臺灣博碩士論文加值系統

稻草桿是台灣地區最大宗生質廢棄物，若能妥善利用，使成為能源來源之一，對於能源極度仰賴外國的台灣而言，將是一舉數得。

本研究首先探討稻草桿前處理方法與培養基的不同，如何影響 T. reesei Rut C30 產生纖維素分解酵素的活性高低。研究發現以低濃度 KH2PO4 與誘導培養前調整 pH 值的培養基所誘導的纖維素分解酵素活性較高。另外在前處理方面，稻草桿粗枝鹼前處理所誘導出的纖維素分解酵素活性最高，可達 1.07 FPU/ml；其次是稻草桿粉末（0.79 FPU/ml）與稻草桿2公分粗枝（0.63 FPU/ml）。與文獻比較，本研究使用簡便的前處理方式即可獲得相當活性的酵素，顯示所建立的誘導培養方法有經濟效益，有工業上應用潛力。

另外，蛋白質體學是後基因時代最重要的研究領域，但真菌類T. reesei Rut C30 的蛋白體學仍屬起步階段。本研究使用蛋白體學中最有力的工具—二維膠體電泳，來分析 T. reesei Rut C30 分泌之胞外酵素的組成。除了確立二維膠體電泳分析的較佳條件外，發現經棉花纖維素 Avicel 與不同前處理稻草桿的誘導產生的胞外酵素組成有明顯的不同。另外，與文獻比較，發現本研究分析胞外酵素所得到的蛋白質點數量明顯增多，這結果有助於尋找纖維素分解酵素的蛋白質新組成，對於 T. reesei 屬蛋白體學的建立也將有助益。最後，本研究將二維膠體電泳圖譜對應到的 CBH I 與 CBH II 百分體積總和與酵素比活性（FPU/mg）作圖、以及 β-glucosidase 百分體積與 CB 比活性（U/mg）作圖，分別皆可得到良好的線性關係，這對於探討纖維素分解酵素中成份蛋白的組成比例有相當的幫助。

Rice straw is a major agricultural waste in Taiwan. The generation of energy from this biomass is very valuable for Taiwan, a country that highly depends on the imported energy.
In this research, different pretreated preparations of rice straw were used to induce the production of cellulase complex by a cellulase-producing strain Trichoderma reesei Rut C30 cultured in different media. Results from medium study indicated that the medium with low KH2PO4 concentration and adjusting pH before inducing cultivation could result in higher enzymatic activities for cellulose hydrolysis. Among these different pretreated preparations of rice straw, the alkaline-treated rice straw fragments could result in the highest cellulase activity, which was determined to be 1.07 FPU/ml. Powdered rice straw could generate a cellulase activity of 0.79 FPU/ml, while the non-pretreated rice straw fragment (2 cm) led a cellulase activity of 0.63 FPU/ml. These results suggested that using rice straw after the proposed pretreatments as the substrate for the production of cellulase and consequently the utilization of rice straw for fuel ethanol production is very potential.
Proteomics is a very important research field in the post-genome era. However, proteomics in T. reesei Rut C30 is still on the very early stage. In this research, two-dimensional gel electrophoresis (2-DE) was employed as a tool for analyzing the composition of enzyme complex secreted by T. reesei Rut C30 that was cultivated on rice straw. In addition to the development of preferable method in 2-DE, the protein profile of enzyme complex secreted by T. reesei Rut C30 that was cultivated on Avicel and other pretreated preparations of rice straw was excavated. Results indicated that the cellulase composition varied with the substrate for the induction of cellulose production. In comparison with those in the literature and database, more protein spots were found in the 2-DE gel, suggesting that they may be new members in cellulase complex. Finally, the expression levels of protein spots appeared in the 2-DE gel were found to be well correlated to the specific activities of corresponding cellulase components. The correlation is useful for the determination of composition ratio in the cellulase complex.

中文摘要 .I
英文摘要 .III
目錄 V
圖目錄 XI
表目錄 XVII
第一章 緒論 1
1.1 前言與研究背景 1
1.2 文獻回顧與實驗原理 2
1.2.1 纖維素分解酵素的相關研究 2
1.2.2 T. reesei 菌株之胞外酵素組成 4
（A）纖維素分解酵素（cellulase） 4
（B）半纖維素分解酵素（hemicellulase） 7
1.2.3 稻草桿前處理之相關研究 8
（A）稻草桿成份探討 8
（B）稻草桿前處理 9
1.2.4 二維膠體電泳的相關研究 13
（A）與纖維素分解酵素有關的二維膠體電泳研究 13
（B）樣品前處理 14
（C）第一維與第二維電泳 16
（D）銀染 17
（E）影像處理 18
1.2.5 第一維電泳—IEF 原理 19
1.2.6 第二維電泳—SDS-PAGE 原理 20
第二章 實驗方法、材料與設備 22
2.1 實驗材料 22
2.1.1 Trichoderma reesei Rut C30 的培養 22
2.1.2 孢子、蛋白質、還原醣與葡萄糖的測定 23
2.1.3 二維膠體電泳之第一維 25
2.1.4 二維膠體電泳之第二維 26
2.1.5 銀染 27
2.2 實驗儀器 28
2.3 實驗方法 30
2.3.1 Trichoderma reesei Rut C30 的培養 31
（A）固態培養 31
（B）液態前培養 32
（C）誘導培養 33
2.3.2 蛋白質濃度的測定 35
（A）蛋白質濃度檢量線 35
（B）測量樣品中蛋白質濃度 35
2.3.3 還原醣濃度的測定 36
（A）還原醣濃度檢量線 36
（B）測量樣品中還原醣濃度 38
2.3.4 葡萄糖濃度的測定 39
（A）葡萄糖濃度檢量線 39
（B）測量樣品中葡萄糖濃度 40
2.3.5 FPA（filter paper assay）測纖維素分解酵素活性 41
2.3.6 內切型纖維素分解酵素（CMCase）活性分析 41
2.3.7 葡萄糖苷酵素活性分析（cellobiose assay） 42
2.3.8 二維膠體電泳之第一維 42
（A）酵素沉澱 42
（B）第一維等電點分離 44
2.3.9 二維膠體電泳之第二維 46
2.3.10 銀染 49
（A）二胺銀法 49
（B）硝酸銀法 51
2.3.11 膠片掃描 52
2.3.12 膠片比對與分析 53
（A）蛋白質點偵測 53
（B）蛋白質點比對 54
第三章 實驗結果與討論 56
3.1 蛋白質濃度測定與其檢量線 56
3.2 還原醣濃度測定與其檢量線 56
3.3 葡萄糖濃度測定與其檢量線 59
3.4 孢子濃度檢量線 61
3.5 各種酵素活性的意義 61
3.6 高濃度 KH2PO4 與未調整 pH 值培養基（原始培養基）的
誘導效果 63
3.6.1 純粹物理性前處理稻草桿誘導效果 63
3.6.2 物理性加化學性前處理稻草桿誘導效果 67
（A）酸前處理 67
（B）鹼前處理 71
3.6.3 無誘導物時酵素分泌情形 75
3.7 低濃度 KH2PO4 與誘導前調整 pH 值培養基（改良培養基）
的誘導效果 76
3.7.1 誘導效果 77
3.7.2 與其他論文的比較 81
3.7.3 無誘導物 82
3.7.4 內切型纖維素分解酵素與葡萄糖苷酵素活性 83
3.8 二維膠體電泳分析 85
3.8.1 經由原始培養基誘導產生胞外酵素之二維膠體電泳圖 85
3.8.2 經由改良培養基誘導產生胞外酵素之二維膠體電泳圖 96
3.8.3 二維膠體電泳條件最佳化 101
（A）沉澱方法比較 101
（B）IEF 條件對於電泳圖譜的影響 104
（C）第二維電泳條件對於電泳圖譜的影響 106
（D）銀染條件對於電泳圖譜的影響 106
3.8.4 電泳圖譜與誘導天數的關係 108
3.8.5 二維膠體電泳資訊與比活性的關係 113
第四章 結論 117
第五章 參考文獻 120
附錄A 不同樣品不同操作時間，二維膠體電泳圖譜重現性分析
附錄B 無誘導物與經 Avicel 誘導產生胞外酵素之二維膠體電泳圖
譜差異
附錄C 其他二維膠體電泳圖譜

【中文文獻與專書】

李如月“纖維二糖之合成及其固定化應用”中原大學化學工程研究所碩士論文（1994）

莊榮輝“酵素化學實驗”國立台灣大學農業化學系（2000）
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