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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 李昆達(Kung-Ta Lee) | |
dc.contributor.author | Bo-Kai Chiu | en |
dc.contributor.author | 邱柏凱 | zh_TW |
dc.date.accessioned | 2021-07-11T14:43:19Z | - |
dc.date.available | 2021-11-02 | |
dc.date.copyright | 2016-11-02 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-12 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78134 | - |
dc.description.abstract | 漆酶 (EC 1.10.3.2) 是自然界廣泛存在的一種藍色多銅氧化酵素,可氧化分解木質素及包括雙酚A (bisphenol A)、鄰苯二酚 (ortho-diphenol)、對苯二酚 (para-diphenol)、胺基苯酚 (aminophenol) 等多種酚類化合物物質,同時將氧氣還原為水,常應用於紙漿工業、工業廢水處理及生質能源開發等。本實驗室先前自納豆菌Bacillus subtilis natto NTU18中選殖漆酶基因cotA,並轉殖至大腸桿菌表現,發現重組納豆菌漆酶的熱穩定性較市售雲芝漆酶為佳。為進一步優化重組大腸桿菌生產納豆菌漆酶之效能與可調控性,減低工業化生產及純化程序成本,本研究利用訊號序列修飾目標基因,及調整胞膜間區 (periplasmic space) 釋放條件等方式,探討重組大腸桿菌外泌生產納豆菌漆酶的可行性。經實驗發現,納豆菌漆酶毋需修飾訊號序列,即能自然轉移至胞膜間區。此外,藉由菌體培養過程中添加 2% glycine及Triton X-100,並採「誘導」與「釋放」分開處理的方式,可有效提升納豆菌漆酶釋放至培養基的效率,使用Hinton’s flask振盪培養可生產納豆菌漆酶活性達1.06±0.007 U/mL ,再配合細胞高密度培養技術,經20小時培養後於胞外釋放之納豆菌漆酶活性可達5.72±0.38 U/mL,較原先胞內表現並破菌的方式所得活性提高近170倍,有利於工業化生產效能之提升。 | zh_TW |
dc.description.abstract | Laccase (EC 1.10.3.2) is one of the blue multicopper oxidases that catalyze the oxidation of lignin and many aromatic substrates including bisphenol A, ortho-diphenol, para-diphenol, and aminophenol with reduction of oxygen to water. It has many applications such as dye removal in textile, industrial wastewater treatment, and biofuel development. The laccase gene cotA was cloned from Bacillus subtilis natto NTU18, and laccase CotA was produced in Escherichia coli expression system by our lab. In previous study, the higher optimum reaction temperature indicated that recombinant CotA was more themostable than commercial fungal (Trametes versicolor) laccase. In this study, 5 kinds of N-terminal and C-terminal signal peptides were added to cotA gene sequence to enhance the translocation of target protein, and the periplasmic protein was released with chemical method to upgrade the efficiency and controllability of recombinant CotA laccase production in E. coli. The results showed that CotA laccase can be transferred to periplasmic space without any signal peptide addition. And using 2% glycine and Triton X-100 after induction would increase CotA laccase release to the culture medium. The released laccase activity reached 1.06±0.007 U/mL in Hinton’s flask, and 5.72±0.38 U/mL in bioreactor with high cell-density culture for 20 hours, which was 170-fold to intracellular production. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T14:43:19Z (GMT). No. of bitstreams: 1 ntu-105-R99B22002-1.pdf: 1995291 bytes, checksum: f35265ff0f78149730b27d3a26913473 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員會審定書 I
謝誌 II 摘要 III Abstract IV 縮寫表 V 中英文對照表 VI 目錄 VII 圖表目錄 IX 第1章 前言 1 1.1. 大腸桿菌 (Escherichia coli) 外泌系統 1 1.1.1. 第一型外泌系統 (Type I secretion system) 1 1.1.2. 第二型外泌系統 (Type II secretion system) 2 1.2. 異源蛋白質的表現與外泌 2 1.3. 漆酶 (laccase) 4 1.3.1. 起源與功能 4 1.3.2. 結構 5 1.3.3. 應用 5 1.4. 漆酶的異源表現 6 1.5. 納豆菌 7 1.6. 研究目的 7 1.7. 研究大綱 8 第2章 材料與方法 9 2.1. 菌株及基因載體 9 2.1.1. 熱休克 (heat shock) 細胞轉形法 10 2.2. 含訊號序列之納豆菌漆酶重組基因與質體建構 10 2.2.1. 納豆菌漆酶基因cotA 10 2.2.2. 訊號序列 (signal peptide, sp) 10 2.2.3. E. coli M15/pQE-30 Xa-cotA-hlyA表現系統建構 12 2.2.4. E. coli M15/pQE-30 Xa-(Type II signal peptide)-cotA質體建構 12 2.2.5. E. coli BL21 (DE3)/pEXP5-CT/TOPO®-(Type II signal peptide)-cotA質體建構 13 2.3. 重組大腸桿菌培養與誘導異源漆酶表現 13 2.3.1. 500 mL Hinton’s flask振盪培養 13 2.3.2. 5 L醱酵槽培養 13 2.4. 冷滲透休克法 (Cold osmotic shock) 15 2.5. Glycine /Triton X-100釋放法 15 2.6. 漆酶活性測定 16 2.7. SDS-PAGE電泳與活性染色分析 16 第3章 結果 18 3.1. 納豆菌漆酶 (CotA laccase) 於E. coli M15/pQE-30 Xa系統之表現 18 3.1.1. 含訊號序列 (signal peptide) 之納豆菌漆酶的建構與表現 18 3.1.2. 納豆菌漆酶在胞膜間區 (periplasmic space) 之累積 18 3.2. 納豆菌漆酶於E. coli BL21 (DE3)/pEXP-5CT/TOPO®系統之表現 19 3.2.1. 含訊號序列納豆菌漆酶的建構與表現 19 3.2.2. 納豆菌漆酶在胞膜間區之累積 19 3.2.3. 胞內納豆菌漆酶的釋放 19 3.3. 漆酶釋放條件的最適化 20 3.3.1. 最適誘導期 21 3.3.2. 最適釋放期 21 3.4. 以細胞高密度培養策略外泌生產納豆菌漆酶 22 第4章 討論 23 4.1. 訊號序列對CotA laccase生產與外泌的影響 23 4.1.1. 訊號序列效能評估 23 4.1.2. CotA laccase在胞膜間區 (periplasmic space) 的累積 23 4.2. CotA laccase的釋放策略 24 4.2.1. 釋放策略的比較 24 4.2.2. 誘導與釋放策略的調整 24 4.3. 細胞高密度培養的成效與策進 26 4.3.1. 細胞高密度培養對CotA laccase外泌生產的提升 26 4.3.2. 未來策進方向 26 4.4. 結論 27 圖表 28 參考文獻 37 附錄 40 | |
dc.language.iso | zh-TW | |
dc.title | 以重組大腸桿菌外泌生產耐熱型漆酶 | zh_TW |
dc.title | Secretory production of thermostable laccase in recombinant Escherichia coli | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 蘇遠志,劉俊民,陳俊任,劉裕國 | |
dc.subject.keyword | 漆?,納豆菌,大腸桿菌,外泌生產, | zh_TW |
dc.subject.keyword | Laccase,Bacillus subtilis natto,Escherichia coli,Extracellular production, | en |
dc.relation.page | 47 | |
dc.identifier.doi | 10.6342/NTU201600857 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-08-12 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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