臺灣博碩士論文加值系統

瘤胃真菌群只占瘤胃微生物族群8%，卻被認為在瘤胃纖維的分解中扮演著重要的角色，主要是因為瘤胃真菌的菌絲可以穿透細胞壁，分解纖維中較堅固頑強的結構。本研究從黃牛瘤胃中分離出瘤胃真菌A10、Á21及水牛瘤胃中分離出瘤胃真菌M08，並研究其分解纖維的能力。在形態鑑定方面，A10具有單鞭毛動孢子、球狀菌體及多中心體，A21具多鞭毛動孢子、絲狀假根及單中心體，M08的單鞭毛動孢子、絲狀假根、及單中心體，此外，再經由核糖體序列親緣關係分析，在形態與親緣關係的雙重結果下，認為A10、A21、M08分別屬於Cyllamyces aberensis、Neocallimastix frontalis、Piromyces 新種的瘤胃真菌。為評估纖維分解能力，將瘤胃真菌培養在粉碎稻草為碳源的培養基，根據養菌前後的稻草，測量NDF(neutral detergent fiber)/ADF(acid detergent fiber)/ADL(acid detergent lignin)變化，以得知瘤胃真菌對稻草中纖維素與半纖維素水解能力，結果顯示A21具有最好的纖維素分解能力，A21與M08對半纖維素的分解則一樣強勢，而A21與M08共培養可以明顯增加纖維素的分解能力，A10與A21共培養則可以明顯增加半纖維素的分解。為了知道A10、A21、M08不同碳原誘導下對不同受質的分解能力，分別以稻草, avicel, CMC作為碳源誘導瘤胃真菌生長後的酵素液，分解β-glucan, avicel, CMC, xylan不同受質的情況，結果顯示利用不同的碳源誘導，確實可以改變瘤胃真菌分泌的酵素活性。為了確定瘤胃真菌是否具有Swollenin蛋白活性，我們以瘤胃真菌Neocallimastix patriciarum W5中所預測Swollenin蛋白的基因設計引子，利用PCR擴增出A21中的Swollenin基因片段，將Swollenin基因片段克隆至pMAL質體再轉殖到表現菌株E. coli-gami表現Swollenin蛋白，Swollenin蛋白純化前後分別與商業酵素Celluclast 1.5L共同反應分解avicel，結果顯示不管純化前後的Swollein蛋白,都沒有增加Celluclast 1.5L分解avicel的能力。

目錄
內容
中文摘要 I
Abstract III
謝誌 V
目錄 VI
圖目錄 X
表目錄 XIII
第一章 前言 1
第二章 文獻回顧 2
2.1 能源的危機與替代能源 2
2.2 生質能源 2
2.2.1 木質纖維的組成 2
2.2.2生物質轉換為生質能源的方法 5
2.2.3木質纖維的水解 6
2.2.4瘤胃與瘤胃微生物 9
2.3 瘤胃真菌 10
2.3.1 瘤胃真菌的生活史 11
2.3.2 瘤胃真菌的鑑別 12
2.3.3瘤胃真菌分解木質纖維素特性 19
2.3.3.1瘤胃真菌分解木質纖維素的評估 20
2.4 瘤胃真菌分解木質纖維的酵素 22
2.5 纖維蓬鬆蛋白 (Swollenin)的發現與其對木質纖維的物理性破壞 23
2.6研究目的 25
第三章 材料與方法 26
3.1 實驗架構 26
3.2 菌株與培養 27
3.2.1 瘤胃真菌分離株來源 27
3.2.2 基礎培養基的置備與培養 27
3.2.3種菌液製備 27
3.3 瘤胃真菌的形態鑑定 27
3.3.1染色與光學顯微鏡的觀察 27
3.3.2螢光染色與光學顯微鏡的觀察 28
3.4 瘤胃真菌的親緣關係 28
3.4.1 Ribosomal DNA的PCR擴增 29
3.4.2 親緣關係的比對 31
3.5 瘤胃真菌在不同碳源下對不同受質的分解能力 31
3.5.1 不同碳源培養基的置備與培養 32
3.5.2 還原醣標準曲線的置備 32
3.5.3在不同受質中反應後的還原醣測定 32
3.6瘤胃真菌分解稻草纖維素與半纖維素的能力 33
3.6.1 纖維素、半纖維素含量的測定 33
3.7瘤胃真菌膨鬆蛋白的基因轉殖、表現、與活性 34
3.7.1瘤胃真菌膨鬆蛋白的基因轉殖 34
3.7.2 瘤胃真菌膨鬆蛋白的誘導與表現 37
3.7.3. 瘤胃真菌膨鬆蛋白的活性測試 40
3.7.4 Swollenin蛋白的純化 42
第四章 結果 44
4.1 瘤胃真菌形態的鑑定與菌親緣關係的分析 44
4.1.1 A10分離株形態特徵觀察結果與親緣關係的分析 47
4.1.2 A21分離株形態特徵觀察結果與親緣關係的分析 51
4.1.3 M08分離株形態特徵觀察結果與親緣關係的分析 54
4.2瘤胃真菌在不同碳源下對不同受質的分解能力 59
4.3瘤胃真菌分解稻草纖維素與半纖維素的能力 61
4.4瘤胃真菌Swollenin蛋白的轉殖與表現 63
4.4.1利用PCR擴增尋找Swollenin基因序列 63
4.4.2Swollenin基因轉型株的確認 64
4.4.3 Swollenin蛋白的最佳誘導條件 65
4.4.4Swollenin粗酵素液的活性測試 66
4.4.5 Swollenin蛋白的純化 67
第五章 討論 72
第六章 結論 75
第七章 參考文獻 76
附註 87
作者簡介 88

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