臺灣博碩士論文加值系統

中文摘要

納豆激酶為納豆菌所分泌之胞外酵素，具有強力溶解血栓活性，在酪蛋白分解活性測試中發現納豆菌SJ與F所測得的比活性相差了有52 % 之多，推估這兩株菌所含納豆激酶在蛋白質的結構上有所差異，而比較其胺基酸序列及蛋白質的二級、三級結構分析結果，推論第53個胺基酸由絲胺酸 ( serine ) 改為脯胺酸 ( proline )可能是結構上造成其活性最主要的原因。本實驗選殖 ( clone ) 定序了九種從納豆所篩選得到的納豆激酶基因，同時也從基因庫中搜尋得到納豆菌之16S核醣體基因片段序列，來建立特殊的引子 ( primer ) ，以建立探討納豆菌在小鼠腸胃道定殖效果的分子檢測技術。小鼠定殖（colonization）試驗結果顯示，利用加熱將非耐熱及非產生芽孢之雜菌去除掉，再配合16S rDNA及納豆激酶引子可以成功的在餵食耐酸納豆菌 ( SJ ) 、耐鹼納豆菌 ( JD11 ) 以及納豆菌孢子 ( SP ) 之小鼠糞便檢體中增幅出其特定的16S rDNA及納豆激酶片段並鑑定其菌種，推論前述這三株菌在餵食後可以通過胃酸、膽汁的分解而成功的定殖在小鼠腸道中，間接證實該三株菌所產生的納豆激酶有機會在腸道被吸收而被小鼠利用。

ABSTRACT

Nattokinase is an extracellular enzyme from Bacillus subtilis var. natto, which has strong fibrinolytic activity. Our studies showed that activities of nattokinase from different strains were different. The activity of nattokinase from natto strain SJ was the highest among nine different strains in caseinolytic activity test. The activity of nattokinase from natto F was lowest and only 52% to that of strain SJ. Nattokinase genes from nine strains were cloned and sequenced. Comparative studies of their predicted primary amino acid sequences, together with the deduced secondary and tertiary structures suggested that the decline of the caseinolytic activity of nattokinase from strain F resulted from the change of serine53 to proline53. Sequences from these nine nattokinase genes and from 16S rDNA obtained from gene bank were used in designing pair of gene specific primers for detecting the presence of DNA fragments in mice’s fecal samples fed with various strains of B.natto. Specific bands of 16S rDNA and nattokinase from strains SJ, JD11 and SP were detected in mice’s fecal samples suggesting that nattokinases from these three strains have better chances in surviving from the digestion of gastric acid and bile. These results provided the molecular evidence in colonization of natto in mice’s intestine.

目錄

封面內頁
簽名頁
授權書 iii
中文摘要 iv
英文摘要 v
誌謝 vi
目錄 vii
圖目錄 xi
表目錄 xiv

第一章　前言 1
第二章　文獻討論 3
2.1 納豆菌簡介 3
2.2 納豆保健成份之作用機轉 3
2.2.1 異黃酮 ( isoflavones ) 之防癌作用 4
2.2.2 骨質疏鬆症之預防 4
2.2.3 抗氧化作用 4
2.2.4 預防心血管疾病 4
2.3 納豆激酶 ( nattokinase ) 5
2.3.1 納豆激酶生化特性 5
2.3.2 納豆激酶溶血栓機轉 6
2.3.3 納豆激酶活性區胺基酸化學修飾之研究 7
2.3.4 納豆激酶對受質之特異性 7
2.3.5 納豆激酶之基因及其蛋白質結構之研究 8
2.4 蛋白質二級及三級結構之預測 9
2.5 16S rDNA及其在菌種分類上的應用 14
2.5.1 細菌核糖體RNA ( ribosomal RNA ) 15
2.5.2 細菌16S rDNA之分析 16
2.6 納豆激酶之腸道吸收 ….17
第三章 材料與方法 19
3.1 實驗材料 19
3.1.1 實驗菌株 19
3.1.2 培養基 19
3.1.3 實驗藥品 19
3.1.4 實驗套組 20
3.1.5 核酸引子序列 20
3.1.6 酵素 21
3.1.7 儀器設備 21
3.1.8 分析軟體 22
3.2 實驗方法 22
3.2.1 納豆菌之培養 22
3.2.2 納豆菌之生長曲線 22
3.2.3 納豆菌胞外酵素液收集 23
3.2.4 酪蛋白分解活性測定法 23
3.2.5 納豆激酶蛋白質定量 24
3.2.6 聚丙烯醯胺凝膠電泳( SDS-PAGE )分析 25
3.2.6.1 聚丙烯醯胺凝膠電泳 ( SDS-PAGE )
膠體配製 25
3.2.6.2 蛋白質樣品前處理 26
3.2.6.3 蛋白質電泳 26
3.2.6.4 染色 ( Staining )、退染 ( Destaining )
27
3.2.7 納豆菌DNA之萃取 27
3.2.8 逢機擴增複製多型性DNA ( Random Amplif
-ied Polymorphic DNA, RAPD ) 28
3.2.9 納豆激酶引子設計 29
3.2.10 以納豆激酶引子進行聚合酶鏈鎖反應 29
3.2.11 枯草桿菌屬16S rDNA引子設計 29
3.2.12 16S rDNA核酸引子進行聚合酶鏈鎖反應
30
3.2.13 膠體電泳回收純化 30
3.2.14 質體DNA之構築 31
3.2.15 質體DNA以熱休克 ( Heat Shock ) 之轉形
( Transformation ) 32
3.2.16 質體DNA之萃取 32
3.2.17 質體DNA定序 33
3.3 納豆激酶序列分析 34
3.3.1 納豆激酶核酸序列與胺基酸序列之分析 34
3.3.2 納豆激酶二級結構之預測 35
3.3.3 納豆激酶三級結構之預測 35
3.4 小鼠餵食納豆菌實驗 36
3.4.1 餵食方法 36
3.4.2 加熱處理小鼠糞便誘導納豆菌產孢子 36
3.4.3 16S rDNA引子對其他菌株之PCR增幅 37
3.4.4 納豆菌小鼠腸道定殖實驗 37
第四章 結果與討論 39
4.1 納豆菌酪蛋白分解活性之測定 39
4.2 納豆激酶基因選質與結構探討 40
4.2.1 納豆激酶基因序列之定序 40
4.2.2 納豆激酶核酸序列及其蛋白質一級、二級
和三級結構 40
4.3 腸道納豆菌之快速檢測技術開發 42
4.3.1 以納豆激酶基因設計引子的納豆菌餵食小
鼠腸道定殖試驗 42
4.3.2 納豆菌之逢機擴增複製多型性DNA 42
4.3.3 以16S rDNA設計引子的納豆菌餵食小鼠
腸道定殖試驗 43
4.3.4 小鼠糞便分子檢測技術開發 45
第五章 結論 46
參考文獻 88





圖目錄

圖1. 血纖維蛋白平板法測試納豆激酶溶血栓活性 47
圖2. 納豆激酶溶解血栓機制 48
圖3. 同源模擬法之流程圖 49
圖4. Nattokinase ( NK ) ; subtilisin E ( SB ) ; subtilisin Carlsberg
( SC ) ; subtilisin lentus ( SE ) 以subtilisin BPN’ ( SS )的
成熟肽胺基酸序列比對與保守結構區 50
圖5. 原核生物16S、23S與5S rDNA位置圖 51
圖6. 酪胺酸標準曲線 52
圖7. 牛血清白蛋白 ( BSA ) 標準曲線 53
圖8. 六株納豆菌之基因組DNA之洋菜凝膠電泳分析圖 54
圖9. yT & A 選殖載體圖譜 55
圖10. SWISS-MODEL之First Approach mode 網頁 56
圖11. SWISS-PDBViewer中ControlPanel、Alignment、Layer
Infos、和Ramachandran Plot等視窗 57
圖12. 九株納豆菌液離心回收上清液之聚丙烯醘胺凝膠電泳 ( SDS-PAGE ) 圖 58
圖13. 以九株納豆菌納豆激酶片段電泳圖 59
圖14. 納豆激酶基因之質體DNA電泳圖 60
圖15. 九株納豆菌pre-pro-mature納豆激酶核酸序列比對 61
圖16. 九株納豆菌成熟納豆激酶與其他枯草桿菌蛋白酶胺基酸
序列之比對 63
圖17. UPGMA群團分析納豆激酶與枯草桿菌蛋白酶胺基酸序列
之親源關係圖 64
圖18. 納豆菌SJ與F之納豆激酶核酸序列比對 65
圖19. 納豆菌SJ與F之納豆激酶胺基酸序列比對 66
圖20. 利用PSIPRED、JNET及SSPRO納豆菌SJ之mature納豆
激酶之二級結構 67
圖21. 利用PSIPRED、JNET及SSPRO納豆菌F之mature納豆
激酶之二級結構 68
圖22. 納豆菌SJ與納豆菌F之第52個胺基酸差異 69
圖23. 納豆菌SJ與納豆菌F之蛋白質三級結構分析 70
圖24. 餵食過納豆菌A以及F小鼠糞便DNA為模板以納豆激酶
引子進行PCR產物之電泳圖 71
圖25. 逢機複製多型性DNA ( random amplified polymorphicDNA
, RAPD ) 電泳圖分析之一 72
圖26. 逢機複製多型性DNA ( random amplified polymorphic DNA
, RAPD ) 電泳圖分析之二 73
圖27. 逢機複製多型性DNA ( random amplified polymorphic DNA
, RAPD ) 電泳圖分析之三 74
圖28. 九株納豆菌基因組 DNA為模板16S rDNA引子進行
PCR之電泳圖分析 75
圖29. 16S rRNA基因片段之質體DNA電泳圖分析 76
圖30. 納豆菌與其他桿菌屬16S rDNA片段之序列分析圖 77
圖31. UPGMA群團分析16S rDNA核酸序列之親源關係圖 78
圖32. 餵食過納豆菌A以及F小鼠糞便DNA為模板以16S
rDNA引子進行PCR之電泳圖 79
圖33. 以16S rDNA引子對加熱過與未加熱之小鼠糞便DNA進
行PCR之電泳圖 80
圖34. 不同細菌DNA以16S rDNA引子進行PCR之電泳圖 81
圖35. 餵食20 % 葡萄糖液之小鼠糞便DNA，以16S rDNA引子
進行PCR之電泳圖( 控制組 ) 82
圖36. 餵食過納豆菌SP之小鼠糞便DNA，以16S rDNA引子進
行PCR之電泳圖 ( 實驗組 ) 83
圖37. 餵食過20 % 葡萄糖液或納豆菌SP之小鼠糞便DNA，
以mature納豆激酶引子進行PCR之電泳圖 84
表目錄

表1. 納豆激酶對不同人工受質之特異性 85
表2. 蛋白質結構模擬常用網站 86
表3. 九株菌與市售之酪蛋白分解活性比較 87

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