等的原料。在本研究之前已有學者將一段由Agrobacterium radiobacter中選殖出的nca基因轉殖入大腸桿菌中,並利用lac啟動子來控制基因表現,可以有效的在宿主細胞體內合成Nca。本研究乃探討重組大腸桿菌E. coli JM109 pQE-30-NCA 之Nca最適生產條件,並成功的建立一套自動控制的饋料批次醱酵系統,藉由高細胞密度培養更進一步提升Nca的生產效率。在500ml三角瓶規模中最適半合成生產培養基組成份為0.5%葡萄糖、0.75%酵母抽出物、0.8%K2HPO4及0.2%KH2PO4,在最適誘導條件下可獲得4.0U/ml的Nca產量,且此帶有nca基因的質體在宿主細胞內相當穩定。在五公升醱酵槽規模中起始培養基組成為1%葡萄糖、0.75%酵母抽出物、0.25%(NH4)2SO4及0.2%KH2PO4,饋料組成分別為75%葡萄糖、30%酵母抽出物及14%氨水。在培養過程中限制葡萄糖在一定濃度以下,並控制pH維持在6.95~7.05區間內,攪拌速率為900rpm、通氣量1.5vvm。培養的第6小時加入0.02mM IPTG誘導nca表現,並將溫度由37℃降到27℃以免生成內涵體。於培養第57小時之菌體量可達42g DCW/L,Nca活性30U/ml。
N-Carbamoyl-D-amino acid amidohydrolase(Nca) is an industrially useful enzyme in the production of D-amino acids for the synthesis of semi-synthetic antibiotics , peptide hormones, pyrethroids, and pesticides. The gene encoding the Nca was cloned from Agrobacterium radiobacter and expressed in E. coli under the control of the lac promoter. The effects of culturing conditions on Nca production by the recombinant E. coli strain were investigated using a fed-batch fermentation system.
The optimal composition of the starter medium was 1.0% glucose, 0.75% yeast extract, 0.25% (NH4)2SO4, and 0.2% KH2PO4. The pH of the culture broth was maintained at between 6.95 and 7.05 by adding 14% ammonia water or the solution containing 75% glucose and 30% yeast extract. The nutrient feeding is also needed in attaining high cell concentration. Following a 6-h cultivation, 0.02mM IPTG was added to induce Nca gene expression and the temperature was shifted from 37 to 27℃ to avoid inclusion body formation. The plasmid harboring Nca gene was found to be stably maintained in the E. coli. Under optimal condition, a cell density of about 42g dry cell weight /L and a volumertric productivity of 0.53U/mL/h could be achieved after 57h culture at agitation and aeration rates of 900 rpm and 1.5vvm, respectively , in a 5-liter jar fermentor.
第一章 前 言--------------------------------------------------------- 1
一、D-Amino acids 與 Antibiotics-------------------------------1
二、N-Carbamoyl-D-amino acid amidohydrolase---------------1
三、利用重組大腸桿菌表現nca基因---------------------------7
四、重組大腸桿菌之高細胞密度培養---------------------------9
五、饋料批次培養之控制原理------------------------------------13
六、研究動機與目的------------------------------------------------14
第二章 材料與方法-------------------------------------------------15
一、菌株---------------------------------------------------------------15
1.1宿主 15
1.2質體之構築與轉型 15
二、Hinton,s三角瓶之培養及誘導表現方法------------------15
2.1 菌種保存 15
2.2 活化及種培養 17
2.3 Hinton,s三角瓶之生長培養 18
2.4 Hinton,s三角瓶之生產培養 19
三、醱酵槽之培養及誘導方法-------------------------------------21
3.1 醱酵槽之構造簡介 21
3.2 醱酵槽之操作程序 21
3.3 醱酵槽之批式生產培養 22
3.4 饋料批次醱酵之高細胞密度培養 23
3.5 高細胞密度培養之誘導表現 25
四、Nca酵素活性分析方法-----------------------------------------26
4.1 酵素活性分析原理 26
4.2 粗酵素的製備 26
4.3 酵素反應步驟 27
五、分析方法-----------------------------------------------------------29
5.1 菌體生長量之測定(OD600、生菌數、乾重) 29
5.2 葡萄糖濃度之測定(DNS法) 30
5.3 Nca酵素純化方法 30
5.4 蛋白質濃度之定量(Bradford protein assay) 31
5.5 蛋白質電泳分析 32
5.6 質體穩定性的測試 35
六、酵素性質測試方法-----------------------------------------------35
6.1 等電交集電泳 36
6.2 最適反應pH之測試 37
6.3 最適反應溫度之測試 38
6.4 Nca酵素之熱穩定性測試 38
6.5酵素動力學之研究 39
第三章 結果與討論-----------------------------------------------40
一、分析方法的建立--------------------------------------------------40
1.1菌體濃度(OD600)之測定條件 40
1.2最適菌體打破條件 40
1.3產物吸收光波長的選擇 41
1.4酵素活性分析的適用範圍 41
1.5最適酵素反應時間 45
1.6Nca之純化方法 45
1.7質體穩定性測試 47
二、Hinton,s三角瓶規模之最適生長條件------------------------51
2.1 培養基種類的選擇 51
2.2 最適生長培養基組成份探討 51
2.3 討論 52
三、Hinton,s三角瓶規模之最適Nca生產條件-------------------56
3.1 最適IPTG誘導濃度探討 56
3.2 最適回收時間的探討 56
3.3 最適生產培養基組成份探討 59
3.4 最適誘導時機的探討 59
3.5 討論 64
四、五公升醱酵槽之高細胞密度培養--------------------------------66
4.1 高細胞密度培養模式的建立 66
4.2 饋料液組成比例的探討 66
4.3 基本饋料模式的建立 67
4.4 培養溫度對高細胞密度的影響 70
4.5 高細胞密度培養條件之最適化 70
4.6 討論 72
五、五公升醱酵槽規模之Nca生產放大實驗----------------------73
5.1 五公升醱酵槽規模之Nca生產放大實驗 73
5.2 利用高細胞密度培養生產之最適誘導時機 73
5.3 減少Nca酵素形成內涵體(Inclusion body) 73
5.4 誘導後菌體濃度及型態的改變 77
5.5 誘導與未誘導之菌體代謝及饋料情形的改變 77
5.6 討論 83
六、合成培養基之應用-------------------------------------------------85
6.1 合成培養基之三角瓶規模生長情形 85
6.2 合成培養基高細胞密度生長 85
6.3 合成培養基高細胞密度生產 89
6.4 討論 89
七、Nca酵素特性探討--------------------------------------------------94
7.1 Nca最適反應pH 94
7.2 Nca最適反應溫度 94
7.3 Nca等電點蛋白質電泳分析 94
7.4 Nca酵素之熱穩定性 94
7.5 Nca酵素動力學之研究 99
7.6 討論 99
第四章 結 論------------------------------------------------------101
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