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系統識別號 U0002-1607200712283600
中文論文名稱 Serratia marcescens TKU011所生產蛋白酶及幾丁聚醣酶之純化與定性
英文論文名稱 Purification and Characterization of Proteases and Chitosanase from Serratia marcescens TKU011
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 95
學期 2
出版年 96
研究生中文姓名 彭若華
研究生英文姓名 Jo-Hui Peng
電子信箱 fafa_0616@yahoo.com.tw
學號 694290080
學位類別 碩士
語文別 中文
口試日期 2007-06-27
論文頁數 73頁
口試委員 指導教授-王三郎
共同指導教授-吳俊弘
委員-陳銘凱
委員-王全祿
委員-王三郎
中文關鍵字 Serratia marcescens  蛋白酶  幾丁聚醣酶  蝦殼粉 
英文關鍵字 Serratia marcescens  protease  chitosanase  shrimp shell wastes 
學科別分類 學科別醫學與生命科學生物學
中文摘要 TKU011係以蝦殼粉為唯一碳/氮源,篩選自台灣中部土壤之一株蛋白酶及幾丁聚醣酶生產菌,經鑑定為Serratia marcescens。TKU011生產蛋白酶和幾丁聚醣酶之較適培養條件為2%蝦殼粉、0.1%K2HPO4及0.05%MgSO4‧7H2O,在25℃、pH7之50mL液態培養基振盪培養2天。發酵所得上清液經硫酸銨沉澱、DEAE-Sepharose及Sephacryl S-100層析步驟後,可分離出一種蛋白酶(P1)及一種幾丁聚醣酶(C1),經SDS-PAGE測得分子量分別為50kDa及21kDa。利用Mascot資料庫比對,P1胺基酸序列分析結果與Serratia protease有8段胜肽相同,P1之N末端胺基酸序列為:AATTGYDAVDDLLHYHER。其最適反應pH、最適反應溫度、pH安定性、熱安定性分別為pH10,50℃,pH5-11,<40℃;其活性會受Cu2+、EDTA所抑制,而2%(v/v) Tween 20和Triton X-100則完全抑制。C1之胺基酸序列分析經與Mascot資料庫比對,與Chitin-Binding Protein Cbp21有3段胜肽相同。其最適反應pH、最適反應溫度、pH安定性、熱安定性分別為pH5,55℃,pH6-8,<50℃;其活性會受Mn2+、Fe2+、EDTA所抑制,而2%(v/v) Tween 40 提升165%。
英文摘要 The protease and chitosanase producing strain, Serratia marcescens TKU011, was isolated from the soil in Taiwan. The optimized culture condition for production of the protease and chitosanase was composed of 2% shrimp shell powder (SSP), 0.1% K2HPO4, 0.05% MgSO4‧7H2O at pH7 and incubated in 250 mL Erlenmeyer flask containing 50 mL kept shaking at 30℃ for 2 days . The protease and chitosanase were purified from the culture supernatant by chromatography procedures of DEAE-Sepharose, and Sephacryl S-100. The molecular mass of protease and chitosanase determined by SDS-PAGE was approximately 50 kDa and 21 kDa, respectively.
According to Mascot there were eight fragments of peptide by the amino acid assay of the protease which were the same with the Serratia protease. The N-terminal amino acid sequence of the protease contains: AATTGYDAVDDLLHYHER. The optimum pH, optimum temperature, pH stability, thermal stability of protease were pH10,
50℃, pH5-11, <40℃, respectively. The protease was inactivated by Cu2+, Fe2+, EDTA, 2% (v/v) Tween 20 and 2% (v/v) Triton X-100.
According to Mascot there were three fragments of peptide by the amino acid sequencing of the chitosanase which were the same with the Chitin-Binding Protein Cbp21. The optimum pH, optimum temperature, pH stability, thermal stability of chitosanase were pH5, 60℃, pH6-8, <50℃, respectively. The chitosanase was inactivated by Mn2+, Fe2+, EDTA, but the chitosanase activated by 2% (v/v) Tween 40.
論文目次 目錄
頁次

簽名頁
授權書
誌謝
中文摘要 I
英文摘要 II
目錄 IV
圖目錄 VIII
表目錄 X

第一章 緒論 1
第二章 文獻回顧 2
2.1黏質沙雷氏菌之應用 2
2.2幾丁質、幾丁聚醣之結構與來源 2
2.2.1 幾丁質 2
2.2.2 幾丁聚醣 3
2.3幾丁質與幾丁聚醣水解酵素 4
2.3.1幾丁質酶 4
2.3.2幾丁聚醣酶 4
2.3.3其他可水解幾丁聚醣之酵素 4
2.4蛋白質水解酵素 5
2.4.1蛋白酶之簡介 5
2.4.2蛋白酶之分類 5
2.4.3蛋白酶之應用 6

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 9
3.5蛋白酶之活性測定 9
3.6幾丁聚醣酶之活性測定 10
3.7蛋白酶之較適培養條件探討 10
3.7.1碳/氮源之選擇 10
3.7.2培養基之酸鹼值 11
3.7.3培養溫度 11
3.7.4培養體積 11
3.7.5較適培養時間 11
3.8酵素之純化分離 11
3.8.1粗酵素液之製備 11
3.8.2離子交換樹脂層析法 12
3.8.3膠體過濾層析 13
3.8.4蛋白質電泳分析(Electrophoresis) 13
3.8.5蛋白酶活性電泳 13
3.9酵素生化特性分析 14
3.9.1酵素最適反應溫度 14
3.9.2酵素熱安定性 14
3.9.3最適反應pH 14
3.9.4酵素pH安定性 14
3.9.5金屬離子與抑制劑對酵素活性之影響 15
3.9.6蛋白酶之基質特異性 15
3.9.7有機溶劑對酵素活性之影響 16
3.10蛋白質轉印 16
第四章 結果與討論 18
4.1蛋白酶與幾丁聚醣酶生產菌之篩選 18
4.2菌株特性 18
4.3碳/氮源之選擇 18
4.4蛋白酶較適生長條件探討 22
4.5蛋白酶及幾丁聚醣酶之純化分離 23
4.5.1粗酵素液製備 23
4.5.2離子交換層析法 24
4.5.3膠體過濾層析法 24
4.5.4 綜合結果 25
4.6蛋白酶及幾丁聚醣酶之分子量判定 25
4.6.1蛋白酶-SDS-PAGE(CBR染色法) 25
4.6.2幾丁聚醣酶- SDS-PAGE(銀染法) 25
4.6.3蛋白酶活性染色 26
4.6.4綜合結果 26
4.7純化後蛋白酶與幾丁聚醣酶之生化特性分析 27
4.7.1蛋白酶之最適反應溫度及熱安定性 27
4.7.2幾丁聚醣酶之最適反應溫度及熱安定性 27
4.7.3蛋白酶之最適pH及pH安定性 27
4.7.4幾丁聚醣酶之最適pH及pH安定性 27
4.7.5金屬離子及抑制劑對蛋白酶與幾丁聚醣酶之影響 27
4.7.6各種界面活性劑對蛋白酶及幾丁聚醣酶之影響 28
4.7.7有機溶劑對蛋白酶與幾丁聚醣酶活性及安定性之影響 28
4.7.7.1有機溶劑對蛋白酶活性及安定性之影響 28
4.7.7.2有機溶劑對幾丁聚醣酶活性及安定性之影響 29
4.7.8蛋白酶之基質特異性 29
4.7.9蛋白酶N端序列分析及蛋白酶與幾丁聚醣酶胜肽質譜鑑定 30
4.7.9.1蛋白酶N端序列分析 30
4.7.9.2蛋白酶與幾丁聚醣酶胜肽質譜鑑定 30
第五章 結論 65
參考文獻 66

圖目錄 頁次

圖4.1 Serratia marcescens TKU011之顯微鏡照片 19
圖4.2 16S rDNA部份核酸序列分析 20
圖4.3 API鑑定系統分析結果 21
圖4.4蝦殼粉末添加對TKU011產生蛋白酶活性之影響 32
圖4.5不同碳源對於TKU011產生蛋白酶活性之影響 33
圖4.6溫度對TKU011產生蛋白酶活性之影響 34
圖4.7 pH對TKU011產生蛋白酶活性之影響 35
圖4.8通氣量對TKU011產生蛋白酶活性之影響 36
圖4.9 S.marcescens TKU011培養於蝦殼培養基所產蛋白酶及幾丁聚
醣酶變化情形 37
圖4.10 S.marcescens TKU011所生產酵素之純化分離流程圖 38
圖4.11 S.marcescens 之DEAE-Sapharose CL-6B 圖譜層析圖 39
圖4.12蛋白酶之Sephacryl S-100層析圖譜 40
圖4.13幾丁聚醣酶之Sephacryl S-100層析圖譜 41
圖4.14蛋白酶於SDS-PAGE之分子量分析 44
圖4.15幾丁聚醣酶於SDS-PAGE之分子量分析 45
圖4.16蛋白酶活性染色 46
圖4.17 TKU011蛋白酶之最適反應溫度及熱安定性 49
圖4.18 TKU011幾丁聚醣之最適反應溫度及熱安定性 50
圖4.19 TKU011蛋白酶之最適pH及pH安定性 51
圖4.20 TKU011幾丁聚醣酶之最適pH及pH安定性 52
圖4.21有機溶劑對蛋白酶活性之影響 57
圖4.22有機溶劑對蛋白酶安定性之影響 58
圖4.23有機溶劑對幾丁聚醣酶活性之影響 59
圖4.24有機溶劑對幾丁聚醣酶安定性之影響 60
圖4.25蛋白酶之胜肽質譜鑑定結果 63
圖4.26幾丁聚醣酶之胜肽質譜鑑定結果 64

表目錄 頁次

表3.1 TKU011生產蛋白酶及幾丁聚醣酶之較適條件 12
表3.2 蛋白質轉印所需溶液配方 17
表4.1 S.marcescens TKU011蛋白酶純化總表 42
表4.2 S.marcescens TKU011幾丁聚醣酶純化總表 43
表4.3 Serratia spp.幾丁聚醣酶特性比較 47
表4.4 S. marcescens蛋白酶特性比較 48
表4.5各種金屬離子對蛋白酶之影響 53
表4.6各種金屬離子對幾丁聚醣酶之影響 54
表4.7各種界面活性劑對蛋白酶之影響 55
表4.8各種界面活性劑對幾丁聚醣酶之影響 56
表4.9蛋白酶之基質特異性 61
表4.10 TKU011生產酵素與其他金屬型蛋白酶之N端序列比較 62
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