State Key Laboratory of Biocatalysis and Enzyme, Engineering Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, Biology Faculty of Hubei University, Hubei University, Wuhan, Hubei Province, 430062, People's Republic of China.
School of Life Sciences, Yunnan Normal University, Kunming, Yunnan, People's Republic of China.
BMC Biotechnol. 2021 Aug 9;21(1):49. doi: 10.1186/s12896-021-00708-4.
Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative.
This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1-7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity.
This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.
纳豆激酶是一种纤维蛋白溶解酶，作为一种促进健康的营养补充剂，具有巨大的市场价值。为了提高纳豆激酶的产量，已经对发酵条件、菌株、培养介质和喂养策略进行了优化。已经使用几种异源表达系统来表达纳豆激酶。毕赤酵母异源表达系统是一种替代方法。
本报告旨在使用毕赤酵母异源表达系统表达来自枯草芽孢杆菌纳豆（NK-Bs）的高水平纳豆激酶，并评估其体内的纤维蛋白溶解活性。构建了携带 aprN 基因 1-7 个拷贝的多拷贝表达菌株。目标蛋白的表达水平在目标基因的五个拷贝时达到最大值。然而，多拷贝表达菌株在摇瓶或高密度发酵中不稳定，导致目标蛋白产量在批次之间存在显著差异。因此，在摇瓶和高密度发酵中使用携带单个 aprN 拷贝的 P. pastoris。在摇瓶发酵中，目标蛋白的产量为 320mg/L，在高密度发酵中约为 9.5g/L。重组纳豆激酶表现出较高的热稳定性和 pH 稳定性。本研究还表明，重组 NK-Bs 具有明显的溶栓活性。
本研究表明，毕赤酵母表达系统是大规模、低成本制备纳豆激酶的理想平台。
