Statistical optimization of fermentation conditions for enhanced production of Nattokinase from Bacillus subtilis MM26

Nattokinase, a potent fibrinolytic serine protease enzyme has drawn significant industrial attention due to its remarkable thrombolytic activity. It is a natural alternative to conventional anticoagulant drugs because of its ability to disintegrate fibrin clots. Conventional fermentation methods frequently provide Nattokinase with poor yields, limiting production efficiency. Hence improving production strategies to enhance Nattokinase are necessary due to the growing demand for safe, efficient, and economical thrombolytic medicines worldwide. This research study aims to optimize the production parameters to enhance the Nattokinase production by Bacillus subtilis MM26, a newly isolated strain. Notably, the strain displays the unique capability to produce Nattokinase in a Menaquinone-7 (MK-7) production medium, demonstrating its adaptability. An optimum production medium for the Nattokinase synthesis by Bacillus subtilis MM26 was identified and produced 155.1 ± 1.24 U/mL of enzyme. The significant variables influencing Nattokinase production were first identified using one factor at a time (OFAT) approach. Response surface methodology (RSM) predicted the statistically optimized conditions, resulting a yield of 3770.66 ± 0.06 U/mL. The purified enzyme was characterized using various analytical techniques including Matrix-assisted laser desorption ionization–tandem time-of-flight mass spectrometry (MALDI TOF), High Performance Liquid Chromatography (HPLC) and Fourier Transform Infrared spectroscopy (FTIR). Fibrin zymography exhibited a clear lytic band corresponding to approximately 27 kDa, confirming the fibrinolytic activity of Nattokinase. The research findings highlight the ability of Bacillus subtilis MM26 for enhanced Nattokinase production. By combining OFAT and RSM, a substantial increase in the activity of enzyme was noted and the characterization validated its identity, purity and fibrinolytic efficiency. The adaptability and high productivity of the strain emphasize its suitability for economical, scalable applications in the nutraceutical or industrial applications.