納豆激酶是由Bacillus subtilis natto所分泌之胞外蛋白酶，具有優異的分解血栓能力。目前已知在腸道內可以被快速吸收且作用時間長達8小時以上，因此在保健醫藥品的應用上格外受到注目。本研究將建構於大腸桿菌質體pET26b(+)的納豆激酶基因，藉由於TB中添加sorbitol和betaine，成功表現產生具有溶解血栓能力的重組納豆激酶。單獨以IPTG誘導時(最佳誘導濃度為1 mM IPTG)，可於誘導後24小時，測得較佳胞外酵素活性約2.71 U/ml;誘導後8小時，測得較佳胞內酵素活性約4.67 U/ml。以lactose誘導(最佳誘導濃度為10 mM)時，可於誘導後16小時，測得較佳胞外酵素活性約1.29 U/ml;誘導後8小時，測得較佳胞內酵素活性約1.75 U/ml。IPTG與lactose共同誘導時，最佳誘導濃度為10 mM lactose和0.125 mM IPTG，可於誘導後20小時，測得較佳胞外酵素活性約3.36 U/ml;誘導後4小時，測得較佳胞內酵素活性約3.5 U/ml。

Nattokinase, an extracellular protease produced by Bacillus subtilis natto exhibits excellent fibrinolytic activity. It can be absorbed quickly by intestine and has prolonged effect over eight hours. Thus, nattokinase has raised great attention for the potential applications in healthy protection and medicine. In this study, the nattokinase gene constructed on pET26b(+) was successfully expressed in TB containing betaine and sorbitol to produce nattokinase with fibrinolytic activity.

Using IPTG as the inducer solely, the maximal intracellular nattokinase activity of 4.67 U/ml was obtained 8 hours after induction by 1 mM IPTG, while the maximal extracellular activity of 2.71 U/ml was obtained 24 hours after induction by the same condition. As lactose was used as inducer instead, the higher intracellular activity of 1.75 U/ml was obtained 8 hours after induction by 10 mM lactose. Meanwhile, the higher extracellular enzyme activity of 1.29 U/ml was obtained 16 hours after induction. With lactose and IPTG as inducers after induction, the maixmal intracellular activity of 3.50 U/ml was obtained 4 hours and the maximal extracellular activity of 3.36 U/ml was detected 20 hours after induction by 10 mM lactose and 0.125 mM IPTG.

誌謝I
中文摘要II
英文摘要III
目錄IV
圖表目錄V
第一章 前言1
1.1 日本人之飲食習慣1
1.2 納豆的起源及製作1
1.3 納豆主要營養成分和生理功效3
1.4 納豆激酶4
1.5 血栓5
1.6 冠狀動脈疾病6
1.7 納豆激酶功效於血栓之驗證7
1.8 納豆激酶功效於高血壓患者之功效8
1.9 預防骨質疏鬆9
1.10 保健食品及藥物服用之準則10
1.11 研究目的11
第二章 實驗材料13
2.1 菌株13
2.2 質體13
2.3 表現載體pET26b之製備13
2.4 實驗藥品及材料14
2.5 實驗儀器14
第三章 實驗方法16
3.1 質體抽取16
3.2 勝任細胞(Competent Cell)的製備17
3.3 轉型作用(Transformation)17
3.4 重組納豆激酶的誘導表現18
3.5 Caseinolytic activity的檢測19
第四章 實驗結果與討論20
4.1 以TB為培養液時重組納豆激酶之表現20
4.2 以含有sorbitol和betaine的TB為培養液、並以IPTG作為誘導劑時之酵素活性表現20
4.3 以含有sorbitol和betaine的TB為培養液、並以lactose作為誘導劑時之酵素活性表現21
4.4 以含有sorbitol和betaine的TB為培養液、並以lactose與IPTG共同誘導時之酵素活性表現22
第五章 結論23
第六章 圖與表25
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