通过敲除聚谷氨酸合成基因提高纳豆杆菌纳豆激酶的生产效率

Improve the production efficiency of Bacillus natto nattokinase by knocking out the γ-PGA synthetic gene

纳豆激酶是纳豆杆菌(Bacillus natto)产生的具有溶栓作用的酶,纳豆杆菌液体发酵时产生的聚谷氨酸(polygutamic acid,γ-PGA)使发酵液黏度升高,导致纳豆激酶分离纯化困难,成本较高,限制了其规模化生产。该研究采用同源重组双交换技术,敲除纳豆杆菌中合成γ-PGA的关键基因pgsB,降低发酵液中γ-PGA的含量和发酵液黏度,提高后续纳豆激酶的分离纯化效率。结果表明pgsB基因缺失株与野生型菌株相比,发酵液中γ-PGA产量显著下降,在发酵24 h时γ-PGA含量下降了57.9%。此外两者的细胞密度无明显差异,说明pgsB基因的缺失可显著降低发酵液黏度且对菌体的生长没有影响。使用超滤法纯化纳豆激酶的过程中,基因缺失株的纳豆激酶回收率比野生型提高了19.2%。因此,敲除pgsB基因后,纳豆激酶分离纯化的效率得到了提高,为纳豆激酶的工业化生产提供了新方法。

Nattokinase is a thrombolytic enzyme produced by Bacillus natto. The polyglutamic acid(γ-PGA) produced during the cultivation of B. natto increases the viscosity of the fermentation broth, which makes the separation and purification of nattokinase difficult. It increases the cost and limits the large-scale production of nattokinase. In this study, the γ-PGA synthetic gene pgsB was knocked out by homologous recombination, to reduce the content of γ-PGA and the viscosity of the fermentation broth, which might improve the efficiency of separation and purification of nattokinase. The result showed that the yield of γ-PGA of pgsB-deleted strain reduced significantly compared to the wild strain. At the 24;hour of fermentation, the yield of γ-PGA of pgsB-deleted strain reduced 57.9%. Besides, there was no significant difference in cell density, indicating that the deletion of pgsB gene reduced the viscosity of the fermentation broth and had no effect on the growth of the bacteria. The recovery rate of nattokinase of the pgsB-deleted strain was increased by 19.2% in the process of purifying nattokinase by ultrafiltration. Therefore, the separation and purification efficiency of nattokinase had been improved after knocking out the pgsB gene, which might provide a new method for the industrial production of nattokinase.