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研究生:謝孟洲
研究生(外文):Meng Chou Hsieh
論文名稱:AspergilluscarneusM34聚木糖酵素與植酸酵素同步生產之評估及聚木糖酵素最適化生產
論文名稱(外文):Estimation of simultaneous production for xylanase/phytase and optimal production of xylanase from Aspergillus carneus M34
指導教授:方繼方繼引用關係
指導教授(外文):Tony J. Fang Ph. D.
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:133
中文關鍵詞:Aspergillus carneus M34聚木糖酵素植酸酵素實驗設計
外文關鍵詞:Aspergillus carneus M34xylanasephytaseexperiment design
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本研究之目的為評估Aspergillus carneus M34同步提升聚木糖酵素與植酸酵素活性的可行性,並進行聚木糖酵素的最適化生產探討。首先了解營養因子對酵素產量的影響,並利用部份因子設計篩選重要的影響因子,再依據結果判斷同步提升酵素活性之可行性。接著,以陡升路徑實驗尋找聚木糖酵素較佳生產條件,並探討磷源、培養溫度與起始pH值的影響。最後,以中心混成實驗設計獲得最大酵素產量。
依據磷源、碳源與實驗設計之結果,顯示聚木糖酵素及植酸酵素兩種酵素,在因子種類的選擇以及階層的調整時,無法藉由同一個因子的改變而達到活性同步提升之目的。中心混成實驗所得到的數學模式,平穩點的形式為鞍點,預估的聚木糖酵素活性為11.66 U/ ml。而在聚木糖酶最適化生產上,當碳源為1.5% 茭白筍殼半纖維素,氮源為0.8% 酵母萃取物與0.4% 氯化銨,起始pH值為7.00,於30℃培養5天後,可獲得的最大酵素活性為27.31±1.14 U/ ml。
The objective of this study was to evaluate the possibility of enhancement of xylanase and phytase activity simultaneously from Aspergillus carneus M34 and to establish the optimal conditions for xylanase production by using experimental design methodology. The effects of nutrition on the enzyme activity were evaluated, and then the important factors were selected through fractional factorial design. The steepest ascent design was used to establish the superior conditions for xylanase production. Effects of the phosphorous source, culture temperature, and initial pH on the xylanase activity were evaluated as well. The optimal enzyme activity was obtained by central composite design.
The results from the phosphorous source, carbon source, and experiment design studies showed that both of xylanase and phytase activity could not be improved simultaneously by selecting the nutritional factors and their concentrations. Xylanase activity was estimated to be 11.66 U/ ml by the mathematic model of central composite design, and the stationary point was found to be a saddle point. The optimal conditions for the xylanase activity from A. carneus M34 are as follows: 1.5% hemicellulose (from coba husk), 0.8 % yeast extract, 0.4% ammonium chloride, initial pH at 7.00, incubation temperature at 30℃. After 5 days of cultivation, the maximal enzyme activity was 27.31±1.14 U/ ml.
目錄
中文摘要 I
英文摘要 II
壹、前言 1
貳、文獻整理 3
一、聚木糖與聚木糖酶 3
(一)植物之聚木糖 3
(二)聚木糖之降解 5
(三)聚木糖酶群 6
二、植酸與植酸酶 10
(一)植酸 10
(二)植酸在營養上扮演的角色 10
(三)植酸之去除 13
(四)植酸酶來源 14
(五)植酸酶的作用機制 16
(六)植酸酶之應用 18
三、實驗設計 18
四、反應曲面法 19
(一)簡介 19
(二)最適化之步驟 20
(三)常用實驗設計類型 22
(四)反應曲面模式之判別 34
(五)反應曲面模式適切性之統計檢驗 37
參、材料與方法 39
一、實驗材料 39
(一)、實驗菌株 39
(二)、培養基 39
1、產孢用培養基 39
2、酵素生產之基礎培養基 39
(三)、化學藥品 40
(四)、實驗設備 42
(五)、電腦套裝軟體 43
二、實驗大綱 44
(一)評估xylanase及phytase同步提升活性之可行性 45
1、磷源影響 45
2、碳源影響 45
3、Plackett-Burman實驗設計 46
4、26-3部份因子設計 46
(二)Xylanase最適化生產 46
1、陡升路徑實驗 46
2、Phytate影響 47
3、溫度與起始pH值的影響 47
4、中心混成設計實驗 47
三、實驗方法 48
(一)菌種保存與活化 48
(二)孢子懸浮液製備 48
(三)批式培養方法與條件 49
四、分析方法 50
(二)pH值測定 51
(三)培養菌液之處理 51
(五)酵素活性測定 52
1、Xylanase 52
2、Phytase 55
(六)總氮之測定 58
五、統計分析及圖形繪製 59
肆、結果與討論 60
一、磷源添加對酵素活性的影響 60
(一)未添加磷酸二氫鉀對酵素活性的影響 60
(二)添加不同濃度磷酸二氫鉀對於酵素活性的影響 62
二、碳源種類對酵素活性的影響 62
三、茭白筍殼半纖維素濃度對酵素活性的影響 67
四、Plackett-Burman 設計 67
五、26-3部份因子設計 77
六、陡升路徑 83
七、Phytate濃度對聚木糖酶的影響 86
八、溫度與起始pH值對聚木糖酶的影響 86
九、中心混成設計 88
伍、結 論 109
一、聚木糖酶與植酸酶之活性同步提升的可行性 109
二、聚木糖酶的最適化生產 110
陸、未來展望 112
柒、參考文獻 114
附錄一、試劑配製方法 128
(一)還原糖含量評估 128
(二)酵素活性分析 129
1、Xylanase 129
2、Phytase 129
(三)茭白筍殼半纖維素之製備 132
圖次目錄
圖2-1、降解聚木糖協同酵素之作用模式 4
圖2-2、植酸之分子結構 11
圖2-3、植酸酶對植酸之脫磷作用 17
圖2-4、反應曲面法進行之流程圖 21
圖2-5、中心混成設計法之星點及中心點補充實驗圖 25
圖2-6、23-1部份實驗設計概念圖 28
圖2-7、三因子的Box-Behnken設計概念圖 31
圖2-8、一階函數之反應曲面圖及反應值(ŷ)變化路徑 33
圖2-9、在一個配適的二階反應曲面中的平穩點 36
圖3-1、搖瓶實驗樣品分析流程 50
圖3-2、DNS法之木糖標準曲線 54
圖3-3、磷酸二氫鉀之標準曲線 57
圖4-1、未添加磷酸二氫鉀對A. carneus M34酵素活性之影響 61
圖4-2、以不同濃度茭白筍殼半纖維素培養A. carnues M34對酵素活性之影響 68
圖4-3、植酸鈉濃度對A. carneus M34生產聚木糖酶的影響 87
圖4-4、常態性或然率圖形 96
圖4-5、茭白筍殼半纖維素與酵母萃取物對聚木糖酶活性之反應曲面圖及等高線圖 98
圖4-6、茭白筍殼半纖維素與氯化銨對聚木糖酶活性之反應曲面圖及等高線圖 99
圖4-7、茭白筍殼半纖維素與起始pH值對聚木糖酶活性之反應曲面圖及等高線圖 100
圖4-8、茭白筍殼半纖維素與培養時間對聚木糖酶活性之反應曲面圖及等高線圖 101
圖4-9、酵母萃取物與氯化銨對聚木糖酶活性之反應曲面圖及等高線圖 102
圖4-10、酵母萃取物與起始pH值對聚木糖酶活性之反應曲面圖及等高線圖 103
圖4-11、酵母萃取物與培養時間對聚木糖酶活性之反應曲面圖及等高線圖 104
圖4-12、氯化銨與起始pH值對聚木糖酶活性之反應曲面圖及等高線圖 105
圖4-13、氯化銨與培養時間對聚木糖酶活性之反應曲面圖及等高線圖 106
圖4-14、起始pH值與培養時間對聚木糖酶活性之反應曲面圖及等高線圖 108
表次目錄
表2-1、半纖維素分解酶之種類 7
表2-2、不同來源植酸酶之基本特性 15
表2-3、23因子設計表 24
表2-4、兩個1/2的23實驗設計 27
表2-5、一個三變數的Box-Behnken設計 29
表3-1、各種碳源之還原榶含量 53
表4-1、以不同磷酸二氫鉀濃度培養A. carneus M34對酵素活性之影響 63
表4-2、以不同碳源培養A. carneus M34之酵素活性與pH值變化 66
表4-3、Plackett-Burman設計中各變數的階層與其對應濃度 71
表4-5、利用Plackett-Burman實驗設計所得聚木糖酶活性之迴歸分析 74
表4-6、利用Plackett-Burman實驗設計所得植酸酶活性之迴歸分析 75
表4-7、26-3部份因子設計之各變數階層與其對應濃度 79
表4-8、26-3部份因子設計及其結果 80
表4-9、利用26-3部份因子實驗設計所得聚木糖酶活性之迴歸分析 81
表4-10、利用26-3部份因子實驗設計所得植酸酶活性之迴歸分析 82
表4-11、陡升路徑之實驗點及其實驗結果 85
表4-12、以不同溫度與起始pH值培養A. carneus M34對聚木糖酶活性之影響 89
表4-13、中心混成設計之各變數階層與其對應濃度 91
表4-14、中心混成設計及其實驗結果 92
表4-15、利用中心混成設計所得xylanase活性之迴歸分析表 95
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