臺灣博碩士論文加值系統

黑殭菌屬(Metarhizium)是昆蟲病原真菌的一屬，常在世界各地農業害蟲及疾病媒介控制方案中，被作為化學殺蟲劑替代品。廣泛使用真菌的二次代謝產物包括黑殭菌作為生物殺蟲劑，已經增強其基本生物學的興趣。黑殭菌已被廣泛用於合成環狀六縮酚酸胜肽—稱為黑殭菌素，乃是由a-羥基酸及五個胺基酸殘基組成。事實上，環縮酚酸胜肽加上天然環胜肽，合併成具有一個以上酯鍵連結為特色之天然產物大家族，這一類天然產物，因其表現出的多樣化生物活性而在科學界引起廣大興趣，包括抗癌、抗生素、抗真菌、免疫抑制劑或抗發炎活性，與其有趣的作用機制及引人注意的分子結構一致。本實驗中以5 L攪拌式發酵槽發酵培養黑殭菌素，再以乙酸乙酯萃取出其成分，並且利用毛細管電泳分析與定量黑殭菌素，半製備型HPLC分離純化。實驗結果顯示，毛細管電泳分析條件為40 mM Sodium broate、pH 9.8、15 kV、215 nm、57 cm×50 μm×37.5 μm即有相當好的分析效果。而質譜方面，destruxin B分子量為593，經由ESI-MS正離子模式可得到m/s 594，ESI-MS/MS分析可得到分子量481之片段離子。

Metarhizium is a genus of entomopathogenic fungi used world-wide as alternatives to chemical insecticides in agricultural pest and disease-vector control programs. The wide utilization of M. anisopliae as bioinsecticide has increased interest in its basic biology, including its fungal secondary metabolites.M. anisopliae has been widely used for synthesis of cyclic hexadepsipeptides,known as destruxins (dtxs) which consist of an a-hydroxyl acid and five amino acid residues.together with natural cyclic peptides, emerge as a broad family of natural products characterized by the occurrence of at least one ester linkage. The great interest that this class of natural products has elicited in the scientific community is explained by the diverse range of biological activities that they exhibit, including antitumoral, antibiotic, antifungi, immunosuppressant, or anti-inflammatory activities, coincidence with intriguing mechanisms of action and attractive molecular architectures. The destruxin used in this study were isolated and purified from submerged culture.Qualitative and quantitative analysis was performed on a Capillary Electrophoresis system.The optimum conditions were pH 9.8、40 mM Sodium broate、10％ Acetonitrile, at voltage15 kV, and detected wavelength 214 nm. The ESI mass spectrum shows an abundant protonated molecule (dxt B), [M+H]+ m/z 594, and MS2 analysis of the m/z 481 product ion of MS of m/z 594.

總目錄
摘要 I
ABSTRACT II
致謝 III
總目錄 IV
圖目錄 VI
表目錄 VII
第一章 緒論 1
1-1 前言 1
1-2 文獻回顧 3
1-2-1 黑殭菌素的介紹 3
第二章 毛細管電泳簡介與黑殭菌素之分析 33
2-1 毛細管電泳簡介與背景 33
2-1-1毛細管電泳之簡介 33
2-1-2毛細管電泳應用 35
2-2電泳與電滲透流 36
2-3 毛細管電泳之各種分離模式 40
2-3-2 毛細管電泳對於蛋白質、胜肽之應用 44
2-3-3 毛細管電泳對於醣類之應用 44
2-3-4 毛細管電泳對於生物鹼、酚酸類化合物之應用 45
2-3-5 毛細管電泳對於維生素、有機酸之應用 45
2-4黑殭菌素之毛細管電泳定量分析 47
2-4-1材料與方法 47
2-4-2菌種與菌種保存 48
2-5結果與討論 50
第三章 黑殭菌素之分離純化 55
3-1 前言 55
3-2 材料與方法 56
3-2-1 發酵程序 56
3-2-2萃取 56
3-2-3 儀器與設備 56
3-3 結果與討論 59
第四章 結論與展望 70
參考文獻 73
作者簡歷 94

圖目錄
圖1-1 黑殭菌素B (11)由十字花科葉片代謝的可能代謝途徑.....................14
圖1-2 大蠟螟幼蟲與DE (30)一起培養時可能的代謝途徑..........................16
圖 2-1 毛細管電泳原理....................................................................................37
圖 2-2 毛細管區帶電泳和高效能液相層析的流動分佈...............................39
圖 2-3 CZE 機制分離圖....................................................................................41
圖 2-4 MEKC 機制分離圖................................................................................43
圖 2-5 不同緩衝溶液濃度下分析黑殭菌素之層析圖譜...............................52
圖 2-6-1 不同pH 值下分析黑殭菌素之層析圖譜.........................................53
圖 2-6-2 不同pH 值下分析黑殭菌素之層析圖譜.........................................54
圖 3-1 半製備型HPLC 層析圖譜圖3-2 黑殭菌素B 之檢量線...................61
圖 3-2 黑殭菌素B 之檢量線...........................................................................62
圖 3-3 分離純化後DB 在CE 之層析圖譜.....................................................63
圖 3-4 黑殭菌素E 之檢量線...........................................................................64
圖 3-5 分離純化後DE 在CE 之層析圖譜.....................................................65
圖 3-6 黑殭菌素B ESI-MS 圖譜.....................................................................67
圖 3-7 黑殭菌素B ESI-MS/MS 之特性圖譜..................................................68
圖 3-8 黑殭菌素B ESI-MS/MS 特性圖譜之解說圖......................................69


表目錄
表1-1 黑殭菌素的共通結構和已知的黑殭菌素............................................... 7
表1-2 黑殭菌素定性和定量分析方法總結....................................................12
表1-3 黑殭菌之黑殭菌素致病性的指標........................................................23
表 2-1 電滲流(Electroosmotic flow, EOF)之變因...........................................38
表 3-1 毛細管電泳對於黑殭菌素之偵測極限及定量極限...........................66

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