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研究生:郭秋媚
研究生(外文):Chiu-Mei Kuo
論文名稱:利用基因重組之大腸桿菌生產盤尼西林醯化酶之研究
論文名稱(外文):Penicillin Acylase Production by Recombinant E. coli
指導教授:劉懷勝劉懷勝引用關係
指導教授(外文):Hwai-Shen Liu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學工程學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:154
中文關鍵詞:甘油脯胺酸白胺酸硫胺素
外文關鍵詞:ThiamineProlineGlycerolLeucine
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盤尼西林醯化酶(penicillin acylase)於製藥工業上常被利用來生產6-APA(6-amionpenicillanic acid)以製造半合成抗生素,而盤尼西林醯化酶是一種微生物酵素,故本研究是利用搖瓶培養基因重組之大腸桿菌HB101(pTrcKnPAC2902)生產盤尼西林醯化酶。實驗首先探討添加Glycerol、Leucine、Proline及Thiamine於培養基中之最適化濃度,接著利用相互混合添加及不同添加方式,尋求酵素產量最多之添加物及添加方式。
由實驗結果得知,個別添加Glycerol、Leucine、Proline及Thiamine之最適化濃度分別為2.5、0.25、0.2及0.003 g/L,其中又以添加Glycerol所生產之酵素為最多。接著利用Glycerol、Leucine、Proline及Thiamine相互混合添加,對於酵素產量並無加成性地增加,且所生產之酵素量皆不如單獨添加Glycerol多,而特別的是當同時添加Glycerol與Leucine時,對於菌體生長及酵素生產皆明顯受到抑制,因此便利用(1)將Leucine延後至Glycerol耗盡時(24 hr)添加。(2)培養至24 hr時,添加NaOH使培養基中的pH產生先降後升之現象。此兩個方法皆對於菌體生長及酵素生產有所改善,又以後者較佳。
除此之外,亦利用Glycerol進行不同添加方式之實驗,其酵素產量皆少於初始培養即加入Glycerol之方式。而由諸多實驗中吾提出一假設:菌體將Glycerol代謝成未知成分X (pH下降),過程中主要弁酮偯飢U生長;待Glycerol耗盡,未知成分X即又被代謝成另一未知成分Y (pH上升),此過程中則主要助益酵素生成,而此假設也隨後於利用不同添加量的醱酵液(不同培養時間)之實驗中獲得證實。
中文摘要…………………………………………………………………...I
英文摘要…………………………………………………………………..II
目錄………………………………………………………………………III
圖目錄…………………………………………………………………VIII
表目錄…………………………………………………………………..XXI



第一章 緒論 1
第二章 文獻回顧 2
2.1 微生物酵素之利用 2
2.2 盤尼西林醯化酶之分類 3
2.3 盤尼西林醯化酶之來源 4
2.4 大腸桿菌合成盤尼西林醯化酶之途徑 6
2.5 利用微生物大量生產盤尼西林醯化酶 7
2.5.1 菌種之改良方法 8
2.5.2 利用培養環境來調控微生物的代謝作用 12
2.6 盤尼西林醯化酶之應用 24
2.6.1 利用盤尼西林醯化酶催化生產6-APA 24
2.6.2 盤尼西林醯化酶其它重要應用 27
第三章 實驗材料與方法 30
3.1 菌株 30
3.2 培養基 31
3.3 實驗藥品 32
3.4 實驗儀器 33
3.5 培養方法 33
3.5.1 預培養 33
3.5.2 主培養 34
3.6 分析方法 39
3.6.1 菌量之測定 39
3.6.2 盤尼西林醯化酶活性之測定 39
3.6.3 甘油濃度之測定 41
第四章 結果與討論 43
4.1 探討Glycerol、Leucine、Proline、Thiamine對菌體生長及酵素活性之影響 43
4.1.1 添加不同濃度之甘油(Glycerol)對菌體生長及酵素活性之影響 43
4.1.2 添加不同濃度之Leucine對菌體生長及酵素活性之影響 49
4.1.2a 【低濃度】添加Leucine濃度為0.1、0.15、0.2及0.25 g/L對菌體生長及酵素活性之影響 52
4.1.2b 【高濃度】添加Leucine濃度為0.2、0.25、0.3及0.35 g/L對菌體生長及酵素活性之影響 55
4.1.3 添加不同濃度之Proline對菌體生長及酵素活性之影響 59
4.1.3a 【低濃度】添加Proline濃度為0.1、0.2、0.3及0.4 g/L對菌體生長及酵素活性之影響 59
4.1.3b 【高濃度】添加Proline濃度為0.4、1.0、1.5及2.0 g/L 對菌體生長及酵素活性之影響 62
4.1.4 添加不同濃度之Thiamine對菌體生長及酵素活性之影響 66
4.1.4a 【低濃度】添加Thiamine濃度為0.001、0.002、0.003及0.004 g/L對菌體生長及酵素活性之影響 66
4.1.4b 【高濃度】添加Thiamine濃度為0.004、0.008、0.016及0.032 g/L對菌體生長及酵素活性之影響 69
4.2 探討培養基中含有Glycerol之條件下,以分別及混合方式添加Leucine、Proline及Thiamine對菌體生長及酵素活性之影響 75
4.2.1 分別添加Leucine、Proline及Thaimine對菌體生長及酵素活性之影響 75
4.2.2 混合添加Leucine & Proline、Leucine & Thiamine及Proline & Thiamine對菌體生長及酵素活性之影響 81
4.2.3 同時添加Leucine、Proline及Thiamine對菌體生長及酵素活性之影響 85
4.3 探討培養基中含有Glycerol之條件下,以培養過程中改變培養基中pH或延後添加的方式對菌體生長及酵素活性之影響 90
4.3.1 初始培養基中亦含有Leucine,培養至24 hr後添加NaOH對菌體生長及酵素活性之影響 90
4.3.2 培養24 hr後添加Leucine對菌體生長及酵素活性影響 95
4.3.3 培養24 hr添加Thiamine對菌體生長及酵素活性影響 100
4.4 探討培養基中含有2.5 g/L甘油之條件下,利用饋料式增加培養基中甘油濃度對菌體生長及酵素活性之影響 105
4.4.1 培養12 hr後進行兩段式添加不同濃度之甘油 105
4.4.2 培養24 hr後每隔12小時添加微量之甘油 111
4.5 探討甘油對於菌體生長及酵素活性明顯助益之緣故 117
4.5.1添加不同培養時間醱酵液對菌體生長及酵素活性影響 117
4.5.1a 添加5 ml醱酵液於50 ml新鮮培養基中 118
4.5.1b 添加11 ml醱酵液於50 ml新鮮培養基中 122

第五章 結論 128
參考文獻 132
附錄 143
附錄A-1 乾菌重對OD600nm之檢量線 143
附錄A-2 E. coli HB101(pTrcKnPAC2902)之生長曲線 144
附錄B-1 盤尼西林醯化酶校正曲線之製作 145
附錄B-2 盤尼西林醯化酶之實際活性與量測值之比較 148
附錄B-3 超音波探針系統對酵素活性之影響 151
附錄B-4 超音波探針系統對釋出細胞內酵素活性之影響 152
附錄C 甘油(Glycerol)校正曲線之製作 153



圖目錄


圖2-1. Providencia rettgeri生產之盤尼西林G醯化酶之結構圖 5
圖2-2. Escherichia coli ATCC11105生產之盤尼西林G醯化酶與盤尼西林G在其活性位置上之示意圖 6
圖2-3. Escherichia coli ATCC11105合成盤尼西林醯化酶之途徑 7
圖2-4 苯乙酸(PAA)濃度對E. coli Ny. I/3-67生產盤尼西林醯化酶活性之影響 13
圖2-5.苯乙酸添加量對E. coli ATCC11105及E. coli G270菌體生長、培養基之pH、酵素活性及比活性之影響 14
圖2-6. IPTG對E. coli JM101(pPA102)生產盤尼西林醯化酶之影響 16
圖2-7. IPTG對 E. coli HB101(pTrcKnPAC2902)生產盤尼西林醯化酶之影響 16
圖2-8. 添加不同濃度之葡萄糖(5~20 g/L)對盤尼西林醯化酶活性生產之影響 18
圖2-9. 甘油對E. coli Ny. I/3-67生產盤尼西林醯化酶活性之影響 18
圖2-10. 葡萄糖及cAMP對於生產盤尼西林醯化酶活性之影響 19
圖2-11. 培養基之pH值對於E. coli ATCC11105與 E. Coli DH5 (pUSAD2)生產盤尼西林醯化酶之影響 20
圖2-12. 溫度(20~40℃)對於E. coli ATCC11105與 E. Coli DH5 (pUSAD2)生產盤尼西林醯化酶之影響 21
圖2.13. Bacillus sp. PGS 10於有、無最適化之培養基中(a)酵素活性, (b)乾菌重, (c)酵素比活性之表現 22
圖2-14. 薄膜回流式醱酵槽示意圖 23
圖2-15. 利用濃縮氧氣通氣法(OEA)培養E. coli 9633(pGL-5)以達到高細胞密度培養 24
圖2-16. 盤尼西林醯化酶水解盤尼西林G 25
圖2-17. pH值對盤尼西林醯化酶活性之影響 25
圖2-18. 化學法與酵素催化法生產6-APA 27
圖2-19. 盤尼西林醯化酶催化進行醯化及去醯化反應 28
圖2-20. 利用盤尼西林G醯化酶合成xemilofiban hydrochloride 29
圖4.1.1(A) 培養基中添加不同甘油濃度時之生長曲線 46
圖4.1.1(B) 培養基中添加不同甘油濃度時,pH隨時間之變化 46
圖4.1.1(C) 培養基中添加不同甘油濃度時,培養基中甘油濃度隨時間之變化 47
圖4.1.1(D) 培養基中添加不同甘油濃度時,胞內酵素體積活性(U/L)隨時間之變化 47


圖4.1.1(E) 培養基中添加不同甘油濃度時,酵素比活性(U/L-OD600nm)隨時間之變化 48
圖4.1.1(F) 培養基中添加不同甘油濃度時,胞外酵素體積活性(U/L)隨時間之變化 48
圖4.1.2(A) 培養基中添加不同Leucine濃度時之生長曲線 50
圖4.1.2 (B) 培養基中添加不同Leucine濃度時,pH隨時間之變化 50
圖4.1.2 (C) 培養基中添加不同Leucine濃度時,胞內酵素體積活性(U/L)隨時間之變化 51
圖4.1.2(D) 培養基中添加不同Leucine濃度時,酵素比活性(U/L-OD600nm)隨時間之變化 51
圖4.1.2(E) 培養基中添加不同Leucine濃度時,胞外酵素體積活性(U/L)隨時間之變化 52
圖4.1.2a(A) 培養基中添加不同Leucine濃度時之生長曲線 53
圖4.1.2a(B) 培養基中添加不同Leucine濃度時,pH隨時間之變化 53
圖4.1.2a(C) 培養基中添加不同Leucine濃度時,胞內酵素體積活性(U/L)隨時間之變化 54
圖4.1.2a(D) 培養基中添加不同Leucine濃度時,酵素比活性(U/L-OD600nm)隨時間之變化 54


圖4.1.2a(E) 培養基中添加不同Leucine濃度時,胞外酵素體積活性(U/L)隨時間之變化 55
圖4.1.2b(A) 培養基中添加不同Leucine濃度時之生長曲線 56
圖4.1.2b(B) 培養基中添加不同Leucine濃度時,pH隨時間之變化 57
圖4.1.2b(C) 培養基中添加不同Leucine濃度時,胞內酵素體積活性(U/L)時間之變化 57
圖4.1.2b(D) 培養基中添加不同Leucine濃度時,酵素比活性(U/L-OD600nm)隨時間之變化 58
圖4.1.2b(E) 培養基中添加不同Leucine濃度時,胞外酵素體積活性(U/L)隨時間之變化 58
圖4.1.3a(A) 培養基中添加不同Proline濃度時之生長曲線 60
圖4.1.3a(B) 培養基中添加不同Proline濃度時,pH隨時間之變化 60
圖4.1.3a(C) 培養基中添加不同Proline濃度時,胞內酵素體積活性(U/L)隨時間之變化 61
圖4.1.3a(D) 培養基中添加不同Proline濃度時,酵素比活性(U/L-OD600nm)隨時間之變化 61
圖4.1.3a(E) 培養基中添加不同Proline濃度時,胞外酵素體積活性(U/L)隨時間之變化 62
圖4.1.3b(A) 培養基中添加不同Proline濃度時之生長曲線 63
圖4.1.3b(B) 培養基中添加不同Proline濃度時,pH隨時間之變化 64
圖4.1.3b(C) 培養基中添加不同Proline濃度時,胞內酵素體積活性(U/L)隨時間之變化 64
圖4.1.3b(D) 培養基中添加不同Proline濃度時,酵素比活性(U/L-OD600nm)隨時間之變化 65
圖4.1.3b(E) 培養基中添加不同Proline濃度時,胞外酵素體積活性(U/L)隨時間之變化 65
圖4.1.4a(A) 培養基中添加不同Thiamine濃度時之生長曲線 67
圖4.1.4a(B) 培養基中添加不同Thiamine濃度時,pH隨時間之變化 67
圖4.1.4a(C) 培養基中添加不同Thiamine濃度時,胞內酵素體積活性(U/L)隨時間之變化 68
圖4.1.4a(D) 培養基中添加不同Thiamine濃度時,酵素比活性(U/L-OD600nm)隨時間之變化 68
圖4.1.4a(E) 培養基中添加不同Thiamine濃度時,胞外酵素體積活性(U/L)隨時間之變化 69
圖4.1.4b(A) 培養基中添加不同Thiamine濃度時之生長曲線 70
圖4.1.4b(B) 培養基中添加不同Thiamine濃度時pH隨時間變化 71
圖4.1.4b(C) 培養基中添加不同Thiamine濃度時,胞內酵素體積活性(U/L)隨時間之變化 71
圖4.1.4b(D) 培養基中添加不同Thiamine濃度時,酵素比活性(U/L-OD600nm)隨時間之變化 72
圖4.1.4b(E) 培養基中添加不同Thiamine濃度時,胞外酵素體積活性(U/L)隨時間之變化 72
圖4.2.1(A) 分別添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基之生長曲線 78
圖4.2.1(B) 分別添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,pH隨時間之變化 78
圖4.2.1(C) 分別添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,甘油濃度隨時間之變化 79
圖4.2.1(D) 分別添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,胞內酵素體積活性(U/L)隨時間之變化 79
圖4.2.1(E) 分別添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,酵素比活性(U/L-OD600nm)隨時間之變化 80
圖4.2.1(F) 分別添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,胞外酵素體積活性(U/L)隨時間之變化 80
圖4.2.2(A) 混合添加Leucine & Proline、Leucine & Thiamine及Proline & Thiamine於含有2.5 g/L甘油培養基之生長曲線 82


圖4.2.2(B) 混合添加Leucine & Proline、Leucine & Thiamine及Proline & Thiamine於含有2.5 g/L甘油培養基pH隨時間變化 82
圖4.2.2(C) 混合添加Leucine & Proline、Leucine & Thiamine及Proline & Thiamine於含有2.5 g/L甘油培養基,甘油濃度隨時間之變化 83
圖4.2.2(D) 混合添加Leucine & Proline、Leucine & Thiamine及Proline & Thiamine於含有2.5 g/L甘油培養基,胞內酵素體積活性(U/L)隨時間之變化 83
圖4.2.2(E) 混合添加Leucine & Proline、Leucine & Thiamine及Proline & Thiamine於含有2.5 g/L甘油培養基,酵素比活性(U/L-OD600nm)隨時間之變化 84
圖4.2.2(F) 混合添加Leucine & Proline、Leucine & Thiamine及Proline & Thiamine於含有2.5 g/L甘油培養基,胞外酵素體積活性(U/L)隨時間之變化 84
圖4.2.3(A) 同時添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基之生長曲線 86
圖4.2.3(B) 同時添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,pH隨時間之變化 86


圖4.2.3(C) 同時添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,甘油濃度隨時間之變化 87
圖4.2.3(D) 同時添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,胞內酵素體積活性(U/L)隨時間之變化 87
圖4.2.3(E) 同時添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,酵素比活性(U/L-OD600nm)隨時間之變化 88
圖4.2.3(F) 同時添加Leucine、Proline及Thiamine於含有2.5 g/L甘油培養基,胞外酵素體積活性(U/L)隨時間之變化 88
圖4.3.1(A) 初始培養基中含有甘油與Leucine,培養至24 hr後添加NaOH之生長曲線 92
圖4.3.1(B) 初始培養基中含有甘油與Leucine,培養至24 hr後添加NaOH,pH隨時間之變化 92
圖4.3.1(C) 初始培養基中含有甘油與Leucine,培養至24 hr後添加NaOH,甘油濃度隨時間之變化 93
圖4.3.1(D) 初始培養基中含有甘油與Leucine,培養至24 hr後添加NaOH,胞內酵素體積活性(U/L)隨時間之變化 93
圖4.3.1(E) 初始培養基中含有甘油與Leucine,培養至24 hr後添加NaOH,酵素比活性(U/L-OD600nm)隨時間之變化 94


圖4.3.1(F) 初始培養基中含有甘油與Leucine,培養至24 hr後添加NaOH,胞外酵素體積活性(U/L)隨時間之變化 94
圖4.3.2(A) 初始培養基中含有甘油,培養至24 hr後添加Leucine之生長曲線 97
圖4.3.2(B) 初始培養基中含有甘油,培養至24 hr後添加Leucine,pH隨時間之變化 97
圖4.3.2(C) 初始培養基中含有甘油,培養至24 hr後添加Leucine,甘油濃度隨時間之變化 98
圖4.3.2(D) 初始培養基中含有甘油,培養至24 hr後添加Leucine,胞內酵素體積活性(U/L)隨時間之變化 98
圖4.3.2(E) 初始培養基中含有甘油,培養至24 hr後添加Leucine,酵素比活性(U/L-OD600nm)隨時間之變化 99
圖4.3.2(F) 初始培養基中含有甘油,培養至24 hr後添加Leucine,胞外酵素體積活性(U/L)隨時間之變化 99
圖4.3.3(A) 初始培養基中含有甘油,培養至24 hr後添加Thiamine之生長曲線 102
圖4.3.3(B) 初始培養基中含有甘油,培養至24 hr後添加Thiamine,pH隨時間之變化 102


圖4.3.3(C) 初始培養基中含有甘油,培養至24 hr後添加Thiamine,甘油濃度隨時間之變化 103
圖4.3.3(D) 初始培養基中含有甘油,培養至24 hr後添加Thiamine,胞內酵素體積活性(U/L)隨時間之變化 103
圖4.3.3(E) 初始培養基中含有甘油,培養至24 hr後添加Thiamine,酵素比活性(U/L-OD600nm)隨時間之變化 104
圖4.3.3(F) 初始培養基中含有甘油,培養至24 hr後添加Thiamine,胞外酵素體積活性(U/L)隨時間之變化 104
圖4.4.1(A) 初始培養基中含有甘油,培養12 hr後進行兩段式添加不同甘油濃度之生長曲線 108
圖4.4.1(B) 初始培養基中含有甘油,培養12 hr後進行兩段式添加不同甘油濃度,pH隨時間之變化 108
圖4.4.1(C) 初始培養基中含有甘油,培養12 hr後進行兩段式添加不同甘油濃度,甘油濃度隨時間之變化 109
圖4.4.1(D) 初始培養基中含有甘油,培養12 hr後進行兩段式添加不同甘油濃度,胞內酵素體積活性(U/L)隨時間之變化 109
圖4.4.1(E) 初始培養基中含有甘油,培養12 hr後進行兩段式添加不同甘油濃度,酵素比活性(U/L-OD600nm)隨時間之變化 110


圖4.4.1(F) 初始培養基中含有甘油,培養12 hr後進行兩段式添加不同甘油濃度,胞外酵素體積活性(U/L)隨時間之變化 110
圖4.4.2(A) 初始培養基中含有甘油,培養24 hr後每隔12小時添加微量之甘油之生長曲線 113
圖4.4.2(B) 初始培養基中含有甘油,培養24 hr後每隔12小時添加微量之甘油,pH隨時間之變化 113
圖4.4.2(C) 初始培養基中含有甘油,培養24 hr後每隔12小時添加微量之甘油,甘油濃度隨時間之變化 114
圖4.4.2(D) 初始培養基中含有甘油,培養24 hr後每隔12小時添加微量之甘油,胞內酵素體積活性(U/L)隨時間之變化 114
圖4.4.2(E) 初始培養基中含有甘油,培養24 hr後每隔12小時添加微量之甘油,酵素比活性(U/L-OD600nm)隨時間之變化 115
圖4.4.2(F) 初始培養基中含有甘油,培養24 hr後每隔12小時添加微量之甘油,胞外酵素體積活性(U/L)隨時間之變化 115
圖4.5.1a(A) 添加不同培養時間醱酵液於新鮮培養基中生長曲線 119
圖4.5.1a(B) 添加不同培養時間之醱酵液於新鮮培養基中,pH隨時間之變化 119
圖4.5.1a(C) 添加培養時間為4 hr之醱酵液於新鮮培養基中,甘油濃度隨時間之變化 120
圖4.5.1a(D) 添加不同培養時間之醱酵液於新鮮培養基中,胞內酵素體積活性(U/L)隨時間之變化 120
圖4.5.1a(E) 添加不同培養時間之醱酵液於新鮮培養基中,酵素比活性(U/L-OD600nm)隨時間之變化 121
圖4.5.1a(F) 添加不同培養時間之醱酵液於新鮮培養基中,胞外酵素體積活性(U/L)隨時間之變化 121
圖4.5.1b(A) 添加不同培養時間醱酵液於新鮮培養基中生長曲線 123
圖4.5.1b(B) 添加不同培養時間之醱酵液於新鮮培養基中,pH隨時間之變化 123
圖4.5.1b(C) 添加培養時間為4 hr之醱酵液於新鮮培養基中,甘油濃度隨時間之變化 124
圖4.5.1b(D) 添加不同培養時間之醱酵液於新鮮培養基中,胞內酵素體積活性(U/L)隨時間之變化 124
圖4.5.1b(E) 添加不同培養時間之醱酵液於新鮮培養基中,酵素比活性(U/L-OD600nm)隨時間之變化 125
圖4.5.1b(F) 添加不同培養時間之醱酵液於新鮮培養基中,胞外酵素體積活性(U/L)隨時間之變化 125
附圖A-1. 乾菌重對OD600nm之檢量線 143
附圖A-2. E. coli HB101(pTrcKnPAC2902)之生長曲線 144
附圖B-1. Penicillin acylase校正曲線 146
附圖B-2. 盤尼西林醯化酶之實際活性與量測值之比較 149
附圖B-3. 超音波探針系統處理時間對PAC酵素活性之影響 151
附圖B-4. 超音波探針系統處理時間對釋出細胞內酵素活性影響 152
附圖C. 甘油濃度之校正曲線 154




表目錄


表2-1. 使用微生物生產工業酵素之優點 2
表2-2. 微生物生產之產品種類 3
表2-3. 盤尼西林G醯化酶之微生物來源 4
表2-4. 1979年~1987年間利用突變或基因重組技術所得之高酵素產能菌株 9
表2-5. IPTG對E. coli DH5?pQEA11)生產盤尼西林醯化酶之影響 17
表3-1. 固態平面培養基之組成 31
表3-2. 預培養與主培養之液態培養基組成 32
表4-1. LB培養基中,添加不同濃度的Glycerol對於菌體濃度及酵素活性之影響(培養後48小時取樣) 73
表4-2. LB培養基中,添加不同濃度的Leucine對於菌體濃度及酵素活性之影響(培養後48小時取樣) 73
表4-3. LB培養基中,添加不同濃度的Proline對於菌體濃度及酵素活性之影響(培養後48小時取樣) 74
表4-4. LB培養基中,添加不同濃度的Thiamine對於菌體濃度及酵素活性之影響(培養後48小時取樣) 74


表4-5. 初始培養基含有2.5 g/L甘油條件下,分別添加0.25 g/L Leucine、0.2 g/L Proline及0.003 g/L Thiamine對於菌體濃度及酵素活性之影響(培養後48小時取樣) 89
表4-6. 初始培養基含有2.5 g/L甘油條件下,混合添加0.25 g/L Leucine、0.2 g/L Proline及0.003 g/L Thiamine對於菌體濃度及酵素活性之影響(培養後48小時取樣) 89
表4-7. 初始培養基含有2.5 g/L甘油條件下,利用饋料式添加甘油對於菌體濃度及酵素活性之影響(培養後72小時取樣) 116
表4-8. 利用添加2.5 g/L甘油於初始培養基中,蒐集培養至4 hr之醱酵液,離心去除細胞後,分別取5 ml及11 ml添加於50 ml新鮮培養基中,對於菌體濃度及酵素活性之影響(培養後72小時取樣) 126
表4-9. 利用添加2.5 g/L甘油於初始培養基中,蒐集培養至30 hr之醱酵液,離心去除細胞後,分別取5 ml及11 ml添加於50 ml新鮮培養基中,對於菌體濃度及酵素活性之影響(培養後72小時取樣) 127
附表B-1. 不同濃度酵素溶液的配法 145
附表B-2. 不同濃度酵素溶G的配法 148
附表C. 不同濃度甘油溶液的配法 153
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