High cell density cultivation of a recombinant Bacillus subtilis for
Several hundred U mL-1 of 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, large-scale
preparation and a simple purification process is required. The present
study utilized a fed-batch process with a pH-stat and
low-glycerol-level-maintain feeding strategy to cultivate a B. subtilis
strain carrying a pHT01 plasmid with an NK-encoding gene (B.
subtilis/pHT01-aprN1). Finally, a NK activity of 7778 ±17.28 U mL-1 was
obtained, which represented a 26-fold increase of NK activity by high
cell density cultivation compared to the flask culture. Furthermore,
fermentation supernatant was successively purified by ammonium sulfate
precipitation and nickel column affinity chromatography with a total NK
recovery rate of 65.2%.