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研究生:蔡佳伶
研究生(外文):Tsai, Chia-Ling
論文名稱:產黃纖維單胞桿菌幾丁質酶基因的人工改造及其蛋白質表現與生化特性
論文名稱(外文):Artificial Engineering of Cellulomonas flavigena NTOU1 Chitinase Gene and Its Protein Expression and Biological Characterization
指導教授:林富邦
指導教授(外文):Lin, Fu-pang
口試委員:吳惠南張繼堯林富邦
口試委員(外文):Wu, Huey-NanCHANG, CHI-YAOLin, Fu-pang
口試日期:2019-11-28
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生命科學暨生物科技學系
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:2019
畢業學年度:108
語文別:中文
論文頁數:84
中文關鍵詞:產黃纖維單胞桿菌幾丁質酶人工改造
外文關鍵詞:Cellulomonas flavigenaChitinaseArtificial Engineering
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產黃纖維單胞桿菌(Cellulomonas flavigena NTOU1)幾丁質酶C.f NTOU1 Chi63分子,已由本研究室從其染色體DNA選殖出基因全長,並於大腸桿菌Rosetta(DE3)pLysS的表現系統中,表現出具有幾丁質酶活性的重組酵素。但所表現的蛋白質量少,且以His-Tag親和性層析純化重組蛋白質效果不佳,尚未完成C.f NTOU1 Chi63之酵素動力學研究。
本研究室多年來以大腸桿菌表現系統表現微生物AcD1ChiAG561幾丁質酶,且用6xHis-Tag親和性層析純化此重組幾丁質酶,均極為成功。故以AcD1ChiAG561為借鏡,全面改寫Cellulomonas flavigena NTOU1 Chi63核苷酸序列,使其更適合大腸桿菌表現系統以取得高純度之C.f NTOU1 Chi63重組幾丁質酶,來完成其酵素動力學的研究。
以AcD1ChiAG561幾丁質酶基因進行改寫之C.f NTOU1 Chi63重組幾丁質酶基因(C.f NTOU1 Chi63 ver.1),其所表現的重組蛋白質量仍然偏少,且以親和性層析純化重組蛋白質的效果不如預期。因此再以大腸桿菌使用之最適化之基因密碼來改寫C.f NTOU1 Chi63重組幾丁質酶基因序列(C.f NTOU1 Chi63 ver.2),可惜仍未有良好的蛋白質表現與純化效果,來完成此酵素動力學的研究。
A full-length chitinase gene encoded in the chromosome DNA from Cellulomonas flavigena NTOU1 has been cloned and its recombinant enzyme has been expressed in E.coli Rosetta(DE3)pLysS with chitinase activity in this laboratory. The kinetic study of this enzyme, C.f NTOU1 Chi63, has not been completed due to the low level of protein expression and its poor His-Tag affinity protein purification.
However, a chitinase gene cloned from another bacteria, AcD1ChiAG561, has been successfully expressed and His-Tag affinity purified with a stable enzyme activity during the past several years in this laboratory.
A modified version of C.f NTOU1 Chi63 gene (C.f NTOU1 Chi63 ver.1) was artificially synthesized based on the codon usage in AcD1chiAG561 and was expressed in the same E.coli expression system. Unfortunately, both its protein expression level and His-Tag affinity protein purification did not have a significant progress. Therefore, a third approach using the E.coli optimized codon usages for C.f NTOU1 Chi63 was chosen (C.f NTOU1 Chi63 ver.2) for this improvement purpose. Again, this trial has not got a significant improvement on the protein expression level and His-Tag affinity protein purification efficiency. The recombinant C.f NTOU1 Chi63 ver.2 remained to keep its enzyme activity.

Keywords:Cellulomonas flavigena, Chitinase, Artificial Engineering
中文摘要.......................................................................................... I
Abstract........................................................................................... II

第一章 前言...................................................................................... 1
1.1 幾丁質及幾丁聚醣........................................................................ 1
1.2 幾丁質水解酵素............................................................................ 2
1.3 Cellulomonas flavigena NTOU 1...................................................... 5
1.4 研究背景與目的............................................................................ 7

第二章 實驗材料與方法.................................................................. 19
2.1 實驗材料及藥品............................................................................ 19
2.2 C. flavigena NTOU 1幾丁質酶人工改造基因................................... 28
2.3 Cellulomonas flavigena NTOU 1 重組幾丁質酶基因表現與純化....... 34

第三章 實驗結果.............................................................................. 49
3.1 C. flavigena NTOU 1 Chi63幾丁質酶的人工改造與其蛋白質表現..... 49
3.2 C. flavigena NTOU 1 Chi63 及其人工改造的基因蛋白質表現的誘導條件...................................................................................................
50
3.3 C. flavigena NTOU1 chi63人工改造幾丁質酶的純化........................ 51
第四章 討論.................................................................................... 74
4.1 C. flavigena NTOU 1 Chi63幾丁質酶的人工改造與其蛋白質表現..... 74
4.2 C. flavigena NTOU 1 Chi63 及其人工改造的基因蛋白質表現的誘導條件...................................................................................................
75
4.3 C. flavigena NTOU1 chi63人工改造幾丁質酶的純化........................ 76

第五章 結論與未來展望................................................................ 77

第六章 參考文獻............................................................................ 79
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