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研究生:楊舒涵
論文名稱:假絲酵母菌脂肪酶(Candida rugosa LIP) 突變基因庫之建構及其活性篩選
論文名稱(外文):Construction and Activity Screening of Lipase Mutagenesis Library from Candida rugosa
指導教授:唐世杰
指導教授(外文):Shye-Jye Tang
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
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:2012
畢業學年度:100
語文別:中文
論文頁數:61
中文關鍵詞:假絲酵母菌脂肪酶
外文關鍵詞:Candida rugosalipase
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Candida rugosa (CRL) 所產生的五種菌體外脂肪酶 (LIP1-LIP5) 在序列上具有高度的相似性,卻有不同的基質特異性,因此實驗室過去以基因重組的方式,重組LIPF和LIP4產生13組新式脂肪酶 (NL-A~NL-M),以研究序列和結構活性間的關係。其中有活性的新式脂肪酶為NL-B、NL-L以及NL-J,其中NL-B的活性較NL-L、NL-J低。後又將NL-B和NL-L以接合產生NL-BL新式脂肪酶,發現NL-BL活性較NL-L低許多。NL-BL以及NL-L在胺基酸序列上只有20個差異,卻對活性產生巨大影響,而這些差異都位於脂肪酶lid的部分。因此本研究於脂肪酶lid的部分建構一個完整的突變基因庫,將所有差異的部分進行突變,並篩選出高脂肪酶催化活性之突變酵素。突變引子概括突變部分長度約為34個胺基酸,將NL-BL lid部分的序列置換成NL-L的序列,進行Quick Change Mutagenesis PCR,建立基因庫,並將之轉殖入S. cerevisiae並且利用tributyrin (C4:0) plate assay初步篩選活性較高之突變株。接著再利用p-Nitrophenyl Laurate (C12:0) 進一步確認活性並將突變株序列定序完成。其結果證實基因庫確實依據設計進行突變,且使得脂肪酶活性上升2-5倍。
The yeast Candida rugosa produces five extracellular lipases (LIP1-LIP5), showing diverse enzyme activity but high gene similarity. The previous researcher recombine LIP F and LIP4, producing 13 chimera lipases: NL-A~NL-M, to study the relationship between sequence and structure-activity. There are only NL-B, NL-J and NL-L have activity.
To understand the substrate specificity and activity between these new lipase, the previous researcher recombinant the NL-B and NL-L trying to higher the activity of new lipase but failed. There are only 20 amino aicds different between NL-BL and NL-L and the difference is on the lid structure.
To understand the different between NL-BL and NL-L, use the Quick Change Mutagenesis PCR to mutant the different sequence from NL-BL to NL-L. The result shows that the activity of mutant are successfully enhance 2-5 folds.

謝誌 i
中文摘要 ii
英文摘要 iii
目錄 iv
表目錄 vi
圖目錄 vii
第一章 序論 1
一、 脂肪酶的應用: 1
1. 食品工業 2
2. 清潔工業: 2
3. 製藥工業: 3
4. 造紙工業: 3
5. 其他工業: 3
二、 脂肪酶的生化特性: 4
1. 催化的反應 4
2. 受質特異性: 6
3. 界面活化作用 (Interfacial Activation): 7
4. 脂肪酶的催化劑和抑制劑: 7
5. 脂肪酶的綜合特性: 8
三、 脂肪酶的結構特性: 9
四、 Candida rugosa lipase (CRL) : 10
五、 Candida rugosa lipase 的結構特性: 10
1. 蓋子(Lid): 10
2. 催化三元體(Active triad): 11
3. 醣基化位置: 11
4. 受質醇結合區: 12
六、 研究源起: 14
1. 重組CRL: 14
2. 使用蛋白質工程製造新式CRL: 14
七、 實驗方向: 15
第二章 材料與方法 16
一、 材料 16
1. 菌種: 16
2. 質體: 16
3. 酵素: 16
4. PCR引子: 16
5. DNA marker: 17
7. 其他: 18
二、 實驗方法 19
1. 突變library構築 19
2. E. coli 勝任細胞製備 20
3. 轉殖至E. coli 20
4. 製備S. cerevisiae 勝任細胞 21
5. 轉殖至S. cerevisiae 21
6. 抽取S. cerevisiae質體 22
7. 脂肪酶活性測定 22
8. 蛋白質結構模擬: 23
第三章 結果 24
一、 建構Mutagenesis Library: 24
二、 突變基因庫在Saccharomyces cerevisiae之表現 24
三、 突變株活性差異: 26
四、 突變株突變數量與活性差異: 26
五、 突變序列結果: 26
六、 突變株結構差異: 28
第四章 討論 29
參考文獻 33
附錄 53

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