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系統識別號 U0002-2407200815272900
中文論文名稱 Serratia sp. TKU017所生產蛋白酶及幾丁質酶之純化與定性
英文論文名稱 Purification and Characterization of Protease and Chitinase from Serratia sp. TKU017
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 96
學期 2
出版年 97
研究生中文姓名 李正宇
研究生英文姓名 JENG-YU LI
學號 695180678
學位類別 碩士
語文別 中文
口試日期 2008-07-16
論文頁數 76頁
口試委員 指導教授-王三郎
委員-陳銘凱
委員-王全祿
中文關鍵字 Serratia sp.  蛋白酶  幾丁質酶  蝦殼粉 
英文關鍵字 Serratia sp.  protease  chitinase  shrimp shell powder 
學科別分類 學科別醫學與生命科學生物學
中文摘要 TKU017 係以蝦殼粉為唯一碳/氮源,篩選自台灣北部土壤之一株蛋白酶及幾丁質酶生產菌,經鑑定為 Serratia sp.。TKU017 生產蛋白酶及幾丁質酶之較適培養條件為含有 2% 蝦殼粉、0.1% K2HPO4 及 0.05% MgSO4.7H2O 之 100 mL 液態培養基 (pH 5) 在 25℃ 搖瓶培養 3 天。所得發酵上清液,經硫酸銨沉澱、DEAE-Sepharose、Phenyl-Sepharose及Sephacryl S-100等層析步驟,純化出一種蛋白酶及一種幾丁質酶,經 SDS-PAGE 測其分子量分別為 53 kDa 及 65 kDa。於最適反應 pH、最適反應溫度、pH安定、熱安定性方面,蛋白酶的為 pH 9、40℃、pH 5-11 及 <40℃;幾丁質酶的為 pH 5、50℃、pH 5-7 及 <50℃。蛋白酶活性受 EDTA 所抑制,為金屬型蛋白酶,幾丁質酶活性則受 Cu2+、Mn2+、SDS 所抑制。
英文摘要 The protease and chitinase producing strain, Serratia sp. TKU017, cultured by using shrimp shell powder as the only carbon/nitrogen source, was isolated from the soil in the northern Taiwan. The optimized condition for the production of the protease and chitinase was found when the culture was shaken at 150 rpm at 25℃for 3 days in 100mL of medium contain 2% shrimp shell powder (SSP), 0.1 % K2HPO4 and 0.05 % MgSO4.7H2O (pH5). One protease and one chitinase were purified by chromatography procedures of DEAE-Sepharose, Phenyl-Sepharose, and Sephacryl S-100. The molecular mass of the protease and the chitinase determined by SDS-PAGE was approximately 53 kDa and 65 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of protease were pH 9, 40℃, pH 5-11, and <40℃, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of chitinase were pH 5, 50℃, pH 5-7, and及<50℃, respectively. The protease activity was inactivated by EDTA;The chitinase activity was inactivated by Cu2+, Mn2+ and SDS.
論文目次 目錄
頁次
授權書
簽名頁
誌謝 I
中文摘要 II
英文摘要 III
目錄 IV
圖目錄 IX
表目錄 XI

第一章 緒言 1
第二章 文獻回顧 2
2.1幾丁質與幾丁聚醣之構造及應用 2
2.1.1幾丁質與幾丁聚醣之構造 2
2.1.2幾丁質與幾丁聚醣之應用 3
2.2 幾丁質酶與幾丁聚醣酶之分布及功能 3
2.2.1 幾丁質酶 3
2.2.2 幾丁聚醣酶 3
2.3蛋白酶 3
2.3.1蛋白酶分類 4
2.3.2蛋白酶之應用 4
2. 4 抗氧化活性 5
第三章 材料與方法 7
3.1 實驗菌株 7
3.2 實驗材料 7
3.3 實驗儀器 8
3.4 生產菌株之篩選 8
3.5幾丁質之製備 9
3.6 幾丁質酶之活性測定 9
3.7蛋白酶之活性測定 10
3.8 酵素較適生產條件探討 10
3.8.1 碳/氮源種類 10
3.8.2 碳/氮源濃度 10
3.8.3培養溫度 11
3.8.4培養基酸鹼值 11
3.8.5 培養體積 11
3.8.6 培養時間 11
3.9 酵素之分離純化 11
3.9.1 粗酵素液之製備 11
3.9.2 陰離子交換層析 12
3.9.3 疏水性層析 12
3.9.4 膠體過濾層析 13
3.10 蛋白質電泳分析 13
3.11蛋白酶活性電泳 13
3.12 蛋白質定量分析 14
3.13 酵素之特性分析 14
3.13.1 酵素最適反應溫度 14
3.13.2 酵素之熱安定性 15
3.13.3 酵素最適反應pH 15
3.13.4 酵素之pH安定性 15
3.13.5 金屬離子與抑制劑對酵素活性之影響 16
3.13.6 界面活性劑對酵素活性之影響 16
3.13.7蛋白酶之基質特異性 16
3.13. 8幾丁質酶之基質特異性 17
3.14 DPPH自由基清除能力之測定 17
3.15薄層色層層析 18
第四章 結果與討論 19
4.1蛋白酶及幾丁質酶生產菌之篩選 19
4.2菌株特性 19
4.3酵素較適生產條件探討 23
4.4蛋白酶及幾丁質酶之分離純化 35
4.4.1 粗酵素液製備 35
4.4.2 陰離子交換層析 35
4.4.3 疏水性層析 35
4.4.4 膠體過濾層析 36
4.4.5 綜合結果 36
4.5蛋白酶及幾丁質酶之分子量測定 37
4.5.1蛋白酶-SDS-PAGE (CBR染色法) 37
4.5.2蛋白酶活性染色 37
4.5.3幾丁質酶- SDS-PAGE (銀染法) 37
4.5.4綜合結果 37
4.6蛋白酶及幾丁質酶之特性分析 51
4.6.1幾丁質酶之最適反應溫度及熱安定性 51
4.6.2蛋白酶之最適反應溫度及熱安定性 51
4.6.3幾丁質酶之最適反應 pH 及 pH 安定性 51
4.6.4蛋白酶之最適反應pH及pH安定性 51
4.6.5金屬離子對幾丁質酶之影響 51
4.6.6金屬離子對蛋白酶之影響 51
4.6.7界面活性劑對幾丁質酶之影響 52
4.6.8界面活性劑對蛋白酶之影響 52
4.7蛋白酶之基質特異性 52
4.8幾丁質酶之基質特異性 53
4.9抗氧化力分析 53
第五章 結論 68
參考文獻 69










圖目錄
頁次
圖2.1 幾丁質、幾丁聚醣及纖維素之構造 6
圖4.1 Serratia sp. TKU017 之顯微鏡照片 20
圖4.2 不同碳/氮源對 TKU017 幾丁質酶生產之影響 25
圖4.3 不同碳/氮源對 TKU017 蛋白酶生產之影響 26
圖4.4 SSP 濃度對 TKU017 幾丁質酶生產之影響 27
圖4.5 SSP 濃度對 TKU017 蛋白酶生產之影響 28
圖4.6溫度對 TKU017 蛋白酶生產之影響 29
圖4.7 pH 對 TKU017 幾丁質酶生產之影響 30
圖4.8 pH 對 TKU017 蛋白酶生產之影響 31
圖4.9培養體積對 TKU017 幾丁質酶生產之影響 32
圖4.10培養體積對 TKU017 蛋白酶生產之影響 33
圖4.11 Serratia sp. TKU017於 SSP 培養基生產蛋白酶及幾丁質酶之生長曲線圖 34
圖4.12 Serratia sp. TKU017 所生產酵素之純化分離流程圖 39
圖4.13 TKU017 之蛋白酶及幾丁質酶 DEAE-Sepharose CL-6B 層析圖譜 40
圖4.14 TKU017 之幾丁質酶 Phenyl-Sepharose 層析圖譜 41
圖4.15 TKU017 之幾丁質酶 Sephacryl S-100 層析圖譜 42
圖4.16 TKU017 之蛋白酶 Phenyl-Sepharose 層析圖譜 43
圖4.17 TKU017 之蛋白酶 Sephacryl S-100 層析圖譜 44
圖4.18 TKU017 蛋白酶 SDS-PAGE 之分子量分析及活性染色 47
圖4.19 TKU017 幾丁質酶 SDS-PAGE 之分子量分析 48
圖4.20 pH對TKU017幾丁質酶酵素活性之影響 55
圖4.21溫度對TKU017幾丁質酶酵素活性之影響 56
圖4.22 pH對TKU017蛋白酶酵素活性之影響 57
圖4.23溫度對TKU017蛋白酶酵素活性之影響 58
圖4.24薄層色層分析TKU017發酵上清液之抗氧化物質 66
圖4.25發酵不同培養基所得DPPH清除率的比較 67

















表目錄
頁次
表4.1 16S rDNA 部份核酸序列分析 21
表4.2 API生化生理試驗分析結果 22
表4.3 Serratia sp. TKU017幾丁質酶純化總表 45
表4.4 Serratia sp. TKU017蛋白酶純化總表 46
表4.5 TKU017蛋白酶胜肽質譜鑑定結果 49
表4.6化學品對TKU017酵素之影響 59
表4.7界面活性劑對TKU017酵素之影響 60
表4.8微生物來源之幾丁質酶/幾丁聚醣酶特性比較 61
表4.9 Serratia spp.之蛋白酶特性比較 62
表4.10 Serratia sp.TKU017所生產幾丁質酶及蛋白酶之特性 63
表4.11 TKU017蛋白酶之基質特異性 64
表4.12 TKU017幾丁質酶之基質特異性 65
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