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研究生:羅珮芸
研究生(外文):Lo,Pei-Yun
論文名稱:利用固定化的 L-阿拉伯糖異構酶及 L-核糖異構酶生產稀有醣類
論文名稱(外文):Production of rare sugars by immobilized L-arabinose isomerase and L-ribose isomerase
指導教授:方翠筠
口試委員:蔡國珍曾文祺
口試日期:2019-06-28
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:91
中文關鍵詞:L-核醣L-核酮糖固定化菌體固定化酵素稀有糖生產
外文關鍵詞:L-riboseL-ribuloseimmobilized cellimmobilized enzymerare sugar production
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目錄
摘要…………………………………………………………………………………I
Abstract……………………………………………………………………………II
目錄………………………………………………………………………………..III
圖目錄……………………………………………………………………………..VI
表目錄…………………………………………………………………………….VII
壹、 前言 1
一、 研究背景 1
二、 研究目的 2
貳、 文獻回顧 3
一、 稀有醣類 3
1. 簡介 3
2. 應用 3
3. L-核酮糖 (L-ribulose) 3
4. L-核糖 (L-ribose) 3
二、 L-核糖 (L-ribose) 之生產 4
三、 生產L-核糖所需之酵素 5
1. L-阿拉伯糖異構酶 (L-AI) 5
2. L-核糖異構酶 (L-RI) 5
四、 重組蛋白在細菌中之蛋白質表現 6
1. ClearColi BL21 (DE3) 6
2. CondonPlus-RIL 7
3. 乳糖操控組 (Lactose operon) 7
五、 固定化 8
1. 簡介 8
2. 方法 8
3. 固定化材料介紹 9
4. 含 L-AI及L-RI之菌體固定化 10
5. L-核酮糖及 L-核糖之生產 11
參、 實驗設計與流程 12
一、 以乳糖誘導可表現 Af-RI 的大腸桿菌菌體 12
二、 以發酵槽生產可分別表現 F270N Ts-AI 及 Af-RI 的大腸桿菌菌體 12
三、 以 BeaverBeadsTM IDA-Nickel 及 Ni SepharoseTM 6 Fast Flow resin固定化酵素 12
四、 利用乳化法製備固定化菌體 12
五、 L-核糖及 L-核酮糖之生產 13
肆、 實驗材料與方法 14
一、 實驗材料 14
1. 菌株與質體 14
2. 抗生素 14
3. 蛋白質標準品 14
4. 市售套組 14
5. 培養基 14
6. 化學藥品 14
7. 實驗設備 16
二、 藥品配置 17
1. Terrific broth (TB) 培養液 17
2. Super Optimal Broth (SOB) 培養液 18
3. Salt-optimized carbon broth (SOC) 培養液 18
4. 抗生素配置 18
5. Cysteine-carbazole 呈色法試劑 18
6. 定量蛋白質之相關試劑 19
7. SDS-PAGE 相關藥劑製備 19
三、 實驗方法 20
1. 製備小量質體 20
2. 轉形作用 21
3. 蛋白質表現 22
4. 發酵槽生產 24
5. 製備粗酵素液 24
6. 重組型 F270N Ts-AI 及 Af-RI 純化 24
7. 蛋白質定量 25
8. 活性分析與特性探討 25
9. 固定化酵素 27
10. 固定化菌體 27
11. 以固定化菌體及酵素生產 L-核酮糖及 L-核糖 29
12. HPLC-ELSD 分析 30
13. 統計分析 31
伍、 結果與討論 32
一、 Af-RI 表現與純化 32
1. 利用乳糖進行誘導 32
2. 以發酵槽進行大量生產 32
二、 F270N Ts-AI 表現與純化 32
三、 固定化 33
1. 固定化酵素 33
2. 固定化菌體 33
3. 純化酵素與固定化菌體特性比較 35
四、 生產 L-核糖與 L-核酮糖 37
1. 批次生產 37
2. 連續生產 37
陸、 結論 39
柒、 參考文獻 40
捌、 附錄 82
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吳泰徵,2017,Actinotalea fermentans ATCC 43279來源重組 L-核糖異構酶之特性與固定化並以蛋白質工程改變其熱穩定性,國立臺灣海洋大學食品科學系碩士學位論文,基隆
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游銘遠,2012,Geodermatophilus obscurus DSM43160 來源之 L-核糖異構酶之基因選殖、表現、純化及特性探討,國立臺灣海洋大學食品科學系碩士學位論文,基隆
黃幸光,2014,熱穩定性阿拉伯糖異構酶之表現、特性探討、稀有醣類生產及蛋白質工程,國立臺灣海洋大學食品科學系博士學位論文,基隆
劉俞均,2018,以蛋白質工程固定化菌體改變Actinotalea fermentans ATCC 43279來源重組L-核糖異構酶之熱穩定性,國立臺灣海洋大學食品科學系碩士學位論文,基隆
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