Forty-eight out of sixty-eight bacterial strains isolated from traditional fermented soybean samples (Tuong Ban) showed moderate to high fibrinolytic enzyme activity. Bacillus sp. HY6 had the highest fibrinolytic enzyme activity of 80.4 ± 1.2 FU/mL. The strain was selected for improving fibrinolytic activity via UV irradiation and chemically induced mutagenesis using Ethidium Bromide (EtBr) and Ethyl Methane Sulfonate (EMS). After UV-irradiation, the HY6-derived mutant, designated as strain U5_90.5, exhibited the highest fibrinolytic enzyme activity of 127 ± 5 FU/mL, about 1.6 times higher than that of the wild type HY6 strain. Strain U5_90.5 after UV mutation was subsequently subject to chemically induced mutation by EtBr and EMS. This double mutation resulted in strain ES4 which has the highest fibrinolytic enzyme activity of 404 ± 4 FU/mL. The enzyme production capability of the strain was stable after 10 generations which renders the strain a potential fibrinolytic enzyme producing strain for commercial applications. This result indicated that the Bacillus sp. ES4 could be a potential commercialized fibrinolytic enzyme producing bacterium.

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.

No abstract