Improvement of Fibrinolytic Enzyme Production from Bacillus sp. ES4 by Response Surface Methodology and Exponential Fed-Batch Fermentation

Abstract

To improve the fibrinolytic enzyme production of the mutant strain Bacillus sp. ES4, the response surface methodology was used to optimize the medium composition. Thirty-four flask scale experiments were carried out, and the results were fitted to quadratic models using Design-Expert software. The ANOVA analysis revealed that medium components differently affect cell density and fibrinolytic enzyme production. At optimal condition for fibrinolytic enzyme production, enzymatic activity attained 686 ± 43 FU/mL, which is increased by 1.7-fold compared to the initial medium (404 ± 4 FU/mL). One step fed-batch fermentation, in which the nutrient for optimal enzymatic activity was exponentially feed to the culture, improved fibrinolytic enzyme activity by 5.91-fold (4,057.14±57.14 FU/mL) and cell density by11.9-fold (101.2±0.005). In two steps fed-batch fermentation, the nutrient for optimal cell density was first used, then followed by feeding for optimal enzyme activity. The highest cell density (OD600nm) and fibrinolytic enzyme activity reached 142.3 ± 0.0125 and 5,300 ± 100 FU/mL, respectively after 13 h fermentation. Ultimately, the medium optimization and fed-batch strategy collectively improved fibrinolytic enzyme activity by 13.12-fold.