臺灣博碩士論文加值系統

本研究以本土篩選的纖維素分解菌株Bacillus sp.為研究對象，嘗試改變培養基的組成，使其表現大量纖維素水解酵素(cellulase)。實驗中發現Bacillus sp.在常用於篩選纖維素分解菌的Mandels-Reese medium中的生長效果不佳，主要原因是其碳源和氮源不適當之故。Mandels-Reese medium中的碳源為一可溶性纖維素---羧酸甲基纖維(carboxymethylcellulose，CMC)，然而此碳源不能很有效地為Bacillus sp.所利用，且其有機氮源(peptone)的含量僅0.1 %，因此雖然在固態培養時仍可培養出明顯的Bacillus sp菌落，但於液態培養時，其菌體濃度偏低，經調整有機氮源後，菌體濃度有明顯的提升。接著進一步瞭解CMC在培養基中的影響，發現Bacillus sp.雖可將CMC當做碳源利用，但培養前30小時只利用了總糖濃度0.5 g/l，總糖利用率低於10 %。之後嘗試以其他碳源替換CMC以提升Bacillus sp.的生長與酵素的表現，實驗結果發現以乳醣長期培養時，可顯著地提升菌體濃度，其cellulase總活性在長時間培養後也可優於CMC，但cellulase比活性和葡萄糖和纖維雙醣一樣皆明顯低於以CMC培養之結果；因此，以乳醣培養所獲得的活性之提升，應是肇因於菌體濃度之大幅增加。此外嘗試以鹽析及電泳的方式分析Bacillus sp.所產胞外纖維素內切型酵素種類，結果發現Bacillus sp.所產酵素可能有兩種以上，電泳結果顯示其分子量約116,000。

In this study, the cellulase-producing ability of an indigenous bacterium, identified as Bacillus sp., was characterized and was enhanced by growth medium improvement. The results show that Mandels-Reese (MR) medium used for screening cellulose-degrading bacteria was not favorable for cell growth of the Bacillus strain due primarily to the inappropriate carbon and nitrogen sources. Carboxymethylcellulose (CMC), the soluble type of cellulose, was the only carbon source in MR medium, but can not be utilized efficiently by the strain. Only 10% of the total sugar was consumed when Bacillus sp. was cultured on MR medium containing CMC as the sole carbon source. In addition, the concentration of the organic nitrogen source (peptone) in MR medium was only 1%, and seems to be not enough to support cell growth of the Bacillus strain. Increasing the concentration of organic nitrogen source appeared to lead to a significant increase in the cell concentration of Bacillus sp. Attempts were made to replace CMC by other carbon sources (such as lactose, glucose, and cellubiose etc.). It was found that cell concentration increased dramatically when Bacillus sp. was cultured on medium containing lactose, giving higher total extracellular cellulase activity than that obtained from using the medium with CMC. However, the specific cellulose activity obtained from medium containing lactose was not enhanced than that obtained from the culture grown on CMC. This seems to suggest that the increase in total cellulase activity by using lactose was mainly due to elevation of cell concentration. Moreover, the salting out results suggest that Bacillus sp. may produce more then one type of endocellulase. Analysis with native protein gel electrophoresis also shows that one of the enzymes has a molecule weight of approximately 116,000.

中文摘要........................................................................2
Abstract........................................................................3
致謝............................................................................5
目錄............................................................................6
表目錄.........................................................................10
圖目錄.........................................................................11
符號...........................................................................14
第一章 緒論....................................................................15
1-1 前言..................................................................15
1-2 研究動機..............................................................15
第二章 文獻回顧................................................................17
2-1 纖維素................................................................17
2-2 纖維素水解酵素........................................................18
2-2-1 內切型纖維素分解酵素(endo-β-1,4-D-glucanase)..................18
2-2-2 外切型纖維素分解酵素(exo-β-1,4-D-glucanase)...................18
2-2-3 β-葡萄糖苷酵素(β-1,4-D-glucosidase)..........................19
2-2-4 Cellusome......................................................19
2-3 具纖維素分解能力的生物................................................24
2-4 纖維素水解酵素的應用..................................................25
2-5 纖維素水解酵素的誘導..................................................25
第三章 實驗與方法..............................................................27
3-1 藥品與培養基..........................................................27
3-1-1 藥品...........................................................27
3-1-2 培養基組成.....................................................29
3-2 實驗儀器..............................................................30
3-3 菌種介紹與其保存和培養................................................30
3-3-1 實驗菌種.......................................................30
3-3-2 菌種保存.......................................................31
3-3-3 菌種培養.......................................................31
3-3-4 菌體定量.......................................................31
3-4 還原醣定量............................................................32
3-5 總糖定量..............................................................33
3-6 以HPLC測量glucose、cellobiose和lactose................................33
3-7 蛋白質定量............................................................34
3-8 剛果紅測試............................................................34
3-9 酵素活性測試..........................................................34
3-10 超微薄膜過濾.........................................................35
3-11 鹽析分割.............................................................36
3-12 蛋白質電泳...........................................................39
3-12-1 藥品配製......................................................39
3-12-2 Mini-gel製作..................................................40
3-12-3 蛋白質電泳....................................................41
3-12-4 活性染色......................................................41
第四章 結果與討論..............................................................43
4-1 剛果紅測試............................................................43
4-2 Mandels-Reese (MR) medium中碳源與氮源的影響...........................44
4-2-1 CMC對Bacillus sp.的生長影響....................................45
4-2-2 氮源對Bacillus sp.生長的影響...................................45
4-3 有機氮源對Bacillus sp.生長與纖維素水解活性的影響......................47
4-3-1 Peptone含量的影響..............................................47
4-3-2 有機氮源種類對Bacillus sp.的影響...............................51
4-4 CMC含量對Bacillus sp.生長的影響.......................................54
4-5 不同碳源對Bacillus sp.生長與酵素生產的影響............................57
4-6 鹽析分劃..............................................................61
4-7 蛋白質電泳............................................................61
第五章 結論....................................................................65
參考文獻.......................................................................66
附錄...........................................................................69

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