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系統識別號 U0002-2807200921330100
中文論文名稱 Bacillus cereus TKU006所生產幾丁質分解酵素及其他抗氧化物質之研究
英文論文名稱 Studies on the production of chitinolytic enzymes and other antioxidants from Bacillus cereus TKU006
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 97
學期 2
出版年 98
研究生中文姓名 陳重志
研究生英文姓名 Chung-Chih Chen
學號 696180255
學位類別 碩士
語文別 中文
口試日期 2009-07-16
論文頁數 74頁
口試委員 指導教授-王三郎
委員-郭曜豪
委員-陳佑汲
中文關鍵字 Bacillus cereus  幾丁質酶  幾丁聚醣酶  抗氧化 
英文關鍵字 Bacillus cereus  chitinase  chitosanase  antioxidants 
學科別分類 學科別醫學與生命科學生物學
中文摘要 本研究係探討Bacillus cereus TKU006所生產幾丁質分解酵素及其他抗氧化質之研究,探討其較適培養條件,進行酵素純化與定性為研究目的。
TKU006以1%烏賊軟骨粉末為碳/氮源培養於100 mL含 0.1% K2HPO4及0.05% MgSO4.7H2O之液態培養基,於25℃搖瓶培養三天可得較高幾丁聚醣酶活性;以2%蝦頭粉末為碳/氮源於25 mL液態培養基培養二天得較高幾丁質酶活性。所得發酵上清液經硫酸銨沉澱、陰離子交換層析、膠體過濾層析步驟純化得幾丁質酶、幾丁聚醣酶。幾丁質酶分子量為39 kDa,Fe2+完全抑制酵素活性,最適反應溫度、pH分別為60℃、pH 5,熱安定性、pH安定性分別為≦50℃、pH 2-8。幾丁聚醣酶分子量為44 kDa,受到Fe2+、Cu2+完全抑制酵素活性,最適反應溫度與pH分別為60℃與pH 7,熱安定性、pH安定性分別為≦50℃與pH 3-10。
以25 mL含1%烏賊軟骨、0.1% K2HPO4及 0.05% MgSO4.7H2O之液態培養基,於25℃搖瓶培養二天,可得較佳DPPH自由基清除能力。發酵上清液之自由基清除能力、亞鐵離子螯合能力、還原力分別為75%、97%與441 μg/mL CE,而酚類化合物含量為815 μg/mL GAE。總酚類化合物與自由基清除能力之相關係數為r=0.92。
英文摘要 The purpose of this thesis is to study the production of chitinolytic enzymes and other antioxidants from Bacillus cereus TKU006.
The optimum culture conditions for chitosanase and other antioxidants production were composed of 1% squid pen powder, 0.1% K2HPO4, 0.05% MgSO4.7H2O shaken at 25℃ in 100 mL of liquid medium in an Erlenmeyer flask (250 mL) for 3 days and 25 mL of liquid medium for 2 days, respectively. The optimum culture carbon/nitrogen source for chitinase production was 2% shrimp head powder incubated for 2 days. A chitinase and a chitosanase were purified by chromatography procedures of ion exchange and gel filtration. The molecular mass of the chitinase and chitosanase determined by SDS-PAGE were approximately 39 kDa and 44 kDa, respectively. The chitinase was completely inactivated by Fe2+, and the chitosanase was completely inactivated by Fe2+ and Cu2+. The optimum temperature, optimum pH, thermal stability and pH stability of chitinase were 60℃, pH 5, ≦50℃, pH 2-8 . The optimum temperature, optimum pH, thermal stability and pH stability of chitosanase were 60℃, pH 7, ≦50℃, pH 3-10.
After squid pen powder fermented, the antioxidant properties by the assays of radical scavenging, metal chelating, reducing power and total phenolic content were reached at 75%, 97%, 441 μg/mL cysteine equivalent and 815 μg/mL gallic acid equivalent, respectively. Correlation between DPPH scavenging ability and total phenolic content were r=0.92.
論文目次 封面內頁
簽名頁
授權書
中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖目錄 IX
表目錄 X

第一章 緒論 1
第二章 文獻回顧 3
2.1 Bacillus cereus TKU006之簡介 3
2.2 幾丁質與幾丁聚醣 3
2.2.1 幾丁質類之分子構型 5
2.2.2 幾丁質類之應用 5
2.3 幾丁質酶 7
2.4 N-乙醯幾丁寡醣/幾丁寡醣 7
2.5 自由基與氧化性傷害 9
2.6 抗氧化劑作用機制 9
第三章 材料與方法 11
3.1 菌株 11
3.2 化學材料 11
3.3 實驗儀器 12
3.4 較適培養條件探討 13
3.4.1 碳/氮源之選擇 13
3.4.2 碳/氮源濃度之影響 13
3.4.3 培養體積 13
3.4.4 培養時間 14
3.5 懸浮態幾丁質之製備 14
3.6 酵素活性測定 14
3.6.1 幾丁質酶之活性測定 14
3.6.2 幾丁聚醣酶之活性測定 15
3.7 酵素之純化分離 15
3.7.1 粗酵素液之製備 15
3.7.2 陰離子交換樹脂層析 16
3.7.3 膠體過濾層析 17
3.8 酵素之特性分析 17
3.8.1 酵素之最適反應溫度與熱安定性 17
3.8.2 酵素之最適反應pH與pH安定性 18
3.8.3 金屬離子與抑制劑對酵素之影響 19
3.8.4 界面活性劑對酵素之影響 19
3.8.5 基質特異性 19
3.9 抗氧化性質分析 20
3.9.1 DPPH自由基清除能力之測定 20
3.9.2 金屬離子螯合能力之測定 20
3.9.3 還原能力之測定 21
3.9.4 酚類化合物之定量分析 22
第四章 結果與討論 23
4.1 較適培養條件探討 23
4.1.1 碳/氮源之選擇 23
4.1.2 碳/氮源濃度之影響 25
4.1.3 培養體積之影響 25
4.1.4 培養時間之影響 28
4.1.5 培養條件總結 28
4.2 酵素之純化分離 31
4.2.1 粗酵素液 31
4.2.2 陰離子交換樹脂層析 33
4.2.3 膠體過濾層析 34
4.2.4 酵素之分子量判定(CBR染色法) 34
4.2.5 綜合結果 35
4.3 酵素之特性分析 44
4.3.1 酵素之最適反應溫度與熱安定性 44
4.3.2 酵素之最適反應pH與pH安定性 44
4.3.3 金屬離子與抑制劑對酵素之影響 45
4.3.4 界面活性劑對酵素活性之影響 46
4.3.5 基質特異性 46
4.3.6 胜肽質譜鑑定 47
4.4 抗氧化性質分析 55
4.4.1 自由基清除能力 55
4.4.2 金屬離子螯合能力 55
4.4.3 還原能力 56
4.4.4 酚類化合物之定量 57
4.4.5 綜合結果 57
參考文獻 60
附錄 73

圖 目 錄
圖2.1 幾丁質、幾丁聚醣之結構 4
圖4.1 不同碳/氮源對TKU006幾丁聚醣酶生產及DPPH自由基清除能力之影響。 24
圖4.2 SPP濃度對TKU006幾丁聚醣酶生產及DPPH自由基清除能力之影響。 26
圖4.3 培養體積對TKU006幾丁質酶生產及DPPH自由基清除能力之影響。 27
圖4.4 幾丁質酶/幾丁聚醣酶生產菌之發酵上清液DPPH自由基清除能力。 30
圖4.5 TKU006幾丁質酶/幾丁聚醣酶之純化分離流程 32
圖4.6 幾丁聚醣酶之DEAE-Sepharose CL-6B層析圖譜 36
圖4.7 幾丁聚醣酶之Macro-Prep DEAE層析圖譜 37
圖4.8 幾丁質酶之Sephacryl-S100層析圖譜 38
圖4.9 幾丁聚醣酶之Sephacryl-S100層析圖譜 39
圖4.10 TKU006幾丁質酶於SDS-PAGE之分子量分析 40
圖4.11 TKU006幾丁聚醣酶於SDS-PAGE之分子量分析 41
圖4.12 幾丁質酶最適反應pH、最適反應溫度及pH安定性與熱安定性 48
圖4.13 幾丁聚醣酶最適反應pH、最適反應溫度及pH安定性與熱安定性 49
圖4.14 TKU006發酵上清液與α-生育醇之DPPH清除能力比較本 58
圖4.15 發酵上清液與EDTA之亞鐵離子螯合能力比較 59

表 目 錄
表2.1 幾丁質/幾丁聚醣之應用 6
表2.2 幾丁質分解酵素之種類 8
表4.1 TKU006生產幾丁質酶/幾丁聚醣酶與DPPH自由基清除能力之較適培養條件 29
表4.2 B. cereus TKU006幾丁質酶純化總表 42
表4.3 B. cereus TKU006 幾丁聚醣酶純化總表 43
表4.4 金屬離子和抑制劑對幾丁質酶/幾丁聚醣酶活性之影響 50
表4.5 不同界面活性劑對酵素活性之影響 51
表4.6 酵素之基質特異性 52
表4.7 TKU006幾丁質酶之胺基酸序列比對 53
表4.8 TKU006幾丁聚醣酶之胺基酸序列比對 54
表4.9 微生物發酵產物之還原力比較 60
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