【作者】 梁剑光；

【导师】 熊晓辉；

【作者基本信息】 南京工业大学 ， 生物工程， 2003， 硕士

【摘要】 纳豆激酶（Nattokinase NK）是纳豆发酵过程中由纳豆枯草杆菌（Bacillus subtils natto）产生的一种丝氨酸蛋白酶。1987年日本学者须见洋行首先发现。研究表明，纳豆激酶具有很强的溶解血栓功能，与临床所用的尿激酶、链激酶等溶栓药物相比，NK具有安全性好，易被人体吸收，作用直接迅速，药效持续时间长，可由纳豆枯草杆菌直接发酵生产因而造价低廉等优点，许多学者预言，NK将是一种很有潜力的新型溶栓药物。 本论文对纳豆激酶液态发酵优化、分离纯化以及酶学特性等几个方面进行了系统的研究，最终得出以下结果： 1．从实验室一株高产纳豆激酶菌株出发，优化了液态发酵条件。研究表明，种子培养基的最佳碳源为葡萄糖，浓度为1％；最佳氮源为蛋白胨，浓度为1.5％。种子培养基的配方组成为：蛋白胨1.5％，葡萄糖1％，酵母膏0.1％，NaCl 0.3％。发酵培养基的最佳碳源为可溶性淀粉，浓度为2％；最佳氮源为胰胨，浓度为0.5％。发酵培养基中加入诱导物A或B有利于纳豆激酶产量的提高，诱导物加入的时间以产酶开始（即第二天）为最好，其加入量为0.1％。金属离子Ca²⁺、Mg²⁺有利于酶的合成。纳豆菌在37℃的生长最好，但产酶量不大，30℃有利于产酶，故选择30℃为最佳发酵温度。pH7.0有利于菌体的生长和产酶；装液量为100ml/500ml即能满足菌对氧的需求，又不影响其产酶；接种量以2％为最佳，最适种龄为20h。经过以上系统优化后，我们所获得的产酶量最高可以达到2644.44IU/ml，是最初产酶量的近2倍。 2．纳豆激酶分离纯化研究表明：纳豆激酶粗酶液经过盐析、透析脱盐、DEAE-Sepharose柱层析、SephadexG-100柱层析等步骤，最终分离的纳豆激酶经过SDS-PAGE电泳检测为单一区带。分离纯化倍数为19.00倍，收得率为42.10％，酶比活为22388.81IU/mg。 3．此外，对纳豆激酶的酶学特性进行了研究，结果是：NK反应的最适温度为45℃—50℃，纳豆激酶在25℃—40℃放置6h—8h稳定性好，残存酶活力为95％以上，温度超过55℃时，酶活下降很快，到70℃时酶活完全消失。最适反应pH为pH7.0—pH7.5，在pH6.0—pH9.0酶活较稳定，Na⁺、K⁺对NK的稳定性无 南京工业大学硕士学位论文多大影响，Cll》、Mn”有较大程度的抑制作用，Hg》呗u可以完全抑制NK的活性，致使NK活性完全丧失。C？＼Mgz”对NK有激活作用，是NK的一种激活剂。更多还原

【Abstract】 Nattokinase(NK) is a fibrinolytic serine enzyme that is extracted from a traditional fermented food of Japan. It is reported that Nk have a strong thrombustic function. Compared with the thrombolytic agents such as urokinase and treptokinase, 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 study on the liquid fermentation of NK, purification,and character of nattokinase..At last ,the main results are as follows:1. The liquid fermentation optimization demonstrate : To the seminal medium, The best carbon source is glucose,the best nitrogen source is peptone . And the composition of the seminal medium is peptone 0.5%,Glu 1%,Yeast extract 0.1% ,NaCl 0.3%. To fermentative medium,the best carbon source is resolutive starch 2%,the best nitrogen source is tryptone 0.5%. inducer A and B can enhance the yield of NK. Ca2+ and Mg2+ are the positive ion which have the greatest effect on NK’s synthesis.The bacteria grows the fast at 37℃,but 30℃ is the most suitable temperature for the production of NK; The bacteria has the biggest speed of growth and the biggest yield of NK under pH7.0.The volume of fermentation medium is 100ml/500ml,2%of 20h-old inoculum is the best condition for NK’s synthesis.2. Purification experiment result: Purifying the enzyme by the ammonium sulfate fraction,DEAE-Sephrose, SephadexG-100 Chromatography and so on,the purified enzyme showed a single protein band in the SDS-PAGE.purified is 19,and the last yield is 42%,Specific activity is 22388.81 IU/mg.3. The character of nattokinase is the follows :the optimum reaction temperature of NK is 45-50℃ ,and the stability of NK is at 25-40℃,which is very good in 6-8 hours.when the temperature exceed 55 ℃,activity of NK drops very fast .when above 70℃ activity of NK forfeits completely.The pH7.0-7.5 is the optimal pH and pH6.0-9.0 is the best condition for NK’s stability Na+ and K+ have less effect onthe NK’s stability ,Cu2+ and Mn2+ have more effect on the NK’s stability;but Hg2+ restraint the activity completely ,Ca2+ Mg2+ have positive effect on NK ,which is a fine activator of NK.更多还原