【作者】 胡升；

【导师】 梅乐和； 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2003， 硕士

【摘要】 筛选得到一株高产纳豆激酶的枯草芽孢杆菌菌株Bacillus subtilis HL-1，对其液体发酵条件进行了优化并在3.7L发酵罐中进行了分批补料发酵，最后对发酵液中的纳豆激酶进行了分离纯化。 采用酪蛋白平板初筛——摇瓶复筛的筛选策略，筛选得到一株野生高产纳豆激酶枯草芽孢杆菌菌株Bacillus subtilis HL-1，摇瓶发酵时该菌株纳豆激酶产量为970 IU·ml^-1。 用响应面方法对Bacillus subtilis HL-1液体发酵生产纳豆激酶的培养条件进行了优化。首先用Plackett-Burman方法对相关影响因素的效应进行评价并筛选出了有显著正效应的胰蛋白胨和有显著负效应的种龄、发酵温度和Na₂HPO₄浓度等四个因素，其他因素对纳豆激酶产量无显著影响。第二步用最陡爬坡路径逼近最大产酶区域。最后由中心组合实验及响应面分析确定了主要影响因素的最佳条件。经优化，该菌株纳豆激酶液体发酵产酶量从优化前的970IU·ml^-1提高到1314IU·ml^-1； 在KLF3.7L（Bioengineering）发酵罐中进行了纳豆激酶大规模发酵实验。首先考察了种子液组成对发酵液产酶的影响，发现在种子液中添加1g·L^-1木糖能提高发酵液产酶活力近30％(1698IU·ml^-1)。在发酵罐中进行了分批发酵实验和补料发酵实验，结果发现：补料发酵能提高纳豆激酶产量，适宜的补料策略是在发酵液菌浓下降后补加新鲜培养基，据此进行分批补料发酵，在装液量为2L时纳豆激酶产量为2348IU·ml^-1。 进行了纳豆激酶的分离和初步纯化实验，确定的分离纯化路径为：发酵液离心除菌→饱和度20％、60％硫酸铵分级沉淀→缓冲液溶解沉淀→Sephadex G-75凝胶层析→Sepharose CM Fast Flow离子交换层析，所得纯化样品经SDS-PAGE电泳检验为单一条带，两步层析的纯化倍数和酶活回收率分别为4.75、74.3％和1.32、77％。更多还原

【Abstract】 Nattokinase is a novel fibrinolytic enzyme with strong and specific thrombolytic activity. In this paper, one strain high yielding nattokinase (NK) was obtained, then the liquid fermentation in shaking flask and fed-batch fermentation in 3.7L fermentator of this strain was optimized, at last isolation and preliminary purification of NK was finished.A wild strain of Bacillus subtil is producing NK named Bacillus subtilis HL-1 was obtained from natto through two-step screening strategy of casein plate selecting producing protease stain first and shaking culture determining secreting NK bacteria finally. The NK activity of B. subtilis HL-1 in liquid culture was 970 IU ?ml-1. Response surface methodology was used to optimize the fermentation condition of NK production of B. subtilis HL-1. In the fist optimization step, a Plackett-Burman design was used to evaluate the influence of related factors. Tryptone influenced NK production positively while NaiHPCU, temperature and age of seed negatively. The others had no significant influence on NK production. The path of steepest ascent was used to approach the optimal region of the fermentation condition subsequently. In the third step the concentrations of tryptone and NaaHPC^ and the age of seed were further optimized using central composite designs and response surface analysis. The optimized condition allowed the NK production to be increased from 970 IU ?ml"1 to 1314 IU ?ml’1.Large-scale liquid fermentation was conducted in Bioengineering KLF 3.7L fermentor. The effect of seed medium on NK production was invested and the result showed that the NK activity in the fermentation culture could be increased 30% (1698 IU ?ml"1) by adding up 1 g ?L"1 xylose into the seed medium. The experiments of batch and fed-batch fermentation revealed that fed-batch benefited NK production and feeding fresh culture medium into fermentator when cell density reduced was preferable, which led to NK activity of 2348 IU ?ml"1 in 2 L culture."7Zhejiang University master paper______________ABSTRACTPurification of NK mainly included four steps: removing cell by centrifugation, 20-60% saturation ammonium sulfate precipitation, Sephadex G-75 gel filtration and Sepharose CM Fast Flow ion-exchange chromatography and purified enzyme appeared single band on SDS-PAGE. The purification factor and NK activity recovery rate of SEC and AIE was 4.75 ^ 74.3 % and 1,32 > 77 %, respectively.更多还原