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系統識別號 U0002-2107200903163600
中文論文名稱 Bacillus subtilis TKU007 所生產納豆激酶之研究
英文論文名稱 Studies on nattokinases from Bacillus subtilis TKU007
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 吳盈瑩
研究生英文姓名 Ying-Ying Wu
學號 696180339
學位類別 碩士
語文別 中文
口試日期 2009-07-02
論文頁數 77頁
口試委員 指導教授-王三郎
委員-劉嚞睿
委員-梁慈雯
中文關鍵字 Bacillus subtilis  納豆激酶  casein  Lactobacillus  促進生長 
英文關鍵字 Bacillus subtilis  nattokinase  casein  Lactobacillus 
學科別分類 學科別醫學與生命科學生物學
中文摘要 Baciilus subtilis TKU007 係一株以蝦頭粉為唯一碳/氮源,篩選自台灣北部土壤之納豆激酶生產菌。TKU007生產納豆激酶之較適培養條件為-含有1%蝦頭粉、0.1% K2HPO4及0.05% MgSO4•7H2O之100 mL 液態培養基,於 pH 7、30℃,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 層析分離步驟,純化出二種納豆激酶,經SDS-PAGE測得分子量分別為 30 kDa (N1)、15 kDa (N2)。於最適反應pH、最適反應溫度、pH安定性、熱安定性方面,N1分別為 pH 8、40℃、pH 6-10、<50℃。納豆激酶 N1 活性會受到Mg2+、Fe2+、Cu2+所促進,然而會受到 Zn2+、PMSF所抑制,為絲胺酸型蛋白酶。
將B. subtilis TKU007發酵所得上清液與酪蛋白於37℃反應2小時,添加所得水解液 (最終濃度2.5 %) 至乳酸菌培養基MRS進行乳酸菌Lactobacillus paracasei TKU010之培養,於25℃、搖瓶培養3天,L. paracasei TKU010菌落數可達到5.9×1012 CFU/mL;較對照組(未添加水解物) 增加2. 3倍,添加未水解的酪蛋白反而會抑制L. paracasei TKU010 生長。
英文摘要 Bacillus subtilis TKU007, cultured by shrimp head powder as the only carbon/nitrogen source, was isolated from the soil in Taiwan. For the production of nattokinase, B. subtilis TKU007 was grown in 100 mL of liquid medium in an Erlenmeyer flask (250 mL) containing 1% SHP, 0.1% K2HPO4 , 0.05%
MgSO4•7H2O and grown in an orbital shaking incubator for 3 days at 30℃ and pH 7. After incubation, the culture broth was centrifuged (4℃ and 10,870 ×g for 15 min) ,
and the supernatant was used for further purification by DEAE-Sepharose, Phenyl Sepharose and Sephacryl S-100. The molecular mass of TKU007 nattokinases determined by SDS-PAGE was approximately 28 kDa (N1) and 15 kDa (N2), respectively. The optimum pH, optimum temperature, pH stability, thermal stability of nattokinase N1 was pH 8, 40℃, pH 6-10、<50℃, respectively. N1 nattokinase activity was activated by Mg2+, Fe2+, Cu2+, inactivated by Zn2+, PMSF, indicating N1 nattokinase was serine protease.
A culture supernatant of B. subtilis TKU007 was mixed with casein and incubated for 2 hour at 37℃. The hydrolysates were added to MRS medium (final concentration 2.5 v/v ) for Lactobacillus paracasei TKU010 culture and incubated for 3 days at 25℃. The growth of L. paracasei TKU010 was 5.9×1012 CFU/mL. Which than the control group (non-add hydrolysates) increase 2.3 fold . However, the addition of unhydrolyzed casein could inhibit the growth of L. paracasei TKU010.
論文目次 目錄
頁次

封面內頁
授權書
簽名頁
誌謝 Ⅰ
中文摘要 II
英文摘要 IV
目錄 VI
圖目錄 XI

表目錄 XIII


第一章 緒論 1
第二章 文獻回顧 3
2.1 蝦、蟹殼與烏賊軟骨廢棄物的組成 3
2.2 納豆激酶 4
2.2.1納豆激酶的發現 4
2.2.2 納豆激酶分解血栓的能力驗證 6
2.2.3納豆激酶溶解血栓的機制 6
2.3 血栓溶解劑 7
2.4益生菌 8
2.5 益生質 11
2.6 活性胜肽 13
第三章 材料與方法 15
3.1 實驗菌株 15
3.2 實驗材料 15
3.3 實驗儀器 16
3.4 納豆激酶之活性測定 17
3.5 蛋白酶之活性測定 17
3.6 納豆激酶較適生產條件探討 18
3.6.1 碳/氮源種類之影響 18
3.6.2 碳/氮源濃度之影響 18
3.6.3 培養基酸鹼值之影響 19
3.6.4 培養體積對酵素產量之影響 19
3.6.5 培養溫度之影響 19
3.6.6 較適培養時間 20
3.6.7 較適培養條件 20
3.7 酵素之純化分離 20
3.7.1 粗酵素液之製備 20
3.7.2 離子交換樹脂層析法 20
3.7.3 疏水性層析法 21
3.7.4 膠體過濾層析 21
3.7.5 蛋白質電泳分析 22
3.8 酵素生化特性分析 22
3.8.1 酵素最適反應溫度 22
3.8.2 酵素熱安定性 22
3.8.3 最適反應 pH 23
3.8.4 酵素 pH 安定性 23
3.8.5 金屬離子與抑制劑對酵素活性之影響 24
3.8.6 界面活性劑對酵素活性的影響 24
3.8.7 納豆激酶之基質特異性 25
3.9 發酵所剩烏賊軟骨粉末 (SPP)之去蛋白處理 25
3.10 還原醣之測定 25
3.11 胜肽促進乳酸菌生長 26
3.11.1 水解時間之探討 26
3.11.2 水解液添加濃度之探討 27
3.11.3 不同酵素之探討 27
3.11.4 活菌計數法 28
第四章 結果與討論 29
4.1酵素較適生產條件探討 29
4.1.1碳/氮源之選擇  29
4.1.2培養之pH值探討 30
4.1.3 培養之通氣量探討 31
4.1.4培養之溫度探討 31
4.1.5 綜合結果 31
4.2納豆激酶之分離純化 32
4.2.1 粗酵素液製備 32
4.2.2離子交換樹脂層析 32
4.2.3疏水性層析 33
4.2.4膠體過濾層析 33
4.2.5 綜合結果 34
4.3蛋白質電泳分析 34
4.3.1分子量測定 34
4.3.2 胺基酸序列鑑定 34
4.3.3 綜合結果 35
4.4納豆激酶之生化特性分析 50
4.4.1納豆激酶之最適反應溫度及熱安定性 50
4.4.2納豆激酶之最適反應 pH及pH安定性 50
4.4.3金屬離子對納豆激酶之影響 50
4.4.4納豆激酶對於基質之特異性 51
4.4.5各種界面活性劑對納豆激酶之影響 52
4.5 發酵所剩烏賊軟骨粉末 (SPP)之去蛋白效果 52
4.6促進乳酸菌生長 59
4.6.1水解時間之探討 60
4.6.2 水解液添加濃度之探討 60
4.6.3不同酵素之探討 61
4.6.4 活菌計數法 61

第五章 結論與未來展望 69
參考文獻 70

圖目錄

圖2.1幾丁質、幾丁聚醣之製備 5
圖2.2納豆激酶在血栓溶解系統之直接與間接作用 9
圖4.1 SHP濃度對B. subtilis TKU007納豆激酶之影響 36
圖4.2不同碳/氮源對B. subtilis TKU007納豆激酶之影響 37
圖4.3 pH對B. subtilis TKU007納豆激酶之影響 38
圖4.4通氣量對B. subtilis TKU007納豆激酶之影響 39
圖4.5溫度對B. subtilis TKU007納豆激酶之影響 40
圖4.6 B. subtilis TKU007 於1% SHP (w/v)生產納豆激酶、蛋白酶、幾丁聚醣之生長曲線圖 42
圖4.7 B. subtilis TKU007所生產酵素之純化分離流程圖 43
圖4.8 B. subtilis TKU007之納豆激酶DEAE-Sepharose CL-6B層析圖譜 44
圖4.9 Phenyl Sepharose之納豆激酶層析圖譜 45
圖4.10 Sephacryl S-100之納豆激酶層析圖譜 46
圖4.11 B. subtilis TKU007納豆激酶SDS–PAGE 之分子量分析 48
圖4.12溫度對B. subtilis TKU007納豆激酶活性之影響 53
圖4.13 pH值對B.subtilisTKU007納豆激酶活性之影響 54
圖4.14 B. subtilis TKU007發酵 1% 烏賊軟骨粉末( w/v )之去蛋白應用 63
圖4.15 B.subtilis TKU007 於1%烏賊軟骨粉末( w/v )生產納豆激
酶、蛋白酶及幾丁聚醣之生長曲線圖 64
圖4.16水解時間對於L. paracasei TKU010生長之影響 65
圖4.17水解液添加濃度對於 L. paracasei TKU010生長之影響 66
圖4.18不同水解液對於 L. paracasei TKU010生長之影響 67

表目錄

表2.1臨床上常見的血栓溶解藥劑 10
表2.2益生菌臨床功效 12
表2.3日本各種胜肽原料的來源與機能 14
表4.1 B. subtilis TKU007生產酵素之較適條件之比較 41
表 4.2 B. subtilis TKU007 納豆激酶純化總表 47
表 4.3 B. subtilis TKU007納豆激酶胜肽質譜鑑定結果 49
表4.4金屬離子對 B. subtilis TKU007納豆激酶活性之影響 55
表4.5 B. subtilis TKU007納豆激酶對於基質之特異性 56
表4.6 界面活性劑對B. subtilis TKU007納豆激酶、蛋白酶活
性之影響 57
表 4.7 Bacillus sp.生產納豆激酶特性比較 58
表4.8 添加2.5% 酪蛋白及酪蛋白水解物對於 L. paracasei
TKU010 菌落數之變化 68
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