心血管疾病一直都是每年全球前幾名死亡原因之一，而造成這些疾病的原因大多是動脈血栓阻塞，因此在相關的治療中，如何降低以及預防血栓的形成，將是避不開的問題。納豆激酶，存在於日本傳統食品納豆中的其中一個成分，已被證實有極強的血栓溶解能力，相比較其他的血栓溶解試劑，安全性較高、可口服
食用作為日常營養補充劑，且眾多的藥理特性也使其被視為是當前預防血栓形成的首要選擇。本研究目的在於開發設計出一套簡化且完整從枯草桿菌發酵液中純化出納豆激酶的製程，以供納豆激酶之量產。首先利用中空纖維膜管搭配切向流過濾系統去除由康達生命科學公司所提供的兩批發酵液中之菌體，所得兩批澄清濾液再進行其中所含納豆激酶之分離純化，由於兩次發酵所使用之條件不同，其中所含蛋白質成分亦不同，因此先以硫酸銨飽和度 50%沉澱處理第一批澄清液，再將上清液飽和度調整為 80%，收集沉澱物進行陽離子交換樹脂吸附純化。第二批發酵液則以硫酸銨飽和度 30%去除雜蛋白沉澱物，再將飽和度 80%所收集沉澱物進一步以陽離子交換樹脂及肝素親和樹脂吸附純化，探討以不同 NaCl 濃度從樹脂上洗脫雜蛋白及純化回收納豆激酶。以 0.2微米之中空纖維膜過濾後可獲得完全無菌之濾液，由逆相高效液相層析分析，則檢測不出代謝產物維生素 K2；所得之純化納豆激酶活性，則以纖維蛋白溶解法測定，第一批發酵液所純化得到活性為 105.167 (FU/mg)，SDS-PAGE 分析純度約 80 ~ 90%；第二批發酵液以陽離子交換樹脂純化的活性為 276.5 (FU/mg)，SDS-PAGE 分析純度約 41.3%，肝素親和樹脂純化得到活性可以達到 292.2 (FU/mg)，純度 80%以上。

Cardiovascular diseases are always on the top causes of death in the world every year, and most of these diseases are caused by arterial thrombosis. Therefore, in related treatments, how to reduce and prevent the formation of thrombosis will be unavoidable problems. Nattokinase, one of the active ingredients in the traditional Japanese food natto, has been proven to have a strong thrombolytic ability. Compared with other thrombolytic reagents, it is safer and can be taken orally as a daily nutritional supplement. Its known pharmacological properties also make nattokinase considered as the first choice for the prevention of thrombosis at present. The purpose of this thesis is to develop and design a simplified and effective process for the purification of nattokinase from Bacillus subtilis fermentation broth that will be feasible for the mass production of nattokinase. First, a 0.2m hollow fiber microfiltration system was employed to completely remove the microorganisms in nattokinase fermentation broth provided by Kangda Life Sciences. The obtained clarified brothes were further used for nattokinase purification. The 1st batch of clarified broth was treated with ammonium sulfate saturation of 50% precipitation to remove the contaminated proteins, then saturation was adjusted to 80% to collect nattokinase containing fraction for further cation exchange resin adsorption. In processing 2nd batch of fermentation broth, ammonium sulfate saturation of 30% was adopted to precipate the impurity proteins, then 80% saturation was again employed to collect nattokinase fraction for cation exchange resin and heparin affinity resin adsorption. By varying NaCl concentrations for eulating the adsorption column, nattokinase could be well purified and recovered. The purified nattokinase activity obtained from 1st fermentation batch as determined by fibrinolysis method was 105.167 (FU/mg), with SDS-PAGE analysis purity was about 90%. From the 2nd batch of fermentation broth, nattiokinase of 276.5 (FU/mg) was IV obtained but with purity about 50%. When heparin affinity resin was employed for purification, nattiokinase activity can reach to 292.2 (FU/mg) with purity > 80%.

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