臺灣博碩士論文加值系統

納豆激酶 （Nattokinase） 又名 subtilisin NAT，由 Bacillus subtilis var. natto （納豆菌） 所分泌出的胞外絲胺酸蛋白酶。之前研究中，發現其對於血纖維蛋白 （fibrin） 具有直接分解的能力，且也能促進體內組織型蛋白酶原活化因子 （tissue plasminogen activtor，t-PA） 的釋放，增加血纖維蛋白酶 （plasmin） 的生成，進而分解血栓 （fibrin clot） 。納豆激酶包含 signal peptide、propeptide 及mature 三個部分組成。本研究為了探討其反應機制，乃於 Escherichia col i系統表達 Bacillus subtilis var. natto 之納豆激酶，同時針對 signal peptide 及propeptide 對其表達、結構摺疊及活性影響之探討，實驗利用聚合酶連鎖反應 （Polymerase chain reaction，PCR） 增幅 Bacillus subtilis var. natto 中三個不同長度片段的納豆激酶基因 （NK） ，分別包含 pre-pro-mature NK基因 （Full-NK） 、 pro-mature NK基因 （Pro-NK） 及 mature NK基因 （Mature-NK） ，並將其接合在表現載體 pET28a ，再轉殖入表現宿主 E.coli BL21（DE3） 中進行重組蛋白之表達。經IPTG 誘導表現之重組蛋白利用 Ni2+-Sepharose bead 親和性管柱進行純化，並進一步測定其酵素活性。結果經由大腸桿菌表逹的 Mature-NK 重組蛋白為不具活性的包涵體 （inclusion body） 。而從 Full-NK 及 Pro-NK 表達之重組納豆激酶則能分解fibrin ，另利用合成受質 Suc-Ala-Ala-Pro-Phe-pNA 測試 Pro-NK 重組蛋白粗酵素液之活性，並根據 Michaelis-Menten 公式計算求得 Km＝0.59 ± 0.01 mΜ 及 Vmax ＝ 1.29 ± 0.01 mΜ / min 。

Nattokinase (also designated Subtilisin NAT) , a potent fibrinolytic enzyme, is extracellular serine protease secreted by Bacillus subtilis var. natto during natto fermentation. Nattokinase not only directly cleaves cross-linked fibrin, but also activates the production of tissue plasminogen activator （t-PA） , resulting in the transformation of inactive plasminogen to the active plasmin. Furthermore, nattokinase enhances its fibrinolysis through cleavage and inactivation of the plasminogen activator inhibitor 1（PAI-1） by limited proteolysis. The nattokinase gene encodes a polypeptide with 29 amino acids signal peptide, 77 amino acids propeptide, and 275 amino acids in the mature enzyme. In this study, a recombinant nattoknase was constructed and characterized. Gene of nattokinase with different sequence （mature, pro- and pre-pro- type） was cloned and expressed in E.coli system. After IPTG （1mM） induction, the overexpressed Mature-NK forms as an inclusion body. The Mature-NK activity is not recovery after solubilized by 6M urea solution, purified by Ni2+-Sepharose bead affinity column and refolding by step-wise dialysis. The lysate of Full-NK and Pro-NK showed fibrinolytic activity. Our results indicate that only the gene of nattokinase with prosequence could produce active nattokinase in E.coli expression system. The lysate of recombinant Pro-NK displays a saturation curve with Km of 0.59 ± 0.01 μΜ and Vmax of 1.29 ± 0.01 mΜ/min for the chromogenic substrate, Suc-Ala-Ala-Pro-Phe-pNA in the kinetic assay.

目錄

頁次
目錄 i
縮寫表 ii
圖目錄 iii
表目錄 v
中文摘要 vi
英文摘要 vii
壹、前言 1
貳、 實驗材料與方法
一、 藥品與試劑 6
二、 實驗菌株 8
三、 儀器設備與器材 9
四、 緩衝溶液及培養液之製備 10
五、 實驗步驟與分析方法
1. 實驗流程 14
2. 構築表達質體 pET28a/Mature-NK、
pET28a / Pro-NK 及 pET28a/Full-NK 15
3. 納豆激酶重組蛋白之表現 20
4. 納豆激酶重組蛋白之純化 21
5. 納豆激酶重組蛋白之酵素活性測定 25
參、實驗結果及討論 27
肆、參考文獻 39
表 46
圖 48
表目錄

頁次
表一、微生物來源之血栓溶解酵素 46
表二、實驗中所使用之引子序列 47

圖目錄

頁次
圖一、蛋白酶活化（maturation） 過程 48
圖二、propeptide幫助蛋白酶活化 （maturation） 過程 49
圖三、納豆激酶血栓溶解作用及相關作用途徑 50
圖四、納豆激酶 （nattokinase） PCR產物及重組質體
DNA agarose gel電泳分析 51
圖五、Mature-NK 重組蛋白於 E.coli BL21 （DE3）
中以不同溫度、IPTG濃度及誘導時間進行表達之
SDS-PAGE（14％）分析圖 52
圖六、Mature-NK 重組蛋白於於 E.coli BL21 （DE3） 中
表達之SDS-PAGE（14％）分析圖 53
圖七、 Mature-NK經 Ni-NTA resin 純化過程 SDS-PAGE
（14％）分析圖 54
圖八、Mature-NK 重組蛋白經質譜（Mass Spectrometry）
分析結果 55
圖九、Mature-NK 重組蛋白及 Pro-NK 重組蛋白於
E.coli BL21（DE3）誘導之western blotting分析圖 56

圖十、誘導時間及溫度不同影響 Pro-NK 重組蛋白誘導
生成之SDS-PAGE（14％）分析 57
圖十一、PMSF 對於Pro-NK重組蛋白在破菌處理
時的影響 58
圖十二、Full-NK重組蛋白和 Pro-NK 重組蛋白誘導之
SDS-PAGE分析圖 59
圖十三、Full-NK 重組蛋白和 Pro-NK 重組蛋白誘導之
western blotting分析圖 60
圖十四、利用 fibrin plate 測定 mature-NK、 Pro-NK 及
Full-NK 重組蛋白的酵素活性
（fibrinolytic activity） 61
圖十五、利用fibrin zymography測定Pro-NK重組蛋白
的酵素活性 （fibrinolytic activity） 62
圖十六、Pro-NK重組蛋白活性分析 63

參考文獻


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