Abstract
To isolate and characterize the cellulolytic microbes, we screen the microbes from the Fan-Yuan compost plant in Changhua, Taiwan at 30 ℃and 50 ℃. Overall 102 colonies have been obtained from the compost at different composting periods. After purification and succession cultivation, forty-nine isolates were survival. They comprised 24 mesophilic microbes (designated as L1 to L24) and 25 thermotolerant microbes (designated as H1 to H25), which were isolated from 30 ℃and 50 ℃, respectively. From the morphological observation, there were 19 bacteria, 8 fungi, and 22 actinomyces. Isolated strains H10 and L14 were finally selected mainly due to their high cellulase activities on solid media with Congo Red Test, and submerged media with the determination of reducing sugars.In the submerged cultivation, the isolate H10 had the optimal growth temperature 45 ℃, optimal pH at 7.0, and optimal cellulase activities at 45 ℃ to 50 ℃. The isolate L14 had the optimal growth temperature 35 ℃, optimal pH at 7.0, and optimal cellulase activities at 35 ℃ to 40 ℃. Optimal reaction temperature of CMCase, avicelase and β-glucosidase of both isolates H10 and L14 was 60 ℃, 50 ℃ and 50 ℃, respectively.In conclusion, the cellulolytic isolates H10 and L14 had optimal growth and cellulase activities both at mid to high temperature. Application of these microbes to compost and other related industry should enhance the added values of cellulose containing waste and thereby reach the goal of recycling.頁次圖目錄 III表目錄 VI壹、 前言 1貳、 材料與方法 16一、試驗菌株 16二、對照菌株 17三、培養基 17四、菌種的培養與保存 20五、菌株篩選策略 20六、固態纖維素分解酵素活性測試 21七、蛋白質分解酵素活性測試 21八、α-澱粉分解酵素活性測試 21九、孢子懸浮液的製備 22十、液態培養 22十一、pH值測定 23十二、細胞乾重測定 23十三、粗酵素液製備 23十四、纖維素分解酵素活性分析－DNS還原糖測定法 23十五、粗酵素液之特性分析 25十六、主要儀器和藥品 26參、 結果與討論 28一、計數纖維素分解酵素菌之總菌數 28二、篩菌 28三、菌株生理特性分析 33四、纖維素分解酵素性質分析 38肆、 結論 42伍、 參考文獻 79圖 目 錄頁次圖一、纖維素的基本結構 44圖二、纖維素的組成 45圖三、早期纖維素分解酵素作用機制假說 46圖四、纖維素分解酵素協同作用機制 47圖五、纖維素分解酵素受產物回饋抑制模式 48圖六、圖六、菌株篩選策略流程圖 49圖七、在50℃下分離菌株No. 88於Mendels-Reese液態培養基之生長情形 50圖八、不同分離菌株在四種固態培養基下培養四天後酵素活性之比較 51圖九、不同溫度下分離菌株H10於Mendels-Reese固態培養基生長四天後之菌落大小和剛果紅試驗透明環大小 52圖十、不同溫度下分離菌株H10於Mendels-Reese液態培養基之生長情形與pH的關係 53圖十一、於Mendels-Reese液態培養中不同溫度對分離菌株H10和分離菌株L14的CMCase酵素活性之影響 54圖十二、於Mendels-Reese液態培養中不同溫度對分離菌株H10和分離菌株L14的avicelase酵素活性之影響 55圖十三、於Mendels-Reese液態培養中不同溫度對分離菌株H10和分離菌株L14的β-glucosidase酵素活性之影響 56圖十四、分離菌株H10於Mendels-Reese液態培養之最適生長溫度（45℃）的生理特性與纖維素分解酵素活性分析 57圖十五、分離菌株H10於Mendels-Reese液態培養中之最適生長pH值 58圖十六、不同溫度下分離菌株L14於Mendels-Reese固態培養基生長四天後之菌落大小和剛果紅試驗透明環大小 59圖十七、不同溫度下分離菌株L14於Mendels-Reese液態培養基之生長情形與pH的關係 60圖十八、分離菌株L14於Mendels-Reese液態培養之最適生長溫度（35℃）的生理特性與纖維素分解酵素活性分析 61圖十九、分離菌株L14於Mendels-Reese液態培養中之最適生長pH值 62圖二十、分離菌株H10粗酵素液中纖維素分解酵素之最適作用溫度 63圖二十一、分離菌株H10之粗酵素液分別置於不同溫度下二十小時後其纖維素分解酵素之殘留酵素活性 64圖二十二、分離菌株L14粗酵素液中纖維素分解酵素之最適作用溫度 65圖二十三、分離菌株H10之粗酵素液分別置於不同溫度下二十小時後其纖維素分解酵素之殘留酵素活性 66附錄一、在放大倍率800倍之顯微鏡下觀察分離菌株H10之特徵型態 85附錄二、在放大倍率800倍之顯微鏡下觀察分離菌株 L14之特徵型態 86表 目 錄頁次表一、具產生纖維素分解酵素能力的微生物 67表二、各類酵素在工業上之應用 68表三、89年6月28日於芳苑堆肥場取樣之樣品中纖維素分解菌株變化 69表四、觀察自芳苑堆肥場中篩選高溫纖維素分解菌株於Mendels-Reese培養基中之菌落特性 70表五、觀察自芳苑堆肥場中篩選中溫纖維素分解菌株於Mendels-Reese培養基中之菌落特性 71表六、篩選高溫纖維素分解菌株於Mendels-Reese培養基中30 ℃及50 ℃下培養四天後菌落和透明環直徑大小 72表七、篩選中溫纖維素分解菌株於Mendels-Reese培養基中30 ℃及50 ℃下培養四天後菌落和透明環直徑大小 73表八、高溫纖維素分解菌株於Mendels-Reese液態培養基中30 ℃及50 ℃下培養四天後纖維素分解酵素活性初步測試 74表九、中溫纖維素分解菌株於Mendels-Reese液態培養基中30 ℃及50 ℃下培養四天後纖維素分解酵素活性初步測試 75表十、不同分離菌株在四種固態培養基下培養四天後酵素活性之比較 76表十一、分離菌株H10和分離菌株L14生理特性與纖維素分解酵素性質 77表十二、不同微生物特性之比較 78