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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 梁博煌(Po-Huang Liang) | |
| dc.contributor.author | Hao-Ming Chen | en |
| dc.contributor.author | 陳浩銘 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:22:43Z | - |
| dc.date.available | 2021-11-08 | |
| dc.date.available | 2022-11-24T03:22:43Z | - |
| dc.date.copyright | 2021-11-08 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-09-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/80937 | - |
| dc.description.abstract | 纖維素和半纖維素是地球上含量豐富的多醣類，透過發酵與酵素作用能夠被轉換成綠能使用。在我們先前的研究中，我們發現了兩種不同功能的酵素，一是來自 Thermotoga maritima 中的雙功能酵素TmCel5A，能夠分解纖維素與聚甘露糖，另一種則是來自Clostridium thermocellum 的CtCel5T，具有分解纖維素、聚木糖、聚甘露糖三種不同糖類的活性。根據這兩酵素的結構比較，我們發現在TmCel5A中210號的色胺酸與CtCel5T中360號的麩胺酸兩者在空間上與受質形成氫鍵作用的位置相似。且210號的色胺酸位於TmCel5A中的Tmloop上，而360號的麩胺酸位在CtCel5T的T2-loop上。於本研究中，我們透過將TmCel5A的210位點突變成麩胺酸，CtCel5T的360位點突變成色胺酸，藉此確認兩者對受質的活性是否也會因兩個關鍵位點的交換而有變化。結果顯示，E360W的突變種失去了催化水解聚木糖的能力但保留了部分的水解聚甘露糖的能力，由此可知麩胺酸對水解聚木糖的活性是很重要的。另一方面，W210E的突變種失去了原本能夠催化水解多糖的能力，由此可知色胺酸的存在對其來說是必要的。而我們也利用的電腦模型與分子對接技術，來顯示突變後受質與酵素間鍵結的變化來試圖觀察為何這些酵素會因此失去活性，事實上是否如此還需其後的實驗證實，本研究提供了交換的方式來加以驗證這兩種胺基酸在各酵素中的需求，相信這也能為日後開發酵素有所助益。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-24T03:22:43Z (GMT). No. of bitstreams: 1 U0001-1309202116323100.pdf: 3790629 bytes, checksum: e209de87402044f671076f80b6771b1e (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "中文摘要： v Abstract: vi ABBREVIATIONS vii Introduction 1 1.1 The demand of renewable energy 1 1.2 Structure of lignocellulosic biomass 1 1.3 Overview of glycoside hydrolases 3 1.4 Overview the development of bioethanol 5 1.5 Aim of this study 5 Method Materials 7 2.1 Reagents 7 2.2 DNA source and bacterial strains 7 2.3 Mutagenesis of CtCel5T and TmCel5A 7 2.4 Expression and purification constructed protein 8 2.5 Protein sequence alignment and structural analysis 9 2.6 Wild-type and mutant protein complexes structural modeling and analysis 10 2.7 Determine of Enzyme Activity 10 2.7.1 preparation of 3, 5-dinitrosalic acid (DNS) reagent 10 2.7.2 Enzyme activity assay 11 2.7.3 Determination of enzyme kinetic 11 Result 13 3.1 Previous study and designed primers for site-directed mutagenesis 13 3.2 Expression and characterization of wild type and mutant protein 14 3.3 Rescue cellulase activity of TmCel5A mutant 16 3.4 Molecular modeling and docking analysis 17 Discussion 19 Tables 21 Figures 25 Reference 43 " | |
| dc.language.iso | en | |
| dc.subject | 分子對接分析 | zh_TW |
| dc.subject | 纖維酵素 | zh_TW |
| dc.subject | 糖苷水解酶第5屬 | zh_TW |
| dc.subject | 蛋白質改造工程 | zh_TW |
| dc.subject | flexible loop | en |
| dc.subject | Cellulase | en |
| dc.subject | GH5 | en |
| dc.subject | docking analysis | en |
| dc.subject | protein engineering | en |
| dc.title | 探討雙功能與三功能纖維水解酵素之受質特異性 | zh_TW |
| dc.title | Substrate specificities of the bi-functional and tri-functional cellulase/xylanase/mannanase enzymes | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 何孟樵(Hsin-Tsai Liu),王彥士(Chih-Yang Tseng) | |
| dc.subject.keyword | 纖維酵素,糖苷水解酶第5屬,蛋白質改造工程,分子對接分析, | zh_TW |
| dc.subject.keyword | Cellulase,GH5,protein engineering,flexible loop,docking analysis, | en |
| dc.relation.page | 51 | |
| dc.identifier.doi | 10.6342/NTU202103150 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-09-15 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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