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研究生:黃振哲
研究生(外文):Huang, Chen-Che
論文名稱:產黃纖維單胞桿菌幾丁質酶Cellulomonas flavigena NTOU1 Chi50 定位突變對其生化特性之影響
論文名稱(外文):Effects of Site-Directed Mutagenesis on the Biochemical Properties of C.f NTOU1 Chi50 from Cellulomonas flavigena NTOU1
指導教授:林富邦
指導教授(外文):Lin, Fu-Pang
口試委員:張繼堯吳惠南
口試委員(外文):Chang, Chi-YaoWu, Huey-Nan
口試日期:2016-07-07
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:2016
畢業學年度:104
語文別:中文
論文頁數:80
中文關鍵詞:產黃纖維單胞桿菌幾丁質酶衍生分子
外文關鍵詞:Cellulomonas flavigenachitinasederivative molecules
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產黃纖維單胞桿菌(Cellulomonas flavigena NTOU1)幾丁質酶 C.f NTOU1 Chi63全長分子其衍生分子為C.f NTOU1 Chi50和 C.f NTOU1 Chi38。為改善降解情況及蛋白質表現量不足的問題,本研究採用定位突變和置換不同表現載體的方式進行。
產黃纖維單胞桿菌的幾丁質酶經前人的研究已取得C.f NTOU1 Chi63的全長分子,並在大腸桿菌的表現系統中表現出具有幾丁質酶活性的重組酵素。C.f NTOU1 Chi63以熱處理或是冷藏處存,均會降解成小分子C.f NTOU1 Chi50及C.f NTOU1 Chi38。C.f NTOU1 Chi50也會降解成C.f NTOU1 Chi38。
目前利用分子量大小及胰蛋白酶trysin的切位預測C.f NTOU1 Chi50在R115胺基酸的位置可能與降解作用有關,因此用Dpn1 site-directed mutagenesis的方式進行定位突變,將R115胺基酸突變為A(alanine)、D(aspartate)、V(valine)、F(phenylalanine)、I(isoleucine)、Y(tyrosine),試圖了解R115X突變對C.f NTOU1 Chi50其蛋白質分子降解之影響。

The full-length chitinase gene, C.f NTOU1 Chi63, has been cloned from Cellulomonas flavigena NTOU1 and has been expressed with its enzymatic activity by the heterologous Escherichia coli expression system. Two different prokaryotic expression systems, pETite® and pET20b(+), both have produced the enzymatically active recombinant chitinase C.f NTOU1 Chi63. Derivative molecules of C.f NTOU1 Chi50 and C.f NTOU1 Chi38 were found under the chitinase activity staining after the SDS-PAGE analysis of the recombinant enzymes.
The protein purification of the recombinant C.f NTOU1 Chi63 was hampered by the serious protein degradation by unknown factors. In order to dissolve this unknown protein degradation, both a site-directed mutagenesis and an alternative expression system were approached. The C.f NTOU1 Chi50 R115 was chosen as the target amino acid after SDS-PAGE analysis based on the relative molecular weights of the derivat-ive chitinase molecules. The Dpn1 site-directed mutagenesis of C.f NTOU1 Chi50 R115X was performed and its mutants with A(alanine)、D(aspartate)、F(phenylalanine)、I(isoleucine)、V(valine) and Y(tyrosine) mutations were successfully obtained. Biocharacterization of these chitinase mutants were followed and the possible protein degradation mechanisms maybe understood.

摘要……………………………………………………………………………………I
Abstract………………………………………………………………………….……II
目錄………………………………………………………………………………….III
圖次………………………………………………………………………….………IV
表次……………………………...………………………………………………..…VI
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……………………………………… 3
1.4研究歷史...........................................4
1.5研究目的………………………………………………………………………6
第二章 實驗材料與方法……………………………………………………………25
2.1實驗材料及實驗藥品………………………………………………………..25
2.2 C. f NTOU1 Chi50R115X幾丁質酶突變分子基因選殖………..………….33
2.3 C. f NTOU1 Chi50R115X重組幾丁質酶基因表現與純化………..……….39
第三章 實驗結果……………………………………………………..……………..48
3.1 C. f NTOU1 Chi50R115X幾丁質酶突變分子基因選殖……..………….…48
3.2 C. f NTOU1 Chi50R115X重組幾丁質酶基因表現與純化……..………….48
3.3 C. f NTOU1 Chi50重組幾丁質酶生化之特性………………….…………..49
第四章 討論…………………………………………………………………………66
4.1 C. f NTOU1 Chi50R115X幾丁質酶突變分子基因選殖……..…………….66
4.2 C. f NTOU1 Chi50R115X重組幾丁質酶基因表現與純化……..………….66
第五章 結論與未來展望……………………………………………………………72
參考文獻……………………………………………………………………………..76

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