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論文名稱: | 以洋菜及其水解產物誘導Pseudomonas vesicularis MA103不同洋菜酶基因表現順序與各洋菜酶水解寡醣組成之探討
| Study on Sequential Expression of Pseudomonas vesicularis MA103 Agarases and the Profiles of Hydrolytic Oligosaccharides Produced by Individual Agarase
研究生: | 蔡品嫻
指導教授: | 潘崇良 Chorng-Liang Pan
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | M98320032
畢業學年度: | 99
語文別: | 中文
關鍵字: | 洋菜
| 洋菜酶
| 寡糖
| 基因表現
全文影像連結: | | (本論文20140729開放校外瀏覽)
| 論文全文(pdf)

論文頁數: | 99
摘要: | 本研究主要在探討以洋菜 (agar)、洋菜糖 (agarose)、半乳糖 (galactose)及新洋菜二糖 (neoagarobiose, N2)誘導Pseudomonas vesiculiaris MA103後，所產生的洋菜酶及基因表現順序之差異。菌株MA103經不同誘導物誘導後，觀察其基因表現之順序及其胞外洋菜酶表現，並以高效能液像層析儀 (high-performance liquid chromatography, HPLC)分析不同時間之被誘導酵素水解液中的產物。Agarose經洋菜酶 (agarase)水解後之水解液，經過超過濾後以HPLC分析可得知產物主要為新洋菜二糖、四糖及微量的六糖，再以膠過濾層析分離純化，以得到後續實驗所需之寡醣。在菌株MA103基因表現部分，設計專一性引子，並以聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 擴增反應觀察各洋菜酶基因表現，添加agar誘導後，培養90 sec 之基因AS4-36和培養30 min 之基因AS4-16表現量較低，在培養1 h時各基因皆明顯的表現；添加agarose誘導後培養90 sec時各基因皆明顯表現，且各時間點的10組洋菜酶基因表現量皆相似；添加galactose誘導之後培養90 sec之基因AS4-28和AS90-28未表現，培養10 min後基因AS90-28亦未表現，培養30 min後AS4-28表現量減弱，培養1 h後各基因皆有表現；以N2誘導時培養90 sec後基因AS4-28和AS90-28未表現，培養10 min後各基因皆開始表現。在洋菜酶表現部分，10個洋菜酶分布於14.9 kDa到143.0 kDa ，在未添加醣類誘導物的對照組樣品中可見到約50 kDa之蛋白質條帶，在各種醣類誘導之胞外粗酵素液，皆可看到約100 kDa、85 kDa和50 kDa等條帶。在以agar誘導30 min後開始見到40 kDa和30 kDa蛋白質條帶；在galactose誘導30 min後觀察到35和20 kDa蛋白質條帶。誘導粗酵素液之水解產物部分，在agarose、galactose 和N2的各時間點都可觀察到新洋菜二糖 (N2)及四糖(N4)的產生，推測是因為粗酵素液產生了聚合作用，將半乳糖及新洋菜二糖 (N2)合成為四糖(N4)及未知的聚合醣類。
論文目次: | 目    錄
| 目錄    i
| 表目錄    v
| 圖目錄    vi
| 附錄目錄    ix
| 中文摘要    1
| 英文摘要 (Abstrate)    2
| 壹、前言    3
| 貳、文獻整理    5
| 一、洋菜酶    5
| 1. 洋菜酶生產菌株與分離來源    5
| 2. 洋菜酶分類    6
| 2-1.    -agarase    6
| 2-2.    -agarase    6
| 3. 溫度和pH 值    7
| 4. 分子質量    7
| 5. 金屬離子    8
| 6. 洋菜酶對洋菜水解的作用機制    9
| 7. 洋菜酶之應用    11
| 7-1.    細胞的分離    11
| 7-2.    DNA的分離    11
| 7-3.    藻類原生質體的製作    12
| 7-4.    海藻多醣的生產與黏度降低    12
| 7-5.    藻類組成的分析    12
| 二、洋菜及洋菜衍生多醣    13
| 1. 洋菜之來源與利用    13
| 2. 洋菜多醣組成    13
| 三、新洋菜寡醣之生理活性    14
| 1. 免疫性    14
| 2. 降血脂    14
| 3. 益菌性    15
| 参、實驗設計    16
| 肆、實驗材料與方法    17
| 一、實驗材料    17
| 1. 實驗菌株    17
| 2. 試驗藥品    17
| 2-1.    藥品    17
| 2-2.    RNA純化及反轉錄套組    18
| 2-3.    引子    18
| 2-4.    聚合酶鏈鎖反應試劑    19
| 2-5.    電泳標準品    19
| 2-6.    培養基組成    20
| 2-7.    洋菜酶反應基質    21
| 2-8.    電泳膠片配製    21
| 2-9.    電泳溶液    22
| 2-10.    DNS 溶液    22
| 3. 儀器設備    22
| 二、實驗方法    23
| 1. 菌株之保存與活化    23
| 1-1.    菌株保存    23
| 1-2.    菌株活化    23
| 2. 洋菜酶之生產與活性測定化    24
| 2-1.    洋菜酶粗酵素液之製備    24
| 2-2.    洋菜酶活性測定    24
| 3. 新洋菜寡醣的純化分離    25
| 3-1.    以Pseudomonas vesicularis MA103和Aeromonas salmonicida MAEF108生產之agarase粗酵素液水解洋菜糖 (Agarose)    25
| 3-2.    以市售agarase (Sigma) 水解洋菜糖    25
| 3-3. 以膠過濾分離新洋菜寡醣    25
| 3-4. 總醣量分析    26
| 4. mRNA表現的探討    26
| 4-1.    Total RNA之萃取    26
| 4-2.    RNA電泳    26
| 4-3.    反轉錄酶作用 (Reverse transcriptase, RT)    27
| 4-4.    聚合酶鏈鎖反應 (Polymerase chain reaction, PCR)    27
| 5. 胞外蛋白質的探討    27
| 5-1.    蛋白質的定量    27
| 5-2.    粗酵素液沉澱    28
| 5-2.    分子量的鑑定    28
| 6. 洋菜酶之水解產物分析    29
| 6-1.    薄層層析法 (Thin-layer chromatography, TLC)    29
| 6-2.    高效能液相層析儀 (High-performance liquid chro- matography, HPLC)    29
| 伍、結果與討論    30
| 一、新洋菜寡醣的生產與純化分離    30
| 1.    Pseudomonas vesicularis MA103和Aeromonas salmonicida MAEF108所生產之Agarase粗酵素液水解洋菜糖  (Agarose)    30
| 2.    以市售Agarase (Sigma)水解洋菜糖    29
| 二、mRNA表現的探討    31
| 1.    胞內RNA的生成變化    31
| 2.    cDNA聚合酶擴增鏈鎖反應    32
| 三、胞外蛋白質的探討    33
| 四、水解產物之分析    34
| 陸、結論    35
| 柒、參考文獻    37
|
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2011 年 9 月 22 日 20:03