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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 黃慶璨 | |
dc.contributor.author | Chang-Chih Chen | en |
dc.contributor.author | 陳長志 | zh_TW |
dc.date.accessioned | 2021-06-13T17:24:13Z | - |
dc.date.available | 2008-03-03 | |
dc.date.copyright | 2005-03-03 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-01-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/39195 | - |
dc.description.abstract | 植酸酶可分解植酸為一常用之飼料添加酵素,本研究以AOX1啟動子系統於重組酵母菌Pichia pastoris大量生產Escherichia coli之植酸酶。除了以甲醇誘導調控之AOX1啟動子系統外,在P. pastoris表現系統中還有另一個持續表現模式之GAP啟動子系統。本研究中成功利用乳糖操縱子系統(lac operon)調控GAP啟動子之表現。利用AOX1啟動子表現E. coli植酸酶基因時,須以BMGY培養基培養至生長定常期後再以含甲醇之誘導培養基BMMY置換生產。若BMGY中不添加蛋白腖、酵母氮源對菌體生長影響不大。而移除誘導培養基BMMY中酵母氮源與蛋白腖並將酵母膏濃度降為原配方十分之一 (0.1%)更有助於植酸酶之生產。將P. pastoris以修飾培養基mBMGHY培養再以誘導培養基mBMMHY置換誘導P. pastoris生產植酸酶,可提高植酸酶產量並降低生產成本。甲醇濃度對P. pastoris在醱酵槽誘導植酸酶生產過程中非常重要,在醱酵槽中利用甲醇控制儀器監控控制培養基中甲醇濃度為0.5%,提供菌體穩定之碳源與誘導物可有效提高植酸酶產量。批次醱酵誘導生產後之菌體可離心回收再次進行誘導生產植酸酶,且以回收菌體進行第二與第三批次之誘導生產,由於回收利用之菌體在誘導過程中已經適應了甲醇之代謝,其植酸酶生產速率皆高於第一批次生產,降低生產成本與時間。P. pastoris以全合成培養基FBSH於5公升醱酵槽中培養,並經過甘油饋料批次培養後,培養液之OD600與生菌數分別為321與2.6 x 1010 CFU/mL。將菌體離心回收,懸浮於mBMMHY誘導培養192小時後,胞外蛋白質產量達6.4 g/L,植酸酶活性則為4,946 U/mL。
P. pastoris之GAP啟動子為持續表現之強力啟動子,可能會在培養過程中影響菌體之生長,故本研究利用E. coli之乳糖操縱子系統來調控GAP啟動子之表現。將乳糖阻遏物(lac repressor, LacI)基因轉形至P. pastoris宿主中,建構一可於胞內表現乳糖阻遏物之菌株P. pastoris KM71I。將乳糖操縱基因(lac operator, lacO)插入GAP啟動子不同位置,並將E. coli植酸酶基因接至GAP-lacO融合啟動子下游作為報導基因。在ATG轉譯起始點前214 bp位置插入lacO序列造成表現量下降50%,顯示此位置對GAP啟動子表現扮演著重要角色。若於GAP啟動子下游之ATG轉譯起始點前插入lacO序列,對GAP啟動子於P. pastoris KM71表現強度影響不大,但於乳糖阻遏物表現宿主P. pastoris KM71I中,則表現活性明顯受到抑制降為22%。培養基中添加IPTG可成功誘導lacO-GAP融合啟動子之表現,而在10 mM IPTG濃度下表現量可回復為88%。本篇為首篇利用乳糖操作子系統成功調控GAP啟動子之研究。 | zh_TW |
dc.description.abstract | Phytase can degrade phytic acid and commonly be used as feed additive. The Escherichia coli phytase was highly expressed in methylotrophic yeast P. pastoris under control of alcohol dehydrogenase (AOX1) promoter. In addition to the inducible AOX1 promoter, the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was used for protein expression in P. pastoris. In this study we successfully regulate the GAP promoter by the lac operator-repressor system. After cultivation of P. pastoris in BMGY until stationary phase, cells were recovered and resuspended in fresh BMMY medium for induction can efficiently improve the protein expression. The formulas of BMGY and BMMY were modified by reducing the concentrations of peptone, yeast nitrogen base (YNB) and yeast extract (YE). Removing the peptone and YNB in BMGY did not affect cell growth significantly. In addition, phytase production in modified induction medium mBMMHY was better than that in BMMY or FBSH. In mBMMHY, peptone as well as YNB in BMMY was removed and YE concentration was reduced to 1/10 (0.1%). Methanol concentration is very important during induction period of P. pastoris fermentation. Using an on-line methanol monitor and controller to maintain the methanol concentration at steady state in fermentor significantly increased the phytase production. After one batch of phytase production, cells may be reused for another batch induction protein production. The phytase production of second and third induction batches were faster than the first batch which may due to the adaptation of methanol metabolism. High cell density was achieved with glycerol fed-batch in minimal medium in 5-L fermentor. The optical density at 600 nm and viable cells reached 321 and 2.6 x 1010 CFU/mL before induction. The cells were resuspended in mBMMHY for induction. After 192 hours induction 6.4 g/L protein was produced in culture supernatant and the phytase activity was 4,946 U/mL.
The constitutive expression of foreign protein under control of GAP promoter in P. pastoris may affect the cell growth. In this study, the E. coli lac operator-repressor system was used to control the expression of GAP promoter. The LacI repressor gene was transformed into P. pastoris host strain and successfully expressed. The lacO operator sequence was integrated into GAP promoter of pGAPZ | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T17:24:13Z (GMT). No. of bitstreams: 1 ntu-94-D88623801-1.pdf: 1169290 bytes, checksum: 4172af380131f40f7f801e2032e23a17 (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 目 錄
壹、 前言 1 一、 植酸與植酸酶之重要性 1 二、 植酸酶之市場價值 5 三、 植酸酶基因選殖與表現相關研究 8 四、 Pichia pastoris表現系統 13 五、 以原核系統操縱子調控真核生物基因表現 19 六、 研究目的 21 貳、 材料與方法 24 一、 菌株、質體與引子 24 二、 藥品、培養基與酵素 24 1. 藥品 24 1.1 一般藥品 24 1.2 植酸酶活性測試所需試劑 29 2. 培養基 29 3. 酵素 33 三、 儀器 33 四、 實驗方法 35 1. 菌種保存 35 2. 植酸酶活性測定 35 3. 以AOX1啟動子系統生產植酸酶 36 3.1 三角瓶培養與酵素生產 36 3.1.1 P. pastoris之前置培養 36 3.1.2 P. pastoris之誘導培養 36 3.2 醱酵槽規模生產植酸酶 36 3.2.1 前置培養 – 批次培養 36 3.2.2 前置培養 – 饋料批式培養(fed-batch) 37 3.2.3 誘導培養 – 甲醇不連續添加 37 3.2.4 誘導培養 – 線上監控甲醇濃度 37 4. GAP啟動子系統之調控 38 4.1 LacI表現宿主建構 38 4.1.1 P. pastoris LacI表現質體pPIC9KI2之建構 38 4.1.2 P. pastoris之轉形(transformation) 41 4.1.3 LacI抗體之製備與純化 42 4.1.3.1 LacI表現載體pET-lacI之建構 42 4.1.3.2 LacI於E. coli中之表現與純化 42 4.1.3.3 LacI 親和性膠體之製備 43 4.1.3.4 LacI抗體之純化 43 4.1.3.5 酵素連結免疫免疫分析法(ELISA) 44 4.1.4 西方免疫雜交法(Western blot hybridization) 44 4.1.4.1 P. pastoris胞內粗萃取液之製備 44 4.1.4.2 蛋白質轉漬 45 4.1.4.3 雜交反應 45 4.2 lacO-GAP融合啟動子建構 46 4.2.1 以PCR將lacO插入pGAPZαA之GAP啟動子區域 46 4.2.2 轉形作用 49 4.2.3 南方雜交確認基因拷貝數目(copy number) 49 4.2.3.1 染色體DNA抽取 49 4.2.3.2 南方漬片(Southern blot)之製備 49 4.2.3.3 探針(Probe)製備 50 4.2.3.4 雜交反應(hybridization) 50 4.3 LacI表現對lacO-GAP融合啟動子之影響 51 4.4 IPTG對誘導lacO-GAP啟動子之影響 52 4.5 不同時間點添加IPTG對植酸酶誘導之影響 52 五、 實驗使用之套組(kit) 52 參、 結果 53 一、 以AOX1啟動子系統生產植酸酶 53 1. 三角瓶規模評估培養基成份最適化 53 1.1 不同前置培養基對P. pastoris生長之影響 53 1.2 不同誘導培養基對植酸酶生產之影響 58 1.3 誘導培養基中YNB濃度對植酸酶生產之影響 60 1.4 誘導培養基中YE濃度對植酸酶生產之影響 62 2. 醱酵槽規模培養與誘導條件測試 64 2.1 不同誘導細胞濃度對植酸酶生產之影響 64 2.2 不同誘導條件對植酸酶生產之影響 66 2.3 菌體再利用對植酸酶生產之影響 68 2.4 高細胞密度培養對植酸酶生產之影響 70 二、 GAP啟動子之調控 72 1. LacI表現宿主之建構 72 1.1 LacI表現質體pPIC9KI2之建構 72 1.2 LacI表現株P. pastoris KM71I之建構 74 1.3 LacI抗體之製備與純化 75 1.3.1 LacI表現與純化 75 1.3.2 LacI 抗體之純化 78 1.4 以西方免疫雜交法偵測LacI於P. pastoris胞內之表現 81 2. lacO-GAP啟動子之建構 83 2.1 GAP啟動子分析 83 2.2 以PCR將lacO序列插入GAP啟動子區域 85 3. 以南方雜交法確認轉殖株基因拷貝數 87 4. lacO插入位置對GAP啟動子之影響 88 5. LacI表現對lacO-GAP融合啟動子之影響 88 6. IPTG對誘導lacO-GAP啟動子之影響 89 7. 不同時間點添加IPTG對植酸酶誘導之影響 92 肆、 討論 94 一、 以AOX1啟動子系統生產植酸酶 94 二、 GAP啟動子之調控 99 伍、 結論 102 一、 以AOX1啟動子系統生產植酸酶 102 二、 GAP啟動子之調控 103 陸、 參考資料 104 圖 次 圖一、 植酸之化學結構 1 圖二、 2002年全球植酸酶市場 7 圖三、 本研究架構圖 23 圖四、 質體pPIC9KI1之構築 39 圖五、 質體pPIC9KI2之構築 40 圖六、 以PCR將lacO序列插入GAP啟動子示意圖 48 圖七、 不同濃度YNB及peptone對P. pastoris生長之影響 56 圖八、 mBMGHY中YE濃度對P. pastoris生長之影響 57 圖九、 不同誘導培養基中對植酸酶生產之影響 59 圖十、 誘導培養基中不同濃度YNB對植酸酶生產之影響 61 圖十一、 誘導培養基中不同濃度YE對植酸酶生產之影響 63 圖十二、 5L醱酵槽中培養與誘導條件對植酸酶生產之影響 65 圖十三、 前置培養基對饋料批式培養中菌體生長之影響 67 圖十四、 菌體再利用對植酸酶生產之影響 69 圖十五、 誘導培養基對高細胞密度培養植酸酶生產之影響 71 圖十六、 lacIq基因之PCR與保存 72 圖十七、 LacI表現質體以HindIII截切確認 73 圖十八、 IPTG濃度對pET-lacI/BL21(DE3)表現LacI之影響 76 圖十九、 以HisTrap column純化LacI 77 圖二十、 以親和性層析管柱純化LacI抗體 79 圖二十一、 LacI抗體純化對西方免疫雜交之影響 80 圖二十二、 以西方免疫雜交法定量KM71I中LacI表現 82 圖二十三、 GAP啟動子序列之分析 84 圖二十四、 以PCR確認lacO插入GAP啟動子位置。 85 圖二十五、 pGP與lacO插入衍生質體示意圖 86 圖二十六、 以南方雜交確認表現套組於轉殖株中拷貝數 87 圖二十七、 lacO-GAP啟動子於KM71與KM71I表現量 90 圖二十八、 不同IPTG濃度對植酸酶誘導之影響 91 圖二十九、 不同時間點添加IPTG之影響 93 表 次 表一、 常用禽類飼料中總磷與植酸磷之含量比較表 2 表二、 不同表現系統之比較 15 表三、 本研究所使用之菌株 25 表四、 本研究所使用之質體 26 表五、 本研究所使用之引子 28 表六、 培養基成本比較表 55 表七、 E. coli appA基因於不同表現宿主之異源表現 94 表八、 P. pastoris密碼子使用率與E. coli appA基因序列比較 98 | |
dc.language.iso | zh-TW | |
dc.title | 大腸桿菌植酸酶於重組酵母菌Pichia pastoris中大量表現之研究 | zh_TW |
dc.title | High level expression of E. coli phytase in recombinant yeast Pichia pastoris | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 許瑞祥,楊盛行,陳浩仁,鄭國展 | |
dc.subject.keyword | ;乳糖操縱子,啟動子,表現,大腸桿菌,植酸酶,酵母菌, | zh_TW |
dc.subject.keyword | Escherichia coli,phytase,Pichia pastoris,expression,promoter,lac opreon, | en |
dc.relation.page | 119 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-01-27 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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