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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王愛玉(Ai -Yu Wang),宋賢一(Hsien-Yi Sung) | |
| dc.contributor.author | Chun-Chi Lin | en |
| dc.contributor.author | 林淳琦 | zh_TW |
| dc.date.accessioned | 2021-06-08T07:17:16Z | - |
| dc.date.copyright | 2011-08-17 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-08-10 | |
| dc.identifier.citation | Akira A, Ayako K (2006) Expression for actinomycete-origin cytochrome P-450 in Escherichia coli. In, Vol US0234337
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26607 | - |
| dc.description.abstract | Cytochrome P450 monooxygenases (P450s) 是由超級家族(superfamily) 基因所表現，普遍存在於生物體中。P450s催化多樣反應，包括carbon hydroxylation、dealkylation、epoxidation及dehalogenation，並且參與藥物的轉化、異種生物化合物的轉換以及生理上重要化合物的生合成。
Nonomuraea angiospora 為放線菌之一株，為探討N. angiospora P450催化之反應，本研究以硫酸銨分劃及DEAE-Sephacel離子交換管柱層析自N. angiospora 中純化P450。然而，純化所得的酵素濃度偏低，不易偵測。因此將N. angiospora之P450基因選殖入表現載體中，轉形至酵母菌Pichia pastioris X-33表現重組P450蛋白質。以TALON® Metal Affinity Resin純化末端帶有His-tag之重組P450蛋白質，可於SDS-PAGE及免疫轉印分析中觀察到重組P450蛋白質色帶。於in vitro的活性分析中未見重組P450蛋白質之轉化活性，可能由於重組P450蛋白質並未與heme結合，或者因為缺少了P450催化反應時所需的適當redox partners所致。 | zh_TW |
| dc.description.abstract | Cytochrome P450 monooxygenases (P450s), which are encoded by a superfamily of genes, are widely distributed in various organisms. They catalyze a broad of versatility reactions such as carbon hydroxylation, dealkylation, epoxidation and dehalogenation, and are involved in biotransformation of drugs, bioconversion of xenobiotics, biosynthesis of physiologically important compounds, and so on.
Nonomuraea angiospora belongs to Actinobacteria.To investigate the reactions catalyzed by P450 of N. angiospora, the enzyme was purified by ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography. However, the enzyme activity could not be detected, which might due to the low level of proteins. The plasmid carrying the open reading frame of a P450 gene of N. angiospora was transformed into Pichia pastioris X-33 for expression of recombinant P450. The His- tagged recombinant proteins were purified by TALON® Metal Affinity Resin. A major protein band was observed when the Co2+-based IMAC- purified proteins were analyzed by SDS-PAGE and immunoblotting. The result of in vitro activity assay showed that the recombinant P450 did not exhibit enzyme activity , which might due to the absence of heme group in the recombinant protein and/or the lack of appropriate redox partners for coupling the reaction catalyzed by P450. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T07:17:16Z (GMT). No. of bitstreams: 1 ntu-100-R98b47212-1.pdf: 2864044 bytes, checksum: a601bab1aaa1e100e3026a10ad9f44ec (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 摘要i
Abstract ii Abbreviation iii 第一章 緒論 1 1 細胞色素P450 (Cytochrome P-450) 1 1.1 細胞色素P450簡介 1 1.2 Cytochrome P450 命名法則 2 1.3 P450 電子傳遞系統及反應機制 3 1.4 Cytochrome P450 之生理功能及其應用 4 2 降膽固醇藥物 5 2.1 膽固醇 (Cholesterol) 簡介及其相關疾病 5 2.2 非statins類降膽固醇藥物發展 6 2.3 Satins 類藥物 7 2.4 Pravastatin 簡介及其應用 10 3 微生物P450 應用於生產pravastatin之發展 11 4 放線菌 (Actinobacteria) 簡介 12 5 研究動機與目的 13 第二章 材料與方法 15 1 實驗材料與藥品 15 1.1 菌種 15 1.2 質體 15 1.3 實驗藥品 16 2 實驗儀器與設備 16 2.1 核酸電泳 16 2.2 蛋白質電泳 16 2.3 離心機 17 2.4 高效液相層析儀 17 2.5 細胞破碎儀 17 2.6 其他儀器 17 3 實驗方法 18 3.1 放線菌培養及分析 18 3.2 放線菌RNA分離與分析 20 3.3 放線菌P450之蛋白質純化 24 3.4 放線菌P450蛋白質分析 25 3.5 DNA 之分離與分析 27 3.6 質體對酵母菌Pichia pastoris X-33之轉形與轉形株篩選 29 3.7 酵母菌 Pichia pastoris X-33轉形株篩選 30 3.8 酵母菌轉形株重組P450蛋白質之表現 31 3.9 重組P450蛋白質之純化 32 3.10 重組P450蛋白質分析 34 第三章 結果與討論 37 1 放線菌N. angiospora 生理探討 37 1.1 生長曲線 37 1.2 Compactin 耐受度探討 37 1.3 P450 基因表現探討 40 2 N. angiospora P450蛋白質之探討 40 2.1 P450蛋白質之誘導及純化 40 2.2 P450 in vitro活性分析 41 2.3 P450之光譜性質 41 3 酵母菌Pichia pastoris X-33表現重組cytochrome P450蛋白質 42 3.1 轉形株鑑定及篩選 43 3.2 重組P450蛋白質最適表現條件探討 43 3.3 重組P450蛋白質之大量表現及純化 44 4 重組cytochrome P450蛋白質探討 45 4.1 P. pastoris X-33轉形株in vitro 活性分析 45 4.2 P. pastoris X-33轉形株in vivo活性分析 45 4.3 光譜性質 46 4.4 外加Heme之活性影響 46 4.5 添加 d-Aminolevulinic acid對活性之影響 47 4.6 以N.angiospora酵素粗抽液作為redox partners之來源in vitro 活性分析 47 5 討論 48 第四章 結論與未來展望 50 1 結論 50 2 未來展望 50 參考資料 53 圖與表61 附錄91 | |
| dc.language.iso | zh-TW | |
| dc.subject | 放線菌 | zh_TW |
| dc.subject | 細胞色素P450 | zh_TW |
| dc.subject | compactin | en |
| dc.subject | actinomyces | en |
| dc.subject | pravastatin | en |
| dc.subject | Cytochrome P450 | en |
| dc.title | 放線菌Nonomuraea angiospora Cytochrome P450 Monooxygenase之純化與檢定 | zh_TW |
| dc.title | Purification and Characterization of Nonomuraea angiospora
Cytochrome P450 Monooxygenase | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳慶三,張珍田,林忠亮 | |
| dc.subject.keyword | 細胞色素P450,放線菌, | zh_TW |
| dc.subject.keyword | Cytochrome P450,compactin,pravastatin,actinomyces, | en |
| dc.relation.page | 100 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2011-08-11 | |
| 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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