Enhanced Nattokinase Production by Bacillus subtilis from Glycerol and Okara: Optimization of Culture Medium via Response Surface Methodology

Yaling DuanWanting DuYang XuGege GuoZhaoxing LiuNing Hao^*

• School of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China

Nattokinase is an alkaline serine protease with potent thrombolytic activity. Due to its safety profile, low cost, and ease of oral administration, it has gained significant attention for therapeutic applications. To meet the demand for cost-effective production of nattokinase via fermentation, this study utilized renewable biomass resources-glycerol and okara-as raw materials for nattokinase production. A metabolic pathway for efficient glycerol utilization in Bacillus subtilis 13932 was engineered using genetic modifications. The co-expression of glpF and glpK significantly enhanced the strain's ability to metabolize glycerol, resulting in improved nattokinase production. Building upon this, fermentation conditions using glycerol as the carbon source and okara as the nitrogen source were optimized. Using a Box-Behnken Design, the optimal medium composition was determined to be: glycerol 23 g/L, okara 96 g/L, MgSO4•7H2O 0.8 g/L, and CaCl2 0.7 g/L, yielding a nattokinase activity of 8709.53 ± 103.45 IU/mL. Finally, fermentation parameters were optimized through a single-factor experiment. The highest nattokinase activity of 10576.28 ± 91.78 IU/mL was achieved under the following conditions: 37 °C, 200 rpm shaking speed, 7% inoculum, and an initial pH of 7.5.