High Cell Density Cultivation of A Recombinant Bacillus Subtilis for Nattokinase Production [post]

Jianhua Zhang, Qing Cui, Bingjun Qian, Xiangjun Sun
2020 unpublished
Background: Nattokinase (NK), a fibrinolytic enzyme, can be produced by culturing recombinant Bacillus subtilis in Luria-Bertani broth in a shaking flask. For use as a nutraceutical, however, a large-scale preparation and a simple purification process are required.Results: The present study utilized a fed-batch process to cultivate a B. subtilis strain carrying a pHT01 plasmid with an NK-encoding gene (B. subtilis/pHT01-aprN1). For batch A (FB A), with a pH-stat two-stage fermentation strategy,
more » ... mentation strategy, we achieved an activity of 2910.5 ± 21.6 U mL-1 and a specific activity of 30.32 U ml-1 OD600-1. Then, we changed the strategy with a later induction and lower feeding rate to pursue higher cell density and thus higher enzyme activity, a 11.9-fold activity of 4521.8 ± 23.8 U mL-1 was acquired, however, the specific activity was lower than FB A. For the third batch, low-glycerol-level-maintain feeding strategy was followed, and finally, a NK activity of 7778 ±17.28 U mL-1 was obtained, according to our knowledge, it was the highest activity assayed by the fibrin plate method ever reported. Furthermore, fermentation supernatant was successively purified by ammonium sulfate precipitation and nickel column affinity chromatography with a total NK recovery rate of 65.2%.Conclusions: Our results indicate that there is a balance between the cell growth rate and NK expression when recombinant Bacillus subtilis is cultured with a fed-batch process. The equilibrium state can be attained by optimizing the induction and feeding strategy, and thus a high cell density and enzyme activity can be achieved.
doi:10.21203/rs.3.rs-91180/v1 fatcat:q2hgytkjgna5pbbhlsau52hjzq