基于摇瓶发酵优化结果,在10 L发酵罐中研究了纳豆芽孢杆菌TN-02产纳豆激酶的发酵控制工艺,分析了发酵产物中纤溶酶的组分纯度。在放大发酵过程中,考察了培养温度和主要碳、氮源的流加补充对产酶的影响,并利用卡那霉素抗性基因kan对纳豆芽孢杆菌TN-02中的纳豆激酶基因aprN进行了部分替换和插入失活,获得了aprN突变的重组菌TN-021。结果表明,通过温度的阶段性控制和甘油、胰蛋白胨补料发酵,获得纳豆激酶的最高表达量为13 978.3 U/mL,是摇瓶发酵最高表达量的1.8倍;在菌株TN-021的摇瓶发酵产物中未检测到纤溶酶活力,证明了纳豆激酶是纳豆芽孢杆菌TN-02发酵产物中唯一纤溶酶。研究结果为纳豆激酶高水平发酵放大工艺控制奠定了基础,同时为纳豆激酶的品质分析方法提供参考。
The fermentation process was optimized to produce nattokinase by Bacillus natto TN-02 in 10 L fermentor. The effects of culture temperature and feeding of major carbon and nitrogen sources on nattokinase production were investigated. A recombinant strain TN-021 in which aprN was disrupted. The results showed that periodic control of temperature and feeding of glycerol and tryptone could elevate the yield of nattokinase to a maximum of 13 978.3 U/mL, which was 1.8 times of that in shake flask fermentation.No fibrinolytic enzyme activity was detected in shake flask fermentation products of strain TN-021, which indicated that nattokinase was the only fibrinolytic enzyme produced by B. natto TN-02. In summary, this research provided a base for high-level scale-up fermentation of nattokinase, as well as for nattokinase quality control.
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