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研究生:吳玫慧
研究生(外文):Wu, Mei-Huey
論文名稱:由lovastatin誘導Clonostachyscompactiuscula產生esterase之純化流程及生化特性分析
論文名稱(外文):Purification and biochemical properties of lovastatin induced esterase from Clonostachys compactiuscula
指導教授:翁秉霖
指導教授(外文):Ong, Ping-Lin
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生化科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
中文關鍵詞:lovastatin酯解酵素
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Lovastatin esterase(LE) 是以lovastatin 誘導Clonostachys compactiuscula所分泌之酯解酵素,對lovastatin C-8 酯鍵具有水解能力而轉化成Triol acid產物。在菌體培養第五天加入5 ml、2.5 mM Lovastatin誘導酵素產生,培養第十天可得LE活性。結合原態電泳分析及Fast Blue B膠體活性染色法確認膠片上四群酯解酵素位置(N1~N4),分別以HPLC酵素活性分析,確定編號N4膠條萃取物含有LE活性。在蛋白質純化流程中,以Q sepharose FF(20 mM Tris pH 8)純化LE,於210 mM氯化鈉濃度始可洗提出具lovastatin 酯解酵素活性。以lovastatin聯結親和層析膠體純化(20 mM Tris pH 8),於480 mM氯化鈉濃度時可洗提出LE活性之酵素蛋白,再以SDS-PAGE分析蛋白質分子量,配合親和層析、Native PAGE純化、gel slice及 HPLC的分析,得知Lovastatin 酯解酵素 (LE)的分子量大小約為55.1 kDa。根據LE蛋白質分子量、功能性及親源性進行資料庫真菌酯解酵素相關序列搜尋及比對,依種類及親源性分為六群,其中fungus esterase part 1、2無法鑑定出序列保留區域,設計出四組引子。經過PCR DNA增幅及DNA序列定序,目前尚無法獲得酯解酵素DNA之相關DNA片斷,未來將再進一步針對LE蛋白質序列解序及精準設計弱化引子,並純化mRNA提高PCR增幅效能,以達量產之目標。
Lovastatin esterase (LE), lovastatin-induced from Clonostachys compactiuscula, acts on the C-8 ester bond of lovastatin to produce triol acid. Mycelium culture was induced with 5ml, 2.5 mM lovastatin at day 5 for LE production and active LE broth was obtained at day 10. There were four groups of esterases (N1~N4) shown on native-PAGE gel stained with fast blue B esterase active staining methods. Only N4 extract contains lovastatin esterase activity analyzed with HPLC enzymatic assay. In protein purification procedure, LE was eluted from Q Sepharose FF (20 mM Tris , pH 8) at 210 mM NaCl. Synthesizes the affinity column by lovastatin as ligand. LE was eluted from affinity column (20 mM Tris , pH 8) at 480 mM. Proteins in N4 extract passing purification with affinity column were separated by SDS-PAGE based on molecular size and the excised gels were tested for LE activity with HPLC analysis. The molecular weight of lovastatin esterase was supposed to be 56.2 kDa. Based on molecular weight, enzymatic function and sequence homology of esterase, the subject searched from NCBI and PIR databases were divided into six groups. Only 4 sets of primers were designed from the conserved region of six groups, except fungus esterase part 1 and 2. After PCR amplification, gene cloning and DNA sequencing, the PCR gene products were not related to any groups of the esterase gene. We will devote to LE protein N-terminal sequencing and more precise degenerate primers design in coming future. Refining mRNA extraction and optimizing PCR amplification will enhance the efficiency of LE gene cloning for protein production.
目錄……………………………………………………………………i
中文摘要………………………………………………………………iv
英文摘要………………………………………………………………v
圖目錄…………………………………………………………………vi
表目錄…………………………………………………………………viii
附錄……………………………………………………………………ix
第一章 前言…………………………………………………………1
1.1 高血脂………………………………………………………1
1.2 高血脂治療藥物-statin…………………………………3
1.3 Simvastatin合成方式………………………………………4
1.4 Clnonstachys compactiuscula簡介………………………5
1.5 酯解酵素的種類……………………………………………6
1.6 基因轉殖……………………………………………………8
1.7 研究動機及目的……………………………………………10
第二章 實驗材料…………………………………………………11
2.1 實驗菌株……………………………………………………11
2.2 實驗試劑……………………………………………………11
2.3 實驗儀器……………………………………………………14
第三章 實驗方法…………………………………………………17
3.1 菌絲體培養…………………………………………………17
3.2 蛋白液粗萃取………………………………………………18
3.3 酯解酵素活性電泳分析法…………………………………18
3.4 Lovastatin 酯解酵素活性電泳定位……………………20
3.5 離子交換層析………………………………………………20
3.6 Lovastatin聯結親和層析 ………………………………22
3.7 以高效能液相層析分析lovastatin 酯解酵素活性……23
3.8 SDS聚丙烯醯銨膠體電泳…………………………………24
3.9 蛋白質銀染色法……………………………………………25
3.10 蛋白質鋅染色法……………………………………………26
3.11 Lovastatin酯解酵素之酵素動力學………………………26
3.12 RNA萃取與製備……………………………………………27
3.13 反轉錄鏈聚合反應 (RT-PCR) 建立cDNA library ……27
3.14 序列檢索及引子設計………………………………………29
3.15 聚合酶鏈鎖反應 (PCR) …………………………………29
3.16 純化PCR產物………………………………………………30
3.17 分離質體DNA………………………………………………30
3.18 接合作用……………………………………………………31
3.19 勝任細胞 ( competent cell )之備製及轉形…………31
3.20 純化質體DNA………………………………………………32
3.21 定序…………………………………………………………33
第四章 結果………………………………………………………34
4.1 菌絲體生長曲線……………………………………………34
4.2 誘導培養之LE活性………………………………………34
4.3 活性電泳分析LE位置之確認……………………………35
4.4 陰離子交換層析……………………………………………35
4.5 陽離子交換層析……………………………………………36
4.6 親和層析……………………………………………………36
4.7 Lovastatin 酯解酵素分子量確認………………………37
4.8 Lovastatin 酯解酵素純化………………………………37
4.9 酵素動力學分析……………………………………………38
4.10 序列檢索及引子設計………………………………………38
4.11 聚合酶鏈鎖反應增幅………………………………………39
4.12 質體定序……………………………………………………39
第五章 討論………………………………………………………40
第六章 參考文獻…………………………………………………46
附圖……………………………………………………………………52
附表……………………………………………………………………68
附錄……………………………………………………………………71
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