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研究生:陳琬瑜
論文名稱:蛋白質工程改良streptomyces clavuligerus擴環酶的熱安定性及青黴素G轉化活性
論文名稱(外文):Engineering of streptomyces clavuligerusDeacetoxycephalosporin C Synthase for Improvement of Thermostability and Penicillin G Conversion
指導教授:魏佳俐魏佳俐引用關係
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生化科技學系研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:100
語文別:中文
中文關鍵詞:Streptomyces clavuligerus擴環酶
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Streptomyces clavuligerus擴環酶(Deacetoxycephalosporin C Synthase)為一個工業上企盼能以生物法取代化學法,將青黴素G(penicillin G)擴環成工業上生產頭孢菌素(Cephalosporins)中間體的目標酵素。本論文利用先前研究篩選出二株具有熱安定性之單點突變株H244R及N268K,以及另一個篩選出在相同位置具有酵素活性提升的突變株H244Q,進一步探討其熱安定性及其對青黴素G的擴環活性。我們首先利用Glutathione S-transferase(GST)蛋白質表現系統及GST親和性管柱將擴環酶表現及純化出來。接著再利用HPLC分析頭孢菌素產量。酵素動力學之結果顯示,與野生型擴環酶相比,H244Q、H244R及N268K突變酵素對青黴素G的kcat/Km分別具有1.8、1.7及1.5倍的提升效果。在熱穩定性分析上,經45℃十分鐘的前處理,H244Q、H244R及N268K的酵素殘留活性分別為26%、37%及45%,而野生型擴環酶則喪失至3%。H244Q、H244R和N268K突變型擴環酶經45℃前處理後的活性半衰期(t1/2),各為8.8、13.0和23.7分鐘,分別為半衰期4.3分鐘的野生型擴環酶的2.0、3.0和5.5倍。再者,野生型擴環酶的最適反應pH值為6.5,其他3種突變型擴環酶均顯示最適反應pH值在5.5~7.5之間。此外,這4種擴環酶的最適反應溫度都是20℃。本論文提供具有較高熱安定性及擴環能力的擴環酶,將有助於固定化酵素技術應用在工業生產頭孢菌素的中間體。
壹、中文摘要 1
貳、英文摘要(Abstract) 3
參、前言 4
一、抗生素 4
二、抗生素之作用機制 4
三、β-lactam類抗生素 6
四、7-胺基去乙醯基頭孢菌素酸 7
五、微生物生成頭孢菌素及青黴素之途徑 8
六、擴環酶之發現與基本特性 11
七、擴環酶之應用與研究現況 14
肆、研究動機 18
伍、材料與方法 19
一、實驗材料: 19
(一)、化學藥品與酵素 19
(二)、培養基配法 20
(三)、化學試劑配法 21
(四)、儀器 23
(五)、菌種及質體 24
二、實驗方法: 26
(一)、自大腸桿菌抽取質體 26
(二)、DNA洋菜膠電泳法 26
(三)、自洋菜膠回收DNA片段 26
(四)、DNA ligation 27
(五)、勝任細胞之製備 27
(六)、電轉形作用 28
(七)、酵素之表現 28
(八)、細胞萃取液之製備 28
(九)、酵素之純化 29
(十)、SDS-PAGE電泳 30
(十一)、蛋白質含量之測定 30
(十二)、擴環酶標準活性之測定 31
(十三)、酵素動力學之測定 31
(十四)、熱安定性之測定 32
(十五)、酵素半衰期之測定 32
(十六)、酵素最適反應pH值之測定 32
(十七)、酵素最適反應溫度之測定 32
(十八)、統計分析 33
(十九)、蛋白質三級結構預測 33
陸、結果 34
一、GST-擴環酶融合蛋白的表現質體建構 34
二、菌株大量表現及純化 34
三、酵素動力學分析 35
五、酵素熱安定性分析 35
六、酵素半衰期分析 36
七、酵素最適反應pH值分析 36
八、酵素最適反應溫度分析 37
柒、討論 39
一、酵素動力學與熱安定性 39
二、酵素最適反應pH值 40
三、酵素最適反應溫度 40
四、酵素結構建模分析 41
五、未來研究 42
捌、參考文獻 43
玖、表 49
拾、圖 50


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