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系統識別號 U0002-1607200713131500
中文論文名稱 Chryseobacterium indologenes TKU014所生產三種蛋白酶之純化及定性
英文論文名稱 Purification and characterization of three proteases from Chryseobacterium indologenes TKU014
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 許菀庭
研究生英文姓名 Wan-Ting Hsu
電子信箱 grace2289@yahoo.com.tw
學號 694290031
學位類別 碩士
語文別 中文
口試日期 2007-06-27
論文頁數 79頁
口試委員 指導教授-王三郎
委員-陳銘凱
委員-王三郎
委員-王全祿
中文關鍵字 Chryseobacterium indologenes  蛋白酶 
英文關鍵字 Chryseobacterium indologenes  protease 
學科別分類 學科別醫學與生命科學生物學
中文摘要 以蝦殼粉為唯一碳/氮源,自台灣土壤篩選到Chryseobacterium indologenes TKU014此株蛋白酶生產菌。
C. indologenes TKU014在含有0.5% 蝦殼粉、0.05 % MgSO4.7H2O及0.1 % K2HPO4(pH6)的液體培養基,於30℃培養一天,可得到較高的蛋白酶活性(0.44U/mL)。
所得發酵液之離心上清液經過硫酸銨沉澱、離子交換樹脂層析法及疏水性層析分離等純化步驟,得到三種蛋白酶P1、P2和P3,經SDS-PAGE測得分子量分別為56kDa、40kDa和40kDa。最適反應溫度分別為30~50℃、40℃和40~50℃,最適pH均偏鹼性分別為pH10、pH8和pH9,其pH安定性為pH5~11、pH6~8和pH8~10,熱安定性則為<50℃、<40℃和<40℃。
三種蛋白酶的活性均受Mn2+、Cu2+、Fe2+的抑制。P1、P2、P3均受EDTA和1,10-phenanthroline的抑制,判定,P1、P2和P3皆屬Zn-金屬型蛋白酶。
基質特異性方面,對酪蛋白、彈性蛋白和角蛋白為基質時,蛋白酶P1、P2和P3具有較佳的活性,對於白蛋白、纖維蛋白、血球蛋白、偶氮白蛋白、偶氮酪蛋白的活性不佳。


英文摘要 Chryseobacterium indologenes TKU014, a protease-producing strain, was isolated from the soil in Taiwan, by using shrimp shell powder (SSP) as the sole carbon/nitrogen source.
The optimized conditions for protease production was found when the culture was shaken at 30℃ for one day in 50mL of medium (pH6 ) containing 0.5% SSP, 0.1% K2HPO4, 0.05% MgSO4.7H2O
Three proteases (P1, P2, and P3) were purified from culture supernatant by ammonium sulfate precipitation, ionic exchange of DEAE-sepharose CL-6B chromatography and Phenyl Sepharose hydrophobic interaction. The molecular mass of TKU014 proteases (P1, P2, and P3) determined by SDS-PAGE was approximately 56 kDa, 40 kDa, and 40 kDa, respectively. The three proteases (P1, P2, and P3) were found to have optimum temperature at30~50℃,40℃,and40~50℃; optimum pH at 10, 8 and 9; thermal stability lower then 50℃, 40℃ and 40℃ ; pH stability at pH 5~11, pH 6~8 and pH 8~10, respectively.
The activity of three proteases (P1, P2, and P3) was completely inactivated by EDTA and 1,10-phenanthroline, so the three proteases (P1, P2, and P3) were Zn-metalloprotease.
As for substrate specificity, these three proteases showed good activity toward casein, elastin and keratin azure as substrates, low activity with hemoglobin, and poor activity with albumin, fibrin, hemoglobin, azocasein, albumin.

論文目次 封面內頁
簽名頁
授權書
中文摘要 I
英文摘要 II
誌謝 IV
目錄 V
圖目錄 IX
表目錄 XI

第一章 緒論 1

第二章 文獻回顧 2
2.1水產廢棄物之微生物利用 2
2.2蛋白酶 2
2.3 彈性蛋白酶 5
2.3.1彈性蛋白酶之簡介 5
2.3.2彈性蛋白酶之應用 6
2.4角蛋白 8
2.4.1角蛋白的簡介 8
2.4.2角蛋白酶的應用 9
2.5 Chryseobacterium indologenes 12
第三章 材料與方法 14
3.1實驗菌株 14
3.2實驗儀器 14
3.3實驗材料 15
3.4生產菌株之篩選與分離 16
3.5蛋白酶活性之測定 16
3.6蛋白質酶最較適生長條件探討 16
3.6.1碳源的影響 16
3.6.2蝦殼粉濃度對酵素產量之影響 17
3.6.3培養溫度之影響 17
3.6.4培養基酸鹼值之影響 17
3.6.5培養體積對酵素產量之影響 17
3.7蛋白酶之純化分離 17
3.7.1粗酵素液的製備 17
3.7.2離子交換樹脂層析法 18
3.7.3疏水性層析法 18
3.7.4蛋白質電泳分析 18
3.7.5蛋白質定量分析 19
3.8胺基酸序列鑑定 19
3.9純化酵素之生化特性分析 19
3.9.1酵素最適溫度 19
3.9.2酵素熱安定 19
3.9.3酵素最適pH的測定 20
3.9.4酵素pH安定性的測定 20
3.9.5金屬離子與抑制劑對酵素活性之影響 20
3.9.6 Zn2+ 的存在與否對於酵素活性的影響 20
3.9.7界面活性劑對酵素活性之影響 21
3.9.8酵素之基質特異性 21
3.10有機溶劑對酵素活性及安定性之影響 22
3.11酵素水解 22

第四章 結果與討論 23
4.1蛋白質酶生產菌之篩選 23
4.2蛋白質酶生產菌株之鑑定 23
4.3蛋白質酶較適生長條件探討 27
4.3.1碳/氮源的種類及濃度之影響 27
4.3.2培養溫度之影響 27
4.3.3培養基酸鹼值之影響 28
4.3.4培養體積對酵素產量之影響 28
4.4蛋白酶之純化分離 36
4.5蛋白質電泳分析 36
4.6胺基酸序列鑑定 37
4.7純化酵素之生化特性分析 44
4.7.1反應pH及溫度對酵素活性之影響 44
4.7.2金屬離子與抑制劑對酵素活性之影響 44
4.7.3 Zn2+ 存在與否對於酵素活性的影響 45
4.7.4界面活性劑對酵素活性之影響 45
4.7.5酵素之基質特異性 46
4.8有機溶劑對酵素活性及安定性之影響 46
4.9酵素水解羽毛和頭髮 47

第五章 結論與未來展望 64

參考文獻 65


圖目錄 頁次
圖4.1Chryseobacterium indologenes TKU014之顯微照片 24
圖4.2Chryseobacterium indologenes TKU014之16SrDNA部分序列25
圖4.3蝦殼粉添加濃度對TKU014生產蛋白酶活性之影響 31
圖4.4培養溫度對TKU014生產蛋白酶活性之影響 32
圖4.5培養基 pH值對TKU014生產蛋白酶活性之影響 33
圖4.6培養體積對TKU014生產蛋白酶活性之影響 34
圖4.7 C. indologenes TKU014所生產蛋白酶之生長曲線圖 35
圖4.8 TKU014所生產蛋白酶之純化分離流程圖 38
圖4.9 TKU014蛋白酶於DEAE-Sepharose CL-6B離子性層析之圖譜39
圖4.10 TKU014蛋白酶於Phenyl Sepharose 6 Fast Flow疏水性層析之圖譜 40
圖4.11純化所得三種TKU014蛋白酶之SDS-PAGE圖 42
圖4.12蛋白酶之最適反應溫度 48
圖4.13蛋白酶之熱安定性 49
圖4.14蛋白酶之最適反應pH 50
圖4.15蛋白酶之pH安定性 51
圖4.16有機溶劑對蛋白酶活性的影響 60
圖4.17蛋白酶對有機溶劑之安定性 61
圖4.18 羽毛粉添加濃度對TKU014水解的影響 62
圖4.19 頭髮添加濃度對TKU014水解的影響 63
圖4.20 C. indologenes TKU014水解羽毛及頭髮的能力 64

表目錄

頁次
表2.1 含幾丁質水產加工廢棄物之微生物再利用 3
表2.2蛋白質酶的應用範圍 4
表2.3 Chryseobacterium spp.之蛋白酶純化 13
表4.1 API鑑定系統分析 26
表4.2不同碳/氮源對於TKU014產生蛋白酶活性之影響………… 29
表4.3 Chryseobacterium spp.生產蛋白酶之最適培養條件 30
表4.4 Chryseobacterium indologenes TKU014蛋白質酶純化概要41
表4.5 Chryseobacterium indologenes TKU014 proteases經胰蛋白酶水解所得胜肽之序列 43
表4.6 Chryseobacterium spp.蛋白酶之部分性質比較 52
表4.7各種物質對P1、P2及P3之蛋白酶活性影響 53
表4.8 Zn2+存在與否對於酵素活性的影響 54
表4.9金屬型蛋白酶之特性比較 55
表4.10各種界面活性劑對蛋白酶之影響 56
表4.11蛋白酶之基質特異性 57
表4.12 TKU014和其他蛋白酶之彈性蛋白酶活性比較 58
表4.13各種菌株生產彈性蛋白酶、角蛋白酶的培養基之比較 59


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