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研究生:瞿志傑
研究生(外文):Chih-Chieh Chu
論文名稱:青花菜葉綠素之活性中心及在Pichiapastoris表達之重組葉綠素之生化特性
論文名稱(外文):The active site of broccoli chlorophyllase and the biochemical properties of the recombinant chlorophyllase expressed in Pichia pastoris
指導教授:蕭介夫蕭介夫引用關係
指導教授(外文):Jei-Fu Shaw
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:102
中文關鍵詞:葉綠素
外文關鍵詞:chlorophyllase
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葉綠素(chlorophyllase, EC 3.1.1.14)將葉綠素水解成chlorophyllide及phytol,被認為是參與葉綠素分解步驟系列反應中的第一個酵素。經由與其他物種的葉綠素胺基酸序列進行比對,我們分別將青花菜葉綠素lipase motif中的Ser以及其他具有高度保守性質的胺基酸Asp與His的位置進行定位點突變,以Ala作為取代。與未突變的BoCLH2相比較,H81A、S141A和D170A突變蛋白質均失去活性,顯示這三個位置對於葉綠素的催化活性相當重要。在BoCLH2內的活性中心將會由催化三元體H81、S141及D170組成。
由於青花菜葉綠素基因在E. coli中之表達量太低,我們嘗試利用Pichia重組蛋白系統進行葉綠素的表現。我們將葉綠素基因構築在pGAPZαC與pPICZαA兩種不同的表現載體中,再將線性化的質體利用電迫法送入SMD1168的genome中進行表現。由Pichia中所分泌出來的重組蛋白質均具有葉綠素的活性,而利用西方點墨實驗也證實其表現。與在E. coli中表現的葉綠素分子量大小相同,顯示在Pichia中表現的葉綠素並沒有經過醣基化的後修飾作用。我們也對其生化特性進行分析。BoCLH1、BoCLH2以及BoCLH3的最適pH值分別是8.0、9.0、7.0。而最適溫度則分別為45℃、45℃和35℃。在60℃加熱10分鐘後,三種BoCLH的活性各為原來活性之70%、40% 與60%。未來將嘗試選用不同的表現載體及菌株,以期獲得大量表達之葉綠素。
Chlorophyllase(EC 3.1.1.14), which catalyzes the hydrolysis of chlorophyll to yield chlorophyllide and phytol, is thought to be the first enzyme in the Chl-degradation pathway. According to amino acid sequence alignment, the Ser residue of lipase motif and the conserved residues of Asp and His were substituted with Ala. Compared to the wild type BoCLH2, the H81A, S141A and D170A variants showed no catalytic activity indicating that H81, S141 and D170 residues are essential for catalysis. Site-directed mutagenesis experiment suggested that three of these residues will form a catalytic traid at BoCLH2 active site.
Since the expression level of BoCLH gene in E. coli was too low, we used Pichia expression system to express the recombinant BoCLH proteins. Two expression vectors pGAPZαC and pPICZαA containing the BoCLH genes were constructed, linearized and introduced into Pichia pastoris SMD1168 by electroporation. The recombinant proteins which expressed extracellularly showed chlorophyllase activity and detected by Western Blot. The secreted proteins had the same molecular weight with those expressed in E. coli, demonstrating that BoCLHs were not glycoslated in Pichia. The optimum pH of BoCLH1, BoCLH2 and BoCLH3 was pH 8.0, pH 9.0 and pH 7.0, respectively. The optimum temperature was 45℃, 45℃ and 35℃ for BoCLH1, BoCLH2 and BoCLH3, respectively. After heating at 60℃ for 10 min, BoCLH1, BoCLH2 and BoCLH3 retained 70%, 40% and 60% activity, respectively.
目 錄
英文摘要 I
中文摘要 II
謝誌 IV
目錄 V
圖表目錄 IX
縮寫表 XI
第一章 緒言 1-13
一、葉綠素簡介 1
二、葉綠素分解途徑 1
三、葉綠素研究緣起 4
四、葉綠素生化特性 4
五、葉綠素的催化反應 5
六、葉綠素之分佈、來源與活性控制 7
七、葉綠素基因的相關研究 9
八、實驗緣起 10
第二章 材料與方法 14-49
一、材料 14
二、常用實驗儀器 18
三、實驗方法 19
(1) 聚合連鎖反應(Polymerase Chain Reaction,PCR) 20
(2) 瓊脂膠體電泳(agarose gel electrophoresis) 21
(3) DNA片段回收 22
(4) 質體接合反應(ligation) 23
(5) E. coli勝任細胞(competent cell)之製備 24
(6) E. coli質體轉化作用(transformation) 24
(7) E. coli轉型菌株(transformant)之篩選 25
(8) 試劑組小量純化DNA質體 26
(9) DNA序列分析(DNA sequencing) 27
(10) 蛋白質電泳 27
(11) 電泳膠體之染色 30
(12) 酯解活性染色法 30
(13) 膠體處理與乾燥 31
(14) 西方點墨分析法(Western blotting) 31
(15) 葉綠素酵素活性測定 34
(16) 蛋白質濃度測定 35
(17) 重組蛋白質的純化 36
(18) 葉綠素分解之生化特性分析 39
四、定位點突變實驗(Site-directed mutagenesis) 40
1、pET-20b(+)表現載體之構築 40
2、基因表現之誘發 41
3、表現蛋白質之確認 42
五、Pichia pastoris系統重組蛋白質之表現 42
1、pGAPZαC表現載體之構築 42
2、pPICZαA表現載體之構築 43
3、試劑組大量純化DNA質體 44
4、Pichia pastoris勝任細胞(competent cell)之製備 46
5、Pichia pastoris的質體轉型 46
6、Pichia pastoris轉型菌株(transformant)之篩選 47
7、Multicopy Recombinant之篩選 48
8、基因表現之誘發 48
9、表現蛋白質的收集 48
10、表現蛋白質的部份純化 49
11、表現蛋白質之確認 49
第三章 結果 50-57
一、青花菜葉綠素定位點突變研究 50
二、青花菜葉綠素在Pichia pastoris中的表現 51
1、表現系統的選擇 51
2、表現系統的構築 53
3、重組蛋白質的表現 53
4、青花菜葉綠素之生化性質分析 55
第四章 討論 58-67
一、青花菜葉綠素定位點突變 58
1、催化三元體的位置 58
2、葉綠素的結構 59
二、青花菜葉綠素重組蛋白質在Pichia pastoris中之表現 60
1、不同表現載體的選用 60
2、葉綠素的酯類水解活性 61
3、His-tag fusion protein的影響 62
4、Glycoslation的現象 63
5、改善重組蛋白質的表現 64
三、青花菜葉綠素之生化特性研究與分析 66
1、最適pH值之研究 66
2、最適溫度之研究 66
3、熱穩定性之研究 66
第五章 參考文獻 68-75
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