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研究生:黃玄舜
研究生(外文):Hsuan-Shuen Huang
論文名稱:黑殭菌素分離純化與毛細管電泳分析方法之探討
論文名稱(外文):Purification and analysis of destruxins from Metarhizium anisopliae.(一)HPLC,(二)CE,(三)CD
指導教授:吳宗正吳宗正引用關係
指導教授(外文):Tzong-Zeng Wu
學位類別:碩士
校院名稱:國立東華大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:98
中文關鍵詞:膠束電動色譜半製備型層析管柱黑殭菌素
外文關鍵詞:destruxinsgradient mobile phaseMEKCPBSCD
相關次數:
  • 被引用被引用:9
  • 點閱點閱:369
  • 評分評分:
  • 下載下載:54
  • 收藏至我的研究室書目清單書目收藏:1
本研究是利用氰甲烷對黑殭菌素做高效率效的萃取,氰甲烷以1:1的比例加入發酵液中均勻的與水相混合為一相,再添加5% 氯化鈉使它分層為兩相,有機相經減壓濃縮20倍後除去絕大部份的氰甲烷後殘留的氯化鈉水溶液會將高濃度的黑殭菌素DB、DMDB、DA、DC析出,利用HPLC以氰甲烷與水混合做梯度分離的移動相並選用半製備型Merck LiChrosorb RP-18(10Î250mm,7μm)之層析管柱可將結晶的黑殭菌素分離並純化出高純度的黑殭菌素DC、DA、DMDB、及DB。黑殭菌素以毛細管電泳膠束電動色譜(MEKC)之分離模式做分析之探討,實驗過程測試了pH、界面活性劑(SDS)濃度、有機修飾劑、電壓及緩衝溶液離子的濃度等幾種影響分析結果的重要因子,實驗結果發現波長214 nm、PH = 9.14、界面活性劑(SDS) 40 mM、有機修飾劑(氰甲烷)40 mM、電壓15 kV、緩衝溶液硼酸鹽溶液40 mM時分析條件最佳。
將四種純化過的黑殭菌素標準品以CD圖譜分析的結果發現黑殭菌素二級結構中具有β-turns的結構,將四種標準品溶於氰甲烷中可得到四種差異極大的圖譜,利用這四種圖譜做基本的母圖,可應用於黑殭菌素的快速鑑定;另外在模擬生物體內環境下,我們將黑殭菌素分別溶於10 mM的PBS中以相同比例混合,結果顯示在PBS溶液中這四種主要的黑殭菌均不會相互作用,表示黑殭菌素會以單體的模式存在來產生生物活性的可能性較高。
The acetonitrile was used to extract destruxins from submerged culture of Metarhizium anisopliae. Sodium chloride was added to the mixture of acetonitrile and broth (1:1 ratio) to the final concentration 5%. There are two phases, the destruxin B、desMB、destruxin A、destruxin C would be salted out from the upper (organic) layer after concentrating 20 fold of the solution by vacuum evaporator. Each components can be well separated by HPLC using Merck LiChrosorb RP-18(10Î250mm,7μm) column with gradient of distilled water and acetonitrile. Also, the separation of each destruxin was done by the MEKC(Micellar Electrokinetic Chromatography Mode).The optimum conditions were pH = 9.14、40 mM SDS、10 mM Acetonitrile、40 mM boric acid, at voltage 15 kV, and detected wavelength 214 nm.
The secondary structure of these destruxins were determine by Circular dichroism(CD). The results indicated that these cyclic peptides have the β-turn conformation . These destruxins have different CD spectra in acetonitrile. This character can be used as fingerprints to identify each destruxin. To further investigate the interactions among destruxins, the combinations of different species of destrusin in PBS were studied by CD. The results supported that destruxins might work independently to each other under in vivo system.
目錄
授權書 i
誌謝 ii
中文摘要 iii
英文摘要 v
目錄 vi
圖目錄 ix
表目錄 xi

第一章 緒論 1
1-1、前言 1
1-2 相關文獻簡介 3
1-2-1黑殭菌素的介紹 3
結構與性質 5
黑殭菌素的用途 6
1-2-2毛細管電泳歷史背景和發展 9
第二章 毛細管電泳簡介與黑殭菌素之分析 12
毛細管電泳簡介 12
2-1儀器構造及介紹 12
2-2 電泳與電滲透流 13
2-3分離效率,解析度(Resolution) 18
2-4毛細管電泳的分類 20
毛細管電泳分析黑殭菌素之探討 23
2-5 前言 23
2-6 材料與方法 24
2-6-1試藥的配製 24
2-6-2 毛細管之處理 24
2-7 結果與討論 26
緩衝溶液選擇 26
表面活性劑之選擇 27
有機修飾劑的選擇 28
pH值的影響 31
第三章 黑殭菌素純化製備之方法 48
3-1前言 48
3-2材料與方法 50
3-2-1 萃取 50
3-2-2 HPLC分離黑殭菌素 50
3-3 結果與討論 54
3-3-1 萃取與純化 54
3-3-2黑殭菌素之定量分析 56
HPLC標準曲線製作 56
UV-visible定量黑殭菌素 56
比耳定律於HPLC中對濃度之計算 57
第四章 Circular dichroism(CD)對黑殭菌素之初步探討 71
4-1前言 71
4-2 材料與方法 73
4-2-1 儀器 73
4-2-2樣品的前處理 73
4-2-3 CD操作程序 74
4-3 結果與討論 74
第五章 結論 85
參考文獻 87
附錄一 95
CD開機及校正 95
附錄二 97
CD樣品偵測及數據儲存 97
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