臺灣博碩士論文加值系統

使用中央研究院植物暨微生物學研究所蕭介夫教授研究室所構築之能外泌 Candida rugosa 脂肪酶 3 (CRL3) 的 Pichia pastoris GS115/CRL3 菌株，首先進行菌株改良，以 Zeocin 作為篩選標記，逐步提升其濃度以篩選具較佳異源基因表現之轉形株。當 Zeocin 濃度為 1500 μg/mL 時，篩得具最高 CRL3 活性之菌株，命名為 Pichia pastoris GS115/CRL3/Z1500。利用 500 mL Hinton`s 搖瓶，採三因子-三水準 Box-Behnken 設計之反應曲面法 (Response surface methodology, RSM) 探討此新轉形株之最適培養條件，當 FM22 培養基碳氮比為 12、接種量 8.8% 及溫度15℃，震盪培養 120 小時之 CRL3 活性為 8.47 U/mL。以該條件於五公升醱酵槽內進行批式培養，控制 pH 5.0 條件下培養之酵素活性比未控制者佳，可達 26.77 U/mL。進一步進行饋料批式培養，以 11.25 mL/h 固定流速饋料培養 216 小時，菌體濃度 (OD600) 與酵素活性分別可達 627.6 與 451.7 U/mL，但培養液中有大量甘油累積；改採仿指數饋料模式結果，甘油累積現象可大幅改善，且培養 168 小時即可得到與固定流速饋料模式相當之酵素活性 (423.2 U/mL)，生產力可提高 20%，達 2.52 U/mL/h。

Pichia pastoris GS115/CRL3, constructed by Prof. Shaw’s lab, Institute of Plant and Microbial Biology, Academia Sinica, was used to produce extracellular Candida rugosa lipase 3 (CRL3). Production strain was further improved by screening and selection on a series of agar plates in stepwise increase of Zeocin concentrations. The highest-yield strain was finally obtained and named as Pichia pastoris GS115/CRL3/Z1500. Using 500-mL Hinton`s flasks, response surface methodology (RSM) based on a three-factor three-level Box-Behnken design of experiment was used to optimize the culture conditions for CRL3 production by P. pastoris GS115/CRL3/Z1500. The optimal culture conditions were found as follows: C/N ratio in FM22, 12; inoculums size, 8.8%; temperature, 15℃. Under the optimal conditions, CRL3 activity of 8.47 U/mL was achieved after 120 h cultivation. Then, batch cultures were carried out in a 5-L bench-top fermentor under the optimal conditions. It was found that pH controlled at 5 was better than pH uncontrolled during cultivation. And a CRL3 activity of 26.77 U/mL was obtained after 96 h cultivation. Based on the result obtained, fed-batch cultures were further performed in order to attain the high cell density culture. While constant feeding rate at 11.25 mL/h was operated, OD600 of 627.6 and CRL3 activity of 451.7 U/mL were obtained, respectively after 216 h cultivation. However, a large amount of glycerol was accumulated in culture broth. While pseudo-exponential feeding mode was used instead, glycerol accumulation was minimized and CRL3 activity of 423.2 U/mL was achieved after 168 h cultivation. Productivity of 2.52 U/mL/h was obtained which was 20% higher than that of constant feeding mode.

摘要 I
Abstract II
目錄 III
圖次 VII
表次 IX
縮寫表 X

第一章 緒論 1
1.1 表現系統 ……1
1.2 嗜甲醇酵母菌 Pichia pastoris 表現系統 3
1.2.1 P. pastoris 蛋白質表現系統的歷史沿革 3
1.2.2 P. pastoris 對甲醇的代謝作用 4
1.2.3 P. pastoris 驅動異源蛋白表現之啟動子 4
1.2.3.1 酒精氧化酶啟動子 (AOX 啟動子) 4
1.2.3.2 甘油醛-3-磷酸脫氫酶啟動子 (GAP 啟動子) 6
1.2.4 P. pastoris 表現生產異源蛋白之優點 6
1.2.5 外泌性訊息胜肽 9
1.3 來自假絲酵母 Candida rugosa 的脂肪酶 9
1.3.1 脂肪酶概述 9
1.3.2 Candida rugosa 脂肪酶 11
1.3.3 Candida rugosa 脂肪酶 3 (CRL3) 13
1.4 後轉形載體放大作用 (Posttransformation vector amplification, PTVA) 15
1.4.1 Zeocin 簡介 17
1.5 反應曲面法 (Response surface methodology, RSM) 17
1.5.1 反應曲面法簡介 17
1.5.2 常用實驗設計類型 19
1.5.2.1 二水準因子設計（two-level factorial design） 19
1.5.2.2 Box-Behnken 設計法 20
1.6 重組酵母菌之高細胞密度培養 20
1.7 饋料批式培養之控制原理 21
1.8 研究動機與目的 24
第二章 材料與方法 26
2.1 使用菌株 26
2.1.1 宿主與質體構築 26
2.1.2 菌株保存方法 26
2.2 藥品與試劑 28
2.2.1 一般試藥 28
2.2.2 抗生素 28
2.2.3 緩衝液 28
2.2.4 培養基 28
2.3 實驗儀器與設備 31
2.4 逐步提升 Zeocin 濃度馴養 P. pastoris GS115/CRL3 以篩選高脂肪活性表現株 32
2.4.1 馴養步驟 32
2.4.2 篩選高脂肪酶活性表現株 33
2.5 Hinton`s 三角瓶之培養與表現 35
2.5.1 菌種活化、種培養及接種前處理 35
2.5.2 培養條件對 P. pastoris GS115/CRL3/Z1500 菌體生長及 CRL3 表現之影響 35
2.5.3 Hinton`s 三角瓶培養之反應曲面法實驗設計 37
2.6 醱酵槽生產 40
2.6.1 醱酵槽操作程序 40
2.6.2 批式培養 (batch culture) 41
2.6.2.1 pH 值控制與否對 P. pastoris GS115/CRL3/Z1500 生長及 CRL3 活性之影響 41
2.6.3 饋料批式培養 (fed-batch culture) 41
2.6.3.1 固定流速饋料模式 (constant feeding mode) 42
2.6.3.2 仿指數饋料模式 (pseudo-exponential feeding mode) 42
2.7 分析方法 43
2.7.1 菌體生長量測定 (OD600) 43
2.7.2 菌體乾重與 OD600 之換算 43
2.7.3 脂肪酶活性分析 43
2.7.4 蛋白質定量法 44
2.7.5 蛋白質膠體電泳 45
2.7.6 甘油濃度分析 47
2.7.6.1 甘油濃度標準曲線製備 48
第三章 結果與討論 49
3.1 脂肪酶活性分析方法的建立 49
3.1.1 最適反應時間的決定 49
3.1.2 最適反應基質濃度 49
3.2 菌株改良之探討 52
3.2.1 建立篩菌方法 52
3.2.2 篩菌結果 52
3.2.2.1 一次篩菌 52
3.2.2.2 透明環半徑/菌落半徑之比值與 CRL3 活性之相關性 54
3.2.2.3 二次篩菌 54
3.3 Hinton`s 三角瓶培養條件之探討 57
3.3.1 P. pastoris GS115/CRL3/Z1500 於全合成培養基 FM22 之生長曲線 57
3.3.2 培養條件對 P. pastoris GS115/CRL3/Z1500 菌體生長及 CRL3 表現之影響 57
3.3.2.1 起始 pH 值 57
3.3.2.2 培養溫度 57
3.3.2.3 培養基碳氮比 61
3.3.2.4 接種量 61
3.4 以反應曲面法探討 Hinton`s 三角瓶培養之最適條件 65
3.4.1 反應曲面法數學模式之建立 65
3.4.2 反應曲面法之最適條件探討 69
3.5 醱酵槽規模之高細胞密度培養 74
3.5.1 批式培養 (batch culture) 74
3.5.2 饋料批式培養 (fed-batch culture) 76
3.5.2.1 固定流速饋料模式 (constant feeding mode) 77
3.5.2.2 仿指數饋料模式 (pseudo-exponential feeding mode) 80
第四章 結論 86
第五章 未來展望 87
第六章 參考文獻 88

圖次
圖 1.1 嗜甲醇酵母菌之甲醇代謝路徑 5
圖 1.2 嗜甲醇酵母菌之甘油代謝路徑 7
圖 1.3 釀酒酵母外泌蛋白路徑 10
圖 1.4 Candida rugosa 脂肪酶之立體結構 14
圖 1.5 單複製數與雙複製數載體 pPICZB-β-lactamase 鑲嵌於 P. pastoris AOX1 啟動子基因座之圖示 16
圖 1.6 Zeocin 結構 18
圖 1.7 本論文之研究架構 25
圖 2.1 表現載體 pGAPZαC-CRL3 27
圖 2.2 逐步提升 Zeocin 濃度馴養 P. pastoris GS115/CRL3 以篩選高脂肪酶活性表現株之示意圖 34
圖 3.1 脂肪酶反應時間與產物 p-nitrophenol 之關係 50
圖 3.2 不同 PNPB 濃度之酵素反應速率變化 51
圖 3.3 以 YPGTA 平板培養基篩選具脂肪酶活性表現 P. pastoris GS115/CRL3 轉形株之圖示 53
圖 3.4 透明環半徑/菌落半徑之比值與 CRL3 活性之相關性 55
圖 3.5 P. pastoris GS115/CRL3/Z1500 之生長曲線 58
圖 3.6 不同溫度培養結果之 10% SDS-PAGE 電泳分析 62
圖 3.7 碳氮比、培養溫度與接種量對 P. pastoris GS115/CRL3/Z1500 表現 CRL3活性之反應曲面圖與等高線圖 70
圖 3.8 接種量、碳氮比與培養溫度對 P. pastoris GS115/CRL3/Z1500 表現 CRL3活性之反應曲面圖與等高線圖 71
圖 3.9 培養溫度、碳氮比與接種量對 P. pastoris GS115/CRL3/Z1500 表現 CRL3活性之反應曲面圖與等高線圖 72
圖 3.10 P. pastoris GS115/CRL3/Z1500 最適化培養之生長曲線 73
圖 3.11 以 Hinton`s 三角瓶培養最適條件進行批式培養 75
圖 3.12 固定流速模式之饋料批式培養 78
圖 3.13 以 10% SDS-PAGE 電泳分析不同培養時間樣品之結果 79
圖 3.14 指數饋料流量模擬圖 81
圖 3.15 仿指數模式之饋料批式培養 82
圖 3.16 以 10% SDS-PAGE 電泳分析不同饋料模式之樣品 84

表次
表 1.1 目前常用表現系統之比較 2
表 1.2 以 Pichia pastoris 為宿主表現各種異源蛋白的產量 8
表 1.3 重要之商業化脂肪酶 12
表 1.4 細菌與酵母菌在各種饋料策略進行高細胞密度醱酵培養結果 22
表 2.1 Hinton`s三角瓶培養 Box-Behnken 設計法之三因子-三水準條件 38
表 2.2 Hinton`s三角瓶培養條件之三因子-三水準 Box-Behnken 設計組合 39
表 3.1 比較藉由不同 Zeocin 濃度篩菌對於菌株外泌脂肪酶活性之影響 56
表 3.2 不同起始 pH 值對 P. pastoris GS115/CRL3/Z1500 菌體濃度及表現異源蛋白 CRL3 活性之影響 59
表 3.3 不同培養溫度對 P. pastoris GS115/CRL3/Z1500 菌體濃度及表現異源蛋白 CRL3 活性之影響 60
表 3.4 不同培養基碳氮比對 P. pastoris GS115/CRL3/Z1500 菌體濃度及表現異源蛋白 CRL3 活性之影響 63
表 3.5 不同接種量對 P. pastoris GS115/CRL3/Z1500 菌體濃度及表現異源蛋白 CRL3 活性之影響 64
表 3.6 Hinton`s三角瓶培養之 Box-Behnken 設計及其實驗結果 66
表 3.7 反應曲面法設計所得 CRL3 酵素活性之變異數分析 67
表 3.8 Hinton`s三角瓶培養之 Box-Behnken 設計迴歸分析表 68
表 3.9 不同饋料模式之培養結果比較 85

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