纳豆芽孢杆菌发酵菜用大豆中纳豆激酶的提取及其性质分析

以菜用大豆为原料，直投式纳豆芽孢杆菌作为发酵剂，纳豆激酶活力作为评价指标，通过单因素和正交试验优化纳豆激酶发酵工艺条件；通过盐溶、硫酸铵分段盐析、二乙氨乙基（DEAE）阴离子交换柱分离纯化纳豆激酶，并采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）确定其分子质量，并进行酶学性质和体外溶栓效果分析。结果表明，菜用大豆发酵生产纳豆激酶的最佳条件为：每100 g蒸煮菜用大豆接入1.15×1011 CFU/g直投发酵剂，发酵温度37 ℃、发酵时间36 h、后熟时间18 h时，此时纳豆激酶活力可达2 326.60 IU/g。纳豆激酶的分子质量介于25～35 kDa之间，最适作用温度、pH值分别为37 ℃、8.0，当温度低于40 ℃、pH值在6.0～8.0时较稳定；体外溶栓实验表明纳豆激酶具有良好的体外溶栓效果。

Extraction and properties of nattokinase from vegetable soybean fermented with Bacillus natto

Using vegetable soybean as the substrate, Directed-Vat-Set Bacillus natto as the starter, and nattokinase activity as evaluation index, the fermentation conditions of nattokinase were optimized by single-factor experiments and orthogonal experiments. Then the extracted nattokinase was isolated and purified by salting-in, salting-out with gradient ammonium sulfate solution and dicthylaminoethyl (DEAE) anion exchange column, and the molecular mass was determined through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the enzymatic property and in vitro thrombolytic properties were also analyzed. The results shown that the optimal conditions for nattokinase production through vegetable soybean fermentation were obtined as follows: On 100 g vegetable soybean base, 1.15×1011 CFU/g Bacillus natto was inoculated as starter with fermentation temperature 37 ℃, fermentation time 36 h and post-ripening time 18 h. The purified nattokinase activity was up to 2 236.60 IU/g with molecular mass between 25-35 kDa. The optimal temperature and pH for nattokinase activity was 37 ℃ and 8.0, respectively. It showed good stability under 40 ℃ and pH 6.0-8.0. The results of in vitro thrombolysis showed that nattokinase had good thrombolysis effect.