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研究生:陳懋慶
研究生(外文):CHEN,MAO-CHING
論文名稱:國術運動者心流體驗與正向情緒之研究
論文名稱(外文):A Study of Flow Experience and Positive Emotion on Chinese Martial Arts Athletes
指導教授:鄭雪玲鄭雪玲引用關係
口試日期:2020-06-10
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技系所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2020
畢業學年度:109
語文別:中文
論文頁數:93
中文關鍵詞:β-葡萄糖苷酶生質能源;瘤胃真菌濾紙測試
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草食動物的消化道中存在著許多可分解植物纖維素之微生物,許多研究指出其中的厭氧性真菌對植物纖維具有良好的分解能力,分解纖維素需要3種酵素的配合,分別是外切型纖維素水解酶、內切型纖維素水解酶以及β-葡萄糖苷酶。β-葡萄糖苷酶是負責催化纖維素分解為葡萄糖的最後一個步驟。於前期研究中,β-葡萄糖苷酶J11及Y102的基因,分別由Neocallimastix frontalis及Orpinomyces.sp的基因體中被選殖出來,而這兩株微生物是由水牛及黃牛瘤胃中所分離出來的絕對厭氧真菌。本研究的目的是分析J11 BGL及Y102 BGL的酵素活性與生化特性,以評估分解纖維素的能力。 J11 BGL及Y102 BGL的基因分別被選殖後,轉殖於大腸桿菌中進行大量表現。純化後的J11 BGL及Y102 BGL 經過分析,發現最佳反應溫度分別為40℃及30℃;最佳反應酸鹼值分別為pH 7.0及pH 6.0。熱穩定性分析發現J11於30℃~40℃之間較穩定,Y102則於30℃以上活性即明顯下降。J11靜置於pH 4.5~10.0之間,仍可保50%以上的活性。而Y102 BGL靜置於pH 4.0~10.0之間,仍可保50%以上的活性。J11及 Y102 的Vmax 分別為129.86±4.01及35.85±2.06 μmole/min/mg,Km 及kcat則分別為1.30±0.21 mM、1.79±0.23 mM 及189.53±5.85 s-1、50.06±13.29 s-1。分析水解濾紙的能力,發現J11 BGL比商業化的β-葡萄糖苷酶Novozyme 188的水解效果更高,所以J11 BGL應用於相關產業上,例如生質酒精的開發等,應頗有潛力。
中文摘要 I
Abstract II
致謝 IV
目錄 V
圖表目錄 IX
第一章 前言 1
第二章 文獻回顧 3
2.1 木質纖維素 3
2.2 纖維素水解酶 5
2.2.1 三大類纖維素水解酶 5
2.2.2 纖維素水解酶的協同作用 6
2.3 β-葡萄糖苷酶的選殖與研究 7
2.4 分析纖維素水解酶活性的方法 9
2.5 纖維素水解酶活性分析之各類受質 10
2.5.1 可溶性受質 11
2.5.2 不可溶性受質 11
2.6 纖維水解酶與生質酒精的開發 12
2.7 研究目的與實驗架構 14
第三章 材料與方法 15
3.1 實驗材料 15
3.1.1 菌株 15
3.1.2 載體 15
3.1.3聚合酶連鎖反應之引子 15
3.1.4分子量標記 15
3.1.5 E.coli培養基 16
3.1.6緩衝溶液及試劑 16
3.1.6.1 5X gel loading buffer 16
3.1.6.2 5 X TBE buffer 16
3.1.6.3 10 X TGS buffer 17
3.1.6.4 2X sample buffer 17
3.1.6.5蛋白質電泳分析之膠體 17
3.1.6.6蛋白質電泳膠體染色液 18
3.1.6.7蛋白質電泳膠體脫色液 19
3.1.6.8 薄層層析呈色液 19
3.1.6.9 DNS 試劑 19
3.1.6.10其他化學藥品 19
3.2實驗方法 19
3.2.1 J11 BGL基因及Y102 BGL基因之次選殖 19
3.2.1.1 J11-pET-21a及Y102-pET-21a 小量純化 20
3.2.1.2 J11 BGL及Y102 BGL基因擴增 20
3.2.1.3 J11 BGL及Y102 BGL基因片段的純化 21
3.2.1.4 質體(pET-21a)之製備 21
3.2.1.5限制酶反應 21
3.2.1.6 J11 BGL及 Y102 BGL基因與pET-21a之接和反應 21
3.2.1.7 J11 BGL-pET-21a及 Y102 BGL-pET-21a 之轉型 22
3.2.2 分析轉型株之最佳表現條件 22
3.2.2.1酵素之最適誘導溫度 22
3.2.2.2酵素之最適誘導時間點 23
3.2.2.3蛋白質定量 23
3.2.2.4 J11 BGL及 Y102 BGL純化 23
3.2.2.5 純化後J11 BGL及Y102 BGL之透析和濃縮 24
3.2.3酵素活性測定 24
3.2.3.1以ρ-nitrophenyl-β-D-glucopyranoside (pNPG)分析酵素活性 24
3.2.3.2以DNS比色法分析酵素活性 25
3.2.4 生化特性分析 25
3.2.4.1 J11 BGL及 Y102 BGL最適反應溫度及酸鹼值分析 25
3.2.4.2 J11 BGL 及 Y102 BGL 酸鹼穩定性分析 25
3.2.4.3 J11 BGL 及 Y102 BGL熱穩定性分析 25
3.2.4.4 J11 BGL及 Y102 BGL 之酸鹼耐受性分析 25
3.2.4.5 J11 BGL及Y102 BGL之介面活性劑與有機溶劑耐受性分析 26
3.2.4.6 J11 BGL 及 Y102 BGL 的受質專一性分析 26
3.2.4.7 J11 BGL及 Y102 BGL酵素之水解產物分析 26
3.2.4.8 J11 BGL 分解纖維濾紙(filter paper)分析 26
3.2.4.9酵素動力學分析 27
第四章 結果 28
4.1 J11 BGL基因及Y102 BGL基因的次選殖與大量表現 28
4.2 J11 BGL及Y102 BGL之純化 28
4.3 J11 BGL及 Y102 BGL之最適反應條件分析 29
4.4 J11 BGL及 Y102 BGL 之酸鹼耐受性分析 29
4.5 J11 BGL及 Y102 BGL之熱穩定性分析 29
4.6 J11 BGL及 Y102 BGL耐鹽性分析 30
4.7 J11 及 Y102 對有機溶劑與介面活性劑之耐受性分析 30
4.8 J11 BGL及Y102 BGL之受質專一性分析 30
4.9 J11 BGL及 Y102 BGL之水解產物分析 30
4.10 J11 BGL及 Y102 BGL 酵素動力學分析 31
4.11 J11 BGL分解纖維濾紙分析 31
第五章 討論 50
第六章 結論 53
參考文獻 54
作者簡介 59

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