Cloning and Expression of the Nattokinase Gene from the Acid-resistant Wild Type Strain Bacillus subtilis natto and Char
黃梁灝、簡宏堅
E-mail: 9511352@mail.dyu.edu.tw
ABSTRACT
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.
Keywords : Bacillus subtilis ; nattokinase ; gene cloning ; enzyme characterization Table of Contents
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?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?基因的表現 16
（1） 納豆激?基因的少量表現 16 （2） 納豆激?基因的大量表現與蛋白回收 17 （3） 納豆激?蛋白的電泳分析 18 第十七節 納豆激?酵素的溶解血栓特性 19 （1） 納豆激?酵素溶解血栓最適pH值的測定 21 （2） 納豆激?酵素溶解血栓pH值耐受性 的測定 21 第十八節 納豆激?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?酵素的溶解 血栓特性 31 （1）納豆激?酵素溶解血栓最適pH值的測定 31 （2）納豆激?酵素溶解血栓pH值耐受性的測定 31 （3）納豆 激?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?酵素溶解血栓活性的影響 35 第五節 納豆激?酵素在E.coli中的分泌性 36 第六節 納豆激?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?酵素 37 第八節 納豆激?的比較 38 圖表 39 參考文獻 62 附錄 65 圖目錄 圖1. 納豆激?在 體內作用的示意圖 39 圖2. 納豆激?的3D立體結構圖（SUB1） 40 圖3. 納豆激?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?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
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