【作者】 刘诚；

【导师】 董明盛；

【作者基本信息】 南京农业大学 ， 食品微生物与生物技术， 2000， 硕士

【摘要】 纳豆激酶（Nattokinase，简称NK）是一种丝氨酸蛋白激酶，最初是从日本传统发酵食品纳豆（Natto）中提取得到的，纳豆激酶具有与链激酶、尿激酶等溶血栓药物同样的溶解血栓作用，比之现用于临床的溶血栓药物，NK具有安全性好，易被人体吸收，作用直接迅速，作用持续时间长，可由微生物发酵生产因而造价低廉等优点，是一种很有潜力的新型溶血栓药物。 本论文在液体发酵生产NK方面进行了研究，得出如下结果： 1．从实验室保存的产纳豆激酶菌种出发，优化液体发酵条件得出：最佳氮源是豆粕粉，最佳碳源玉米粉，碳氮比以1∶1为合适，离子中以Ca²⁺的影响最大，以0.02％为最佳浓度，Mg²⁺的是否添加影响不大，磷酸盐以Na₂HP₄ 0.2％∶NaH₂PO₄ 0.1％为最优，Mn²⁺抑制酶的合成，表面活性剂的添加不影响酶的分泌。细菌在37℃时生长最为迅速，细胞量最大，但对产酶来讲，30℃为最佳温度，故选择30℃作为发酵温度，pH7是细菌生长和产酶的最适pH；该菌种是好氧菌，装液量小利于细菌生长和产酶，但装液量过小，水分挥发过快，不利于NK总产量的提高，故选择100ml/500ml。接种量以2％为最佳，最适种龄为24h。 在以上优化的条件下液体发酵产酶，获得的产酶量为1280IU/ml，是最初产酶量的3倍。 2．NK的最适反应温度为45℃～50℃，酶的稳定性在25℃和37℃时较好，25℃下放置24h残存酶活力为95％，37℃下放置24h残存酶活为78％，55℃以上酶活迅速消失；最适反应pH为7.0～7.5，在pH7～8范围内酶活最为稳定，Hg²⁺可导致酶的迅速失活，Mg²⁺对酶有激活作用；牛血清蛋白、甘油、丙二醇、海藻酸钠、蛋白胨，明胶都能提高酶的稳定性，以酪蛋白为底物时的Vm=7.76μg/ml min，Km=10.87×10³μg/ml。更多还原

【Abstract】 Nattokinase (NK) is a fibrinolytic enzyme that is extracted from natto(a traditional fermented food in Japan). in fact it has enzymatic property of protease. Compared with the thrombolytic agents such as urokinase and streptokinase, Nattokinase is safer, easier to be absorbed by body because of its low molecular weight, and has more direct and more persistent effect on thrombosis, and It is more important that Nattokinase can be fermented by bacteria, which will lower the production cost. So nattokinase can be developed as a new fibrinolytic medicine. This paper investigated the liquid fermentation of NK. The main results were as follows. 1 .Using Bacillus sp.stored in lab, which can produce NK, the liquid fermentation condition was optimized: The best nitrogen source is the defatted soybean, the best carbon source is maize powder, C N==l 1 was the best choice; Ca2~ was the positive ion which had the greatest effect on NK抯 synthesis, while whether Mg2~ was added or not was not very important, 0.02% of Ca2~ and Na2HPO4 0.2% NaH2PO4 0.1% were the optimal conditions for NK~s yield; Mn2~ inhibit NK抯 biosynthesis; Surface-active agents could not increase the secretion of NK: The bacteria grew the fast in 37(2, but 300(2 was the most suitable temperature for the production of NK; The strain had the biggest speed of growth and the biggest yield of NK under PH7; This strain was oxybiotic, So less volume of fermentation medium fit its growth and sythesis of NK better; But because of the volatilization of water was more when volume of media is too little, lOOml/500m1 was choosen for the biggest gross production of NK; 2% of 24h-old inoculum was the best condition for NK抯 synthesis. 2. The optimum reaction temperature of NK was 45 ~?50~C and the stability of NK was very good at 250(2 and 3TC. The relative activity of NK is V Abstract 95% after 24h at 25C and is 78% at 370C; NK inactivated quickly above 55C. The PH7.O~?.5 was the optimal PH and PHTh-8 is the best condition for NK抯 stability; Hg2~ could inhibit NK抯 activity and Mg2~ promoted it , Serum albumin, glycerol, propylene glycol, sodium alginate, peptone, gelatin could improve the stability of NK. When used casein as substrate, Vm=7.76 u gIml mm, Km10.87X iO%t g/ml.更多还原