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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 潘子明(Tzu-Ming Pan) | |
| dc.contributor.author | Yueh-Chang Huang | en |
| dc.contributor.author | 黃岳章 | zh_TW |
| dc.date.accessioned | 2021-05-17T09:14:04Z | - |
| dc.date.available | 2017-08-22 | |
| dc.date.available | 2021-05-17T09:14:04Z | - |
| dc.date.copyright | 2012-08-22 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6475 | - |
| dc.description.abstract | 近年原油價格高漲，替代能源開發備受重視，其中生質柴油 (biodiesel) 為一種可再生性能源，可作為石化柴油的替代品。生產生質柴油之製程，使用全細胞生物觸媒 (whole-cell biocatalyst) 之脂肪酶催化法，可克服鹼催化法之催化劑無法回收和產率不高等缺點，且降低酵素純化與分離過程之成本，然而胞內酵素之全細胞生物觸媒受限於細胞壁和細胞膜之屏障而降低轉化效率，研究以細胞表面表現酵素之系統，應用於全細胞生物之生物轉化為近年研究發展之方向。
本研究使用 Lpp-OmpA 融合方式，建構重組 Geobacillus thermocatenulatus NTU 03 脂肪酶 (r03Lip) 之大腸桿菌表面表現系統，表現 Lpp-OmpA-r03Lip 之融合蛋白至大腸桿菌 BL21(DE3) 細胞表面 (cell surface expressed r03Lip, CSEr03Lip)。由西方點墨法、流式細胞儀 (FACS) 和螢光顯微鏡證實 CSEr03Lip 表現於大腸桿菌細胞表面，且不會影響大腸桿菌細胞之生長及外細胞膜結構的穩定性。酵素活性分析證實 CSEr03Lip 具完整之酵素活性，研究結果 CSEr03Lip 之最適反應溫度為 55oC、最適反應 pH 值為 9，熱穩定性和 pH 值穩定性皆與游離態 r03Lip 相似，且部分之金屬離子、界面活性劑和有機溶劑不影響其之活性，顯示 CSEr03Lip 保有高度之活性與穩定性。然而研究發現 CHAPS 提升 CSEr03Lip 活性與穩定性 21.9% 和 28.9%，鎂離子和丙酮 (acetone) 分別降低其活性及穩定性 26.4% 和 55.2%，此現象未見於游離態 r03Lip。 以 CSEr03Lip 與胞內表現 r03Lip (cytosolic expressed r03Lip, CEr03Lip) 作為全細胞生物觸媒進行脂肪酸異丙酯 (FAIPEs) 之轉化。兩者之適合轉化條件為油醇莫耳比 1：4、水含量小於 10% (w/w of oil)、反應溫度 55oC 和生物觸媒使用量 10% (w/w of oil) 以上，分別反應 20 和 28 小時轉化率可達 90% 以上。使用油炸廢棄油為原料，油脂中酸價、過氧化價隨高溫烹調時間而增加，但不影響全細胞生物觸媒對 FAIPEs 之轉化率，將 CSEr03Lip 與 CEr03Lip 之全細胞生物觸媒回收再利用，兩者使用前 4 次內轉化率達 80% 以上。 本研究使用 CSEr03Lip 之全細胞生物觸媒，相較於 CEr03Lip 系統，結果顯示 CSEr03Lip 確實增加轉化速率並降低生產 FAIPEs 之成本，但酵素提升再利用率為未來發展之重點。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2021-05-17T09:14:04Z (GMT). No. of bitstreams: 1 ntu-101-R99b22008-1.pdf: 3116229 bytes, checksum: 6f2d331eb81b5c4e4e90e229f26e36e3 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 中英文對照縮寫表 i
中文摘要 iii Abstract v 目錄 vii 圖目錄 x 表目錄 xii 第一章 研究背景 1 一、 脂肪酶 1 (一) 脂肪酶簡介 1 (二) 脂肪酶結構 1 (三) 脂肪酶分類 2 (四) 脂肪酶之應用 3 二、 生質柴油 8 (一) 簡介 8 (二) 生產製程 9 (三) 生質柴油相關議題 16 三、 細胞表面工程 18 (一) 研究源起 18 (二) 細胞表面表現系統之組成 18 (三) 微生物表面表現系統 21 (四) 細胞表面表現系統之應用 28 研究目的 31 研究架構 32 第二章 實驗材料及方法 33 一、 實驗材料 33 (一) 藥品試劑 33 (二) 儀器設備 33 二、 實驗方法 34 (一) 建構細胞表面表現脂肪酶系統 34 (二) 表現融合蛋白 40 (三) 全細胞之生物轉化 46 第三章 結果 48 一、 建構細胞表面表現脂肪酶系統 48 (一) 建構 pETOmpA 與 pETOmpA-r03Lip 質體 48 (二) Lpp-OmpA-r03Lip 融合蛋白之結構模擬 48 二、 表現融合蛋白 55 (一) 誘導表現融合蛋白 55 (二) 融合蛋白之表現位置 55 (三) 大腸桿菌生長曲線和細胞膜穩定性分析 58 (四) r03Lip 與 CSEr03Lip 之酵素活性分析 62 三、 全細胞之生物轉化 74 (一) 探討 FAIPEs 之反應條件 74 (二) 以油炸廢棄油生產 FAIPEs 80 (三) 全菌體生物觸媒回收再利用 80 討論 85 結論 89 參考文獻 90 | |
| dc.language.iso | zh-TW | |
| dc.subject | 全菌體生物觸媒 | zh_TW |
| dc.subject | 生質柴油 | zh_TW |
| dc.subject | Lpp-OmpA | zh_TW |
| dc.subject | 脂肪酸異丙酯 | zh_TW |
| dc.subject | 熱穩定性脂肪酶 | zh_TW |
| dc.subject | 細胞表面表現系統 | zh_TW |
| dc.subject | thermostable lipase | en |
| dc.subject | Lpp-OmpA | en |
| dc.subject | cell surface expression system | en |
| dc.subject | biodiesel | en |
| dc.subject | whole-cell biocatalyst | en |
| dc.subject | fatty acid isopropyl ester | en |
| dc.title | 建構 Geobacillus thermocatenulatus NTU 03 熱穩定性脂肪酶在大腸桿菌細胞表面之表現系統及生產生質柴油之應用 | zh_TW |
| dc.title | Cell surface display of Geobacillus thermocatenulatus NTU 03 thermostable lipase on Escherichia coli and its applications for biodiesel production | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蘇遠志,黃健雄,黃顯宗,林志輝 | |
| dc.subject.keyword | Lpp-OmpA,熱穩定性脂肪酶,細胞表面表現系統,生質柴油,全菌體生物觸媒,脂肪酸異丙酯, | zh_TW |
| dc.subject.keyword | Lpp-OmpA,thermostable lipase,cell surface expression system,biodiesel,whole-cell biocatalyst,fatty acid isopropyl ester, | en |
| dc.relation.page | 103 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2012-08-17 | |
| 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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