臺灣博碩士論文加值系統

利用酵母菌（Pichia pastoris）表現系統表現來自大腸桿菌之植酸酶基因，重組植酸酶純化並探討其生化特性。首先以聚合酶鏈鎖反應篩選出大腸桿菌(Escherichia coli BL21 DE3) 之植酸酶基因，確認基因序列正確後將此基因接合到pGAPZC 表現載體，轉形入 Pichia pastoris SMD1168H 宿主中表現，重組植酸酶轉殖株在培養後可於胞外表現15.9 U/mL的酵素活性，經由 nickel 親和性層析進行純化，重組植酸酶之最適 pH 及溫度分別為 5.0 及 50oC，pH 與溫度安定性範圍為 pH 3.0-6.0 與 20-40oC 。植酸酶活性會被 Cu2+、Fe2+、Hg2+、Fe3+、phenylmethyl sulfonylfluoride (PMSF) 及N-tosyl-L-lysine chloromethyl ketone (TLCK) 抑制；但 Ba2+、Mg2+、Ca2+、Sr2+ 具活化效果，純化後的酵素較易被胃蛋白酶分解。
為瞭解植酸酶基因序列的最適化 (gene optimization) 對於 P. pastoris 表現系統中提高蛋白質表現量的影響，重組植酸酶純化並探討其生化特性。從大腸桿菌來源植酸酶基因，依據 P. pastoris 使用頻率較高的特定同義密碼子，設計並合成適合 P. pastoris 表現植酸酶基因的最適化基因序列 (op-phyA)，再接合到 pGAPZαC 載體中以 P. pastoris SMD1168H 表現，轉殖株經由 5L 發酵槽培養114小時後活性可達201.1U/mL。將粗酵素液經濃縮及親和性管柱純化後可得到具有活性之植酸酶，經 SDS-PAGE 電泳分析其分子量為 46 kDa。生化特性分析顯示其最適 pH 為5.0，在 pH 3.0-8.0之間有較好的安定性。最適溫度為50oC，在25-40oC 範圍有較好的溫度安定性，對於胰蛋白酶具有較佳的抵抗能力，此重組植酸酶對於植酸鈣有較高的親和力。

To obtain the phytase gene from Escherichia coli BL21 DE3, express in Pichia pastoris system and characterize their bioproperties, the gene encoding E. coli BL21 DE3 phytase was amplified by PCR. The sequence of phytase was confirmed and ligated into pGAPZaC expression vector. The plasmid (pGAPZaC-phyA) transformed into Pichia pastoris SMD1168H expression host. The recombinant phytase was expressed and secreted into broth by P. pastoris SMD1168H after 96 h cultivation and the activity was reached to 15.9 U/mL. The phytase was purified by nickel affinity chromatography and characterization of recombinant phytase. The recombinant protein with optimal pH and temperature at 5.0 and 50 oC, respectively. It was stable at pH 3.0-6.0 and 20-40 oC. The purified recombinant phytase was strongly inhibited by Cu2+, Fe2+, Hg2+, Fe3+, phenylmethyl sulfonylfluoride (PMSF) and N-tosyl-L-lysine chloromethyl ketone (TLCK), but activated by Ba2+, Mg2+, Ca2+, Sr2+. The purified recombinant phytase was sensitive to pepsin.
To optimize the phytase gene expression in Pichia pastoris system and characterize the recombinant phytase, a mature phytase cDNA of E. coli being altered according to the codons usage preference of Pichia pastoris was artificially synthesized. Pichia pastoris mutant with E. coli phytase optimized gene was cloned into expression vector of pGAPZaC. The phytase activity was 201.1 U/mL after 114 h fermentation in 5L fermentor. The recombinant phytase, with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after Ni Sepharose 6 Fast Flow chromatography. The phytase had an optimal pH and temperature of 5.0 and 50 oC, respectively. It was stable at pH 3.0-8.0 and 25-40 oC. The purified recombinant phytase was resistant to trypsin and revealed higher affinity to calcium phytate than to other phosphate conjugates.

Abbreviations 1
中文摘要 2
Abstract 4
研究動機與目的 6
實驗設計 7
第一章 ：文獻整理 9
壹、 植酸簡介 9
貳、 植酸酶簡介 11
參、 植酸酶特性 15
肆、 植酸酶應用及發展 17
一、 肥料與飼料之應用 17
二、 於食品上的應用 17
1. 應用於麵包製作 17
2. 生產植物蛋白分離物 18
3. 玉米濕磨 18
4. 分離麥麩 19
伍、 重組植酸酶之研究 19
陸、 大腸桿菌植酸分解酶 20
柒、 微生物表現系統 21
一、 Pichia pastoris表現系統 22
二、 Pichia pastoris 驅動異源蛋白質表現之啟動子 23
三、 嗜甲醇酵母菌Pichia pastoris 之分泌性訊息胜肽 25
四、 密碼子使用偏好 (codon usage preference) 26
第二章 ：於酵母菌 (Pichia pastoris SMD1168H) 中表現大腸桿菌 (Escherichia coli) 植酸酶、純化及特性分析 29
壹、 中文摘要 29
貳、 英文摘要 30
參、 前言 31
肆、 實驗設計 32
伍、 實驗材料 32
一、 菌種 32
二、 質體 32
三、 藥品 32
四、 儀器 35
五、 藥品製備 37
陸、 實驗方法 42
一、 E. coli BL21(DE3)植酸酶基因之選殖及序列鑑定 42
1. 植酸酶基因專一性引子(primers)設計 42
2. Genomic DNA之萃取 43
3. 聚合酶鏈鎖反應(PCR) 44
4. 重組植酸酶基因 45
5. E. coli strain通透性細胞的製備 47
6. 質體轉形入 E. coli Top 10F' 通透性細胞 48
7. 重組植酸酶之質體DNA的抽取 49
8. DNA的定序(Sequencing) 51
二、 構築重組植酸酶基因於Pichia pastoris表現系統 51
1. 限制酶作用 51
2. 目標基因膠體純化 52
3. 質體接合作用 (ligation) 52
4. 質體轉形入E.coli TOP10F´通透性細胞 53
5. P. pastoris SMD1168H通透性細胞的製備 53
6. 質體電轉形入P. pastoris SMD1168H通透性細胞 54
7. Pichia pastoris SMD1168H轉殖株之鑑定 55
8. 電轉形 P. pastoris SMD1168H之質體穩定性 56
三、 重組植酸酶基因於Pichia pastoris系統的表現 56
1. 重組植酸酶菌株培養 57
2. 菌數、植酸酶活性與培養液pH之監測 57
四、 重組植酸酶的純化 60
1. 粗酵素液的製備 60
2. 超過濾濃縮 (Ultra-filtration) 60
3. Ni Sepharose 6 Fast Flow 親和性層析 61
4. 蛋白質濃度測定 61
5. 重組植酸酶之分子量 62
五、 重組植酸酶之生化特性分析 63
1. 最適反應溫度 63
2. 最適反應pH值 63
3. 溫度安定性 64
4. pH值安定性 64
5. 抑制劑對重組植酸酶的影響 65
6. 還原劑對重組植酸酶的影響 65
7. 金屬離子對重組植酸酶的影響 65
8. 基質之特異性 66
9. Pepsin 與 trypsin耐受性試驗 66
柒、 結果與討論 66
一、 pGAPZC-phyA酵母菌表現系統之構築、篩選與表現 66
二、 重組植酸酶之純化 70
三、 重組植酸酶之生化特性 72
1. 最適溫度之測定 72
2. 溫度安定性之測定 72
3. 最適酸鹼度試驗 73
4. 酸鹼度安定性試驗 74
5. 金屬鹽類的影響 74
6. 抑制劑與還原劑之影響 75
7. 基質之特異性 76
8. Pepsin 與 trypsin耐受性試驗 77
第三章 ：於酵母菌 (Pichia pastoris SMD1168H) 中表現大腸桿菌 (Escherichia coli) 最適化植酸酶序列與其純化 79
壹、 中文摘要 79
貳、 英文摘要 81
參、 前言 82
肆、 實驗設計 83
伍、 實驗材料 83
一、 菌種 83
二、 質體 83
三、 藥品、儀器及藥品製備 83
陸、 實驗方法 83
一、 植酸酶基因之選殖 84
1. 植酸酶基因序列之設計與合成 84
2. E. coli strain通透性細胞的製備 85
3. 質體轉形入 E. coli Top 10F' 通透性細胞 85
4. 重組植酸酶之質體DNA的抽取 85
二、 構築重組植酸酶基因於Pichia pastoris表現系統 86
1. 限制酶作用 86
2. 目標基因膠體純化 86
3. 質體接合作用 (ligation) 86
4. 質體轉形入E.coli TOP10F´通透性細胞 87
5. DNA的定序(Sequencing) 87
6. P. pastoris SMD1168H通透性細胞的製備 88
7. 質體電轉形入P. pastoris SMD1168H通透性細胞 88
8. Pichia pastoris SMD1168H轉殖株之鑑定 89
9. 電轉形 P. pastoris SMD1168H 之質體穩定性 89
三、 重組植酸酶基因於Pichia pastoris系統的表現 89
1. 重組植酸酶菌株培養 89
2. 生菌數、植酸酶活性與培養液pH之監測 90
四、 重組植酸酶的純化 90
1. 粗酵素液的製備 90
2. 超過濾濃縮(Ultra-filtration) 90
3. Ni Sepharose 6 Fast Flow 親和性層析 91
4. 蛋白質濃度測定 91
5. 重組植酸酶之分子量、醣基染色 91
五、 重組植酸酶之生化特性分析 93
1. 最適反應溫度 93
2. 最適反應pH值 93
3. 溫度安定性 93
4. pH值安定性 93
5. 抑制劑對重組植酸酶的影響 93
6. 還原劑對重組植酸酶的影響 93
7. 金屬離子對重組植酸酶的影響 93
8. 基質之特異性 93
9. 酵素動力學 93
10. Pepsin 與 trypsin 耐受性試驗 94
柒、 結果與討論 94
一、 pGAPZC-op-phyA 酵母菌表現系統之構築、篩選與表 現 ..94
二、 重組植酸酶之純化 98
三、 重組植酸酶之生化特性 100
1. 最適溫度之測定 100
2. 溫度安定性之測定 101
3. 最適酸鹼度試驗 101
4. 酸鹼度安定性試驗 102
5. 金屬鹽類的影響 103
6. 抑制劑與還原劑之影響 103
7. 基質特異性 104
8. Pepsin 與 trypsin 耐受性試驗 105
9. 酵素動力學 105
捌、結論與展望 106
玖、參考文獻 150
投稿論文 160
壹、已發表之論文 160
貳、研討會壁報論文 161

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