臺灣博碩士論文加值系統

Chryseobacterium 又稱Flavobacterium，為非發酵、無運動性之菌株，係屬革蘭氏陰性桿菌。C. taeanense TKU001 以綠豆為主要碳氮源，經發酵後可生產澱粉酶，並進一步探討其酵素純化及定性。澱粉酶之較適培養條件為1.5％綠豆粉（mung bean powder；MBP）、0.1％ K2HPO4 及0.05％ MgSO4．7H2O 之100 mL 液態培養基(pH 9)，於30℃振盪(150 rpm)培養5 天後可得較佳澱粉酶活性。
將發酵上清液經由冷凍乾燥（或減壓濃縮）、DEAE Sepharose CL-6B、Phenyl Sepharose 6 Fast Flow 等一系列管柱層析後進行生化性質分析。最適反應溫度與熱安定性分別為50℃與<60℃，最適反應pH 與pH 安定性分別為pH 9 與pH 7-11。在化學試劑穩定性測試中，Ca2+與Tween 40 可明顯提升酵素活性，但Fe2+、Cu2+及Mn2+則會完全抑制其活性。
抗氧化分析方面，以DPPH 自由基清除力、亞鐵離子螯合力、及還原力三種方式檢測，並測定其總酚含量。自由基清除試驗可達79%以上之有效清除率(1st 天)，亞鐵離子螯合力則為0.34 mg/mL EDTA equivalents (6th 天)，其總酚含量達0.38 mg/mL (7th 天)，其還原力相當於0.36 mg/mL cysteine equivalents (7th 天)。

Chryseobacterium, formerly known as Flavobacterium, is a nonfermenting, nonmotile, gram-negative aerobic rod. An amylase was produced from the culture supernatant of C. taeanense TKU001 with mung bean powder as the main nutirion source. The optimized conditions for amylase production was found when the culture was shaken at 30℃ for five day (150 rpm) in 100mL of medium containing 1.5% mung bean powder, 0.1% K2HPO4, 0.05% MgSO4．7H2O (pH 9).
The amylase was purified from culture by lyophilization (or evaporation), DEAE sepharose CL-6B, and Phenyl Sepharose 6 Fast Flow column chromatography. The optimal temperature and thermal stability of this enzyme were 50℃ and <60℃. The optimal pH and pH stability of this enzyme were pH 9 and pH 7-11. This amylase was activated by Ca2+ ions and Tween 40, but completely inactivated by Fe2+, Cu2+, and Mn2+ ions.
The antioxidative evaluations were analyzed by free radical-scavenging activity, metal-chelating ability, reducing power, and total phenolic contents. The DPPH scavenging rate, Fe2+- chelating activity, reducing power, and total phenolics of TKU001 culture supernatant were revealed 56% (1st day), 0.34 mg/mL EDTA equivalents (6th day), 0.38 mg/mL (7th day), and 0.36 mg/mL cysteine equivalents (7th day), respectively.

封面內頁
授權書
簽名頁
中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VIII
表目錄 IX
第一章 緒論 1
第二章 文獻回顧 2
2.1 Chryseobacterium taeanense TKU001之簡介 2
2.2澱粉之分布及組成 2
2.3綠豆之組成及功用 3
2.4澱粉酶 4
2.5澱粉酶之分類 7
2.5.1 α澱粉酶 7
2.5.2 β澱粉酶 8
2.5.3 γ澱粉酶 8
2.6抗氧化 9
2.6.1自由基終止劑 9
2.6.2還原劑或氧清除劑 9
2.6.3金屬螯合劑 10
第三章 材料與方法 11
3.1實驗菌株 11
3.2實驗材料 11
3.3實驗儀器 12
3.4實驗方法 13
3.4.1澱粉酶活性分析 13
3.4.2酵素較適生產條件探討 14
3.4.2.1碳氮源種類之選擇 14
3.4.2.2培養溫度 15
3.4.2.3碳氮源濃度之影響 15
3.4.2.4培養基酸鹼值 15
3.4.2.5培養體積 15
3.4.2.6較適培養時間 16
3.4.3酵素之純化分離 16
3.4.3.1粗酵素液之製備 16
3.4.3.2陰離子交換樹脂層析 17
3.4.3.3疏水性層析 17
3.4.3.4蛋白質電泳 17
3.4.4酵素生化特性分析 18
3.4.4.1最適反應溫度 18
3.4.4.2熱安定 18
3.4.4.3最適反應pH 18
3.4.4.4 pH安定性 19
3.4.4.5金屬離子與抑制劑對酵素之影響 19
3.4.4.6界面活性劑對酵素之影響 19
3.4.5澱粉酶類型判定 20
3.4.5.1 α澱粉酶活性分析 20
3.4.5.2 β澱粉酶活性分析 20
3.4.6抗氧化分析 21
3.4.6.1 DPPH自由基清除能力 21
3.4.6.2亞鐵離子螯合能力 21
3.4.6.3還原力 22
3.4.6.4總酚含量 22
3.4.7乳酸菌促進生長分析 23
第四章 結果與討論 24
4.1澱粉酶較適生產條件探討 24
4.1.1碳氮源種類之選擇 24
4.1.2培養溫度 24
4.1.3碳氮源濃度之影響 25
4.1.4培養基酸鹼值 25
4.1.5培養體積 25
4.1.6較適培養時間 26
4.1.7較適培養條件 26
4.2澱粉酶之純化分離 33
4.2.1粗酵素液之製備 33
4.2.2陰離子交換樹脂層析 34
4.2.3疏水性層析 34
4.2.4蛋白質電泳 35
4.2.5綜合結果 35
4.3酵素之生化特性分析 40
4.3.1澱粉酶之最適反應溫度及熱安定性 40
4.3.2澱粉酶之最適反應pH及pH安定性 41
4.3.3金屬離子對酵素之影響 41
4.3.4界面活性劑對酵素之影響 42
4.3.5澱粉酶之型態判定 42
4.4抗氧化分析 51
4.4.1 DPPH自由基清除能力 51
4.4.2亞鐵離子螯合能力 51
4.4.3還原力 52
4.4.4總酚含量 52
4.4.5乳酸菌促進生長分析 53
第五章 結論 59
參考文獻 60
圖2.1不同類型澱粉酶之作用位置 5
圖2.2各類澱粉酶之分類 5
圖4.1不同碳源對於TKU001生產澱粉酶活性之影響 27
圖4.2培養溫度對於TKU001生產澱粉酶活性之影響 28
圖4.3 MBP添加濃度對於TKU001生產澱粉酶活性之影響 29
圖4.4培養基pH對於TKU001生產澱粉酶活性之影響 30
圖4.5培養體積對於TKU001生產澱粉酶活性之影響 31
圖4.6 C. taeanense TKU001於1.5% MBP生產澱粉酶、還原醣及其生長曲線圖 32
圖4.7 TKU001所生產澱粉酶之純化分離流程圖 36
圖4.8澱粉酶之DEAE-Sepharose CL-6B層析圖譜 37
圖4.9澱粉酶之Phenyl Sepharose 6 Fast Flow層析圖譜 38
圖4.10 TKU001澱粉酶SDS -PAGE之分子量分析 39
圖4.11 TKU001澱粉酶之最適反應溫度及熱安定性 43
圖4.12 TKU001澱粉酶之最適反應pH及pH安定性 44
圖4.13不同碳源對於DPPH自由基清除能力之影響 54
圖4.14不同碳源對於亞鐵離子螯合能力之影響 55
圖4.15不同碳源對於還原力之影響 56
圖4.16不同碳源對於生產總酚含量之影響 57
圖4.17不同添加濃度對於L. paracasei TKU010生長之影響 58
表2.1澱粉酶在工業上的應用 6
表3.1 DNS試劑的組成 14
表4.1 TKU001生產澱粉酶之較適培養條件 33
表4.2 C. taeanense TKU001澱粉酶之純化總表 40
表4.3金屬離子與抑制劑對澱粉酶之影響 45
表4.4界面活性劑對澱粉酶之影響 46
表4.5各種微生物之澱粉分解酶特性比較 47

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