完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.author | 邱鈺安 | en_US |
dc.contributor.author | Chiu, Yu-An | en_US |
dc.contributor.author | 李耀坤 | en_US |
dc.contributor.author | Li, Yaw-Kuen | en_US |
dc.date.accessioned | 2014-12-12T01:53:01Z | - |
dc.date.available | 2014-12-12T01:53:01Z | - |
dc.date.issued | 2010 | en_US |
dc.identifier.uri | http://140.113.39.130/cdrfb3/record/nctu/#GT079858508 | en_US |
dc.identifier.uri | http://hdl.handle.net/11536/48486 | - |
dc.description.abstract | 醣基化對於生物體的生化合成反應相當重要,在近期的疾病治療上扮演重要的角色,如能快速並高通量的偵測醣基轉移酶酵素活性,便可開啟此類酵素的應用。本研究旨於探討Bacillus cereus之UDP-glycosyltransferase(BcGT-1)的催化反應,我們藉由基因工程將BcGT-1建構在大腸桿菌之BL21(DE3)的表現系統上,並經過DEAE管柱純化,得到大量酵素。本研究因酵素受質的特性,得以利用BcGT-1對4-nitrophenol的轉醣能力,使用一便利的光學測定法,以直接偵測酵素BcGT-1的活性,此法具備快速、方便且能即時偵測等特色。 首先,我們觀察不同硝基苯酚(醣基接受者)的親核性與酵素反應的關係。研究顯示硝基苯酚之pKa對於BcGT-1的催化反應有密切關聯性,酵素的轉醣能力會隨醣基接受者親核力之下降而減弱。 此外,我們亦研究BcGT-1在合成應用之潛力,利用純化後的酵素與不同醣基提供者進行轉醣反應,發現酵素對於UDP-glucose具有高度專一性,而酵素對不同的醣基接受者,如:苯酚、苯硫醇、苯胺類進行轉醣反應,發現能對其成功接上葡萄醣基,此研究顯示此BcGT-1能用來合成各種新穎葡萄醣苷化合物,如O-glucoside、S-glucoside及N-glucoside等,未來將具有廣泛的應用性。 | zh_TW |
dc.description.abstract | Glycosyltransferases have many important biological functions in all species and in the use of disease treatment. Thus we need a real time and high-throughput method to analyze glycosyltransferase activity. In this research, we focus on the UDP-glycosyltransferase from Bacillus cereus (BcGT-1) catalytic reaction. BcGT-1 was successfully over-expressed in E. coli BL21 (DE3) and purified by DEAE column. In this research, we use a quick colorimetric activity assay for glycosyltransferase BcGT-1. The assay principle is based on the glycosylation activity of BcGT-1 on 4-nitrophenol. It provides a timesaving, simple and high-throughput for analyzing glycosyltransferase activity. First, the nitrophenolic compounds with different nucleophilicity were used to be the glucose acceptors in BcGT-1 glycosylation reaction. The nucleophilicity of nitrophenolic compounds was observed to play an important role in enzymatic reaction. The activity of BcGT-1 was decreased when nucleophilicity of acceptor was reduced. In addition, we also investigate the potential of BcGT-1 to be used in synthesis. Using the purified BcGT-1 to catalyze the glycosylation with different donors shows that this enzyme has high specificity to UDP-glucose. However, this enzyme can convert phenolic, thiolphenolic and aromatic amine to their respective glucosides successfully. It means that BcGT-1, has high potential to be used to synthesize novel glycosides like O-glucoside、S-glucoside and N-glucoside. | en_US |
dc.language.iso | zh_TW | en_US |
dc.subject | 醣基轉移酶 | zh_TW |
dc.subject | 醣苷 | zh_TW |
dc.subject | 受質專一性 | zh_TW |
dc.subject | 酵素活性偵測法 | zh_TW |
dc.subject | 枯草桿菌 | zh_TW |
dc.subject | UDP-glycosyltransferase | en_US |
dc.subject | glycoside | en_US |
dc.subject | substrate specificity | en_US |
dc.subject | colorimetric activity assay | en_US |
dc.subject | Bacillus cereus | en_US |
dc.title | 枯草桿菌UDP-glycosyltransferase之受質專一性研究與快速活性偵測法建立 | zh_TW |
dc.title | Research of substrate specificity and a quick colorimetric activity assay method of UDP-glycosyltransferase from Bacillus cereus | en_US |
dc.type | Thesis | en_US |
dc.contributor.department | 應用化學系分子科學碩博士班 | zh_TW |
顯示於類別: | 畢業論文 |