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研究生:黃梁灝
研究生(外文):Liang-Hao Huang
論文名稱:耐酸性納豆菌之納豆激酶基因選殖、表現與酵素活性分析
論文名稱(外文):Cloning and Expression of the Nattokinase Gene from the Acid-resistant Wild Type Strain Bacillus subtilis natto and Characterization of Its Enzyme
指導教授:簡宏堅簡宏堅引用關係
指導教授(外文):Hung-Chien Roger Chien
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:71
中文關鍵詞:枯草桿菌基因選殖納豆激酶酵素活性分析
外文關鍵詞:Bacillus subtilisnattokinasegene cloningenzyme characterization
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由陳明造老師及王正仁老師來的DYU1001及DYU1002經由16 S rDNA鑑定為Bacillus subtilis 枯草桿菌。歸納納豆激酶的資料後,發現納豆激酶可分為三段,pre 區段,pro 區段,以及mature 區段,而mature是納豆激酶主要的功能區,但其耐酸的能力不好,pre-pro是以犧牲打的方式代替mature被胃酸水解,所以依pre-pro-mature nattokinase的胺基酸序列來設計引子,以B. subtilis DYU1002的染色體做為模版,利用PCR,合成長度為1146 bp的DNA片段,將其與B. subtilis的pre-pro-mature nattokinase胺基酸序列比對之相同度為100%。由於目前市面上的納豆激酶幾乎都是以膠囊或散劑的形式來販賣,其對溶解血栓來預防動脈硬化疾病的功能不是很好,可能納豆激酶經過胃酸水解後,其活性就被破壞。而DYU1002這株納豆菌,據王正仁老師說其可在酸性的環境下生長(pH 3.0),所以篩選出DYU1002含有pre-pro-mature nattokinase基因的純株(clone),將此段基因選殖入Escherichia coli菌體內可表現納豆激酶的活性。此ORF全長為1146 bp可轉譯出含382個胺基酸殘基的蛋白質,分子量為42 kDa,是為一種血栓溶解的酵素,其可自體分解為3.2 kDa 的pre-peptide、8.5 kDa 的pro-peptide 及28 kDa的mature nattokinase,回收此酵素,測酵素溶解血栓的活性,發現此酵素最適溶解血栓的pH值為pH 8.0,其在菌體內的酵素溶解血栓的活性為0.96×103 FU/mg。另有以0-30%硫酸銨濃縮後,跑SDS-PAGE,在SDS-PAGE上發現僅有一明顯的蛋白質產物,其位置大約在42 kDa,可能就是選殖的基因所表現出來的蛋白pre-pro-mature nattokinase,所以pre-pro-mature nattokinase在E. coli中可能被分泌到培養液中,推測為一種胞外酵素蛋白,而此分泌到培養液的酵素溶解血栓的活性為5.66×105 FU/mg。此酵素溶解血栓活性的 pH 值耐受性為 pH3.0~pH 10.0。
The databases of DYU1001 and DYU1002 strains from Dr. Chen and Dr. Wang are bioassayed as Bacillus subtilis with 16 S rDNA. After generalizing the data of nattokinase, we find that nattokinase can be divided into three fragments:pre , pro and mature. Mature is the primary functional domain of nattokinase, but its acid-resistant ability is not good. The fragment “pre-pro” protects “mature” from hydrolysis by gastric acid with the sacrificial way. The primer pair was designed in compliance with the amino acid sequence of pre-pro-mature nattokinase. The chromosome of Bacillus subtilis DYU1002 was used as a template to synthesize 1146 bp DNA fragment with PCR amplification, then compare the gene fragment with the amino acid sequence of the Bacillus subtilis’ pre-pro-mature nattokinase. The identities is 100%. The nattokinases are marketed presently most sold in capsules or powders. Their thrombolytic function to against arteriosclerosis is not very effective, because their activity may be destructed through gastric acid. According that Dr. Wang considered the Bacillus subtilis DYU1002 can grow in acidic environment (pH 3.0), we screen the gene of Bacillus subtilis DYU1002 which have the pre-pro-mature nattokinase activity. Then we transformed this gene into E.coli, and expressed the enzymatic activity of nattokinase. The ORF of nattokinase gene is 1146 bp and it translated into a peptide of 382 residues. The molecular weight of the thrombolytic enzyme is 42 kDa. The enzyme was recoveried and measured their thrombolytic activity. We found that
the enzyme has the optimal thrombolytic activity in pH 8.0;the activity in the germ body of enzyme can achieve 0.96×103 FU/mg. In the SDS-PAGE, one obvious protein product from 0-30% ammonium sulfate precipitation was found. Its molecular weight is about 42 kDa. The only one secreted protein from E. coli nattokinase recombinant is called exoenzyme. The thrombolytic activity of this secreted nattokinase was 5.66 × 105 FU/mg. We found that the enzyme has the dural thrombolytic activity in pH3.0~
pH 10.0.
目錄

封面內頁
簽名頁
授權書 iii
中文摘要 iv
英文摘要 vi
誌謝 viii
目錄 ix
圖目錄 xiv
表目錄 xvi
附錄目錄 xvii

第一章 前言 1
第二章 材料與方法 5
第一節 菌株及質體 5
第二節 藥品 5
第三節 培養基 5
第四節 DYU1001及DYU1002 染色體DNA的分離 6
第五節 E.coli質體(plasmid)DNA的抽取 7
第六節 引子(primer)的設計 8
第七節 聚合酶鏈鎖反應(polymerase Chain reaction;PCR) 9
第八節 瓊脂凝膠電泳 (agarose gel electrophoresis) 10
第九節 DNA 片段的回收及純化 11
第十節 限制酵素剪切(enzyme digestion) 12
第十一節 DNA 的黏接反應(ligation) 13
第十二節 大腸桿菌電勝任細胞之製備 13
第十三節 轉型作用(transformation) 14
第十四節 萃取質體DNA 15
第十五節 DNA定序與序列比對 16
第十六節 納豆激酶基因的表現 16
(1) 納豆激酶基因的少量表現 16
(2) 納豆激酶基因的大量表現與蛋白回收 17
(3) 納豆激酶蛋白的電泳分析 18
第十七節 納豆激酶酵素的溶解血栓特性 19
(1) 納豆激酶酵素溶解血栓最適pH值的測定 21
(2) 納豆激酶酵素溶解血栓pH值耐受性的測定 21
第十八節 納豆激酶的濃縮純化 22
第三章 結果 26
第一節 設計pre-pro-mature nattokinase、pro-mature nattokinase
mature nattokinase的引子 26
第二節 抽取B.subtilis DYU1001及B.subtilis DYU1002的染色體 26
第三節 利用polymerase chain reaction( PCR ) 鑑定Bacillus sp.DYU1001及
Bacillus sp.DYU1002的16S rDNA基因 27
第四節 Bacillus sp.DYU1001與Bacillus sp.DYU1002 16S rDNA在E.coli之表
現 27
第五節 利用polymerase chain reaction( PCR ) 選殖pre-pro-mature
nattokinase的基因 29
第六節 pre-pro-mature nattokinase基因的表現 29
(1) pre-pro-mature nattokinase基因在E.coli的表現 29
(2) 純化回收pre-pro-mature nattokinase的酵素 30
第七節 pre-pro-mature nattokinase基因定序及序列比對 30
第八節 納豆激酶酵素的溶解血栓特性 31
(1)納豆激酶酵素溶解血栓最適pH值的測定 31
(2)納豆激酶酵素溶解血栓pH值耐受性的測定 31
(3)納豆激酶酵素在E.coli中的分泌性 32
第四章 結論 34
第一節 Bacillus subtilis DYU1001與Bacillus subtilis DYU1002的16 S rDNA
序列 34
第二節 pre-pro-mature nattokinase的胺基酸序列 34
第三節 pre-pro-mature nattokinase基因的大量表現 35
第四節 pH值對納豆激酶酵素溶解血栓活性的影響 35
第五節 納豆激酶酵素在E.coli中的分泌性 36
第六節 納豆激酶酵素溶解血栓pH值的耐受性 37
第七節 在胞內及胞外的納豆激酶酵素 37
第八節 納豆激酶的比較 38
圖表 39
參考文獻 62
附錄 65
圖目錄

圖1. 納豆激酶在體內作用的示意圖 39
圖2. 納豆激酶的3D立體結構圖(SUB1) 40
圖3. 納豆激酶的3D立體結構圖(SUB2) 41
圖4. 設計pre-pro-mature nattokinase引子 42
圖5. 構築質體 43
圖6. Bacillus subtilis DYU1001與Bacillus subtilis DYU1002染色體電泳圖44
圖7. Bacillus subtilis DYU1001 16 S rDNA PCR電泳圖 45
圖8. Bacillus subtilis DYU1002 16 S rDNA PCR電泳圖 46
圖9. 限制酵素檢測Bacillus subtilis DYU1001 16 S rDNA 47
圖10.限制酵素檢測Bacillus subtilis DYU1002 16 S rDNA 48
圖11.pre-pro-mature nattokinase PCR電泳圖 49
圖12.pre-pro-mature nattokinase抽質體電泳圖 50
圖13.限制酵素檢測pre-pro-mature nattokinase 51
圖14.pre-pro-mature nattokinase的SDS-PAGE 52
圖15.pre-pro-mature nattokinase基因序列比對 53
圖16.pH值對B.subtilis DYU1002 pre-pro-mature nattokinase溶解血栓活性的影
響 54
圖17.pQE-ppm硫酸銨濃縮後溶解血栓的活性 55
圖18.pQE-ppm硫酸銨濃縮後的SDS-PAGE 56
圖19.未濃縮pQE-ppm菌液溶解血栓的活性 57
圖20.納豆激酶酵素的pH值耐受性 58
圖21.Bacillus subtilis DYU1001 16 S rDNA基因
序列比對 59
圖22.Bacillus subtilis DYU1002 16 S rDNA基因
序列比對 60
表目錄

表1. 比較來自不同菌株的血栓溶解酵素 61
附錄目錄

附錄1. 本實驗所使用的菌株與質體 65
附錄2. 本實驗所使用的藥劑配方 66
參考文獻

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and biophysical research communications 197 (3):1340-1347.

3. Fujita M, Ito Y, Hong K, Nishimuro S, (1995a)Characterization of Nattokinase-degraded products from human fibrinogen or cross-linked fibrin.
Fibrinolysis 9, 157–164.

4. Fujita M, Hong K, Ito Y, Misawa S, Takeuchi N, Kariya K, Nishimuro S.(1995)Transport of nattokinase across the rat intestinal tract. Biol Pharm
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14. Sambrook J and Russell DW (2001) Molecular Cloning: a laboratory manual, third edition. Cold Spring Harbor Laboratory Press, New York.

15. Sumi H, Nakajima N, Yatagai C.(1995)A unique strong fibrinolytic enzyme (katsuwokinase) in skipjack "Shiokara," a Japanese traditional fermented food.
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16. Suzuki Y, Kondo K, Ichise H, Tsukamoto Y, Urano T, Umemura K.(2003)Dietary supplementation by fermented soybean, natto, suppresses intimal thickening after endothelial injury in rat femoral artery. Nutrition 19:261–264.

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