Utilizing Box-Behnken Design to Improve the Production of α-amylase via Bacillus Velezensis

Objective: To improve the production yield of α-amylase from Bacillus velezensis sp. by utilizing the Box-Behnken Design (BBD) methodology. Methods: The research process involved a multi-stage approach with different media types selected for specific purposes based on their nutrient composition and the needs of Bacillus velezensis sp. at each stage. Initially, the seed media for Bacillus velezensis sp. was prepared using Luria Bertani (LB) broth, providing a broad range of nutrients for preliminary growth assessment and enzyme activity baseline. The LB broth, rich in proteins, carbohydrates, vitamins, and minerals, supported rapid bacterial growth and provided initial insights into α-amylase production. The growth medium was formulated using starch, NH4NO3, K2HPO4, MgSO4·7H2O, FeCl3, and CaCl2, with chosen components aimed at optimizing enzyme production. Starch served as a primary carbon source, challenging the bacteria to produce α-amylase to break down complex carbohydrates. NH4NO3 supplied essential nitrogen for protein synthesis, while minerals and vitamins (K2HPO4, MgSO4·7H2O, FeCl3, CaCl2) supported optimal bacterial growth and enzyme activity. In the submerged fermentation stage, 2% of the seed media was inoculated into a basal medium containing starch to simulate controlled fermentation conditions. This approach minimized variability and focused on optimizing enzyme production. The initial α-amylase activity recorded was 2.78 U/mL, which indicated that process parameters were not yet optimized. Additional enhancement of enzyme activity was accomplished through the Box-Behnken Design (BBD). This method involved varying four significant parameters at two levels (24) to optimize the yield of α-amylase. Results: A peak of α-amylase concentration of 1092.92 U/mL. under optimized conditions, demonstrating the effectiveness of the staged media approach. The key process parameters influencing enzyme production include pH, temperature, moong husk (P < 0.0001). An optimal α-amylase activity of 1092.92 U/mL is achieved. Conclusion: The research demonstrates that a multi-stage approach, combined with Box-Behnken Design (BBD), can significantly enhance α-amylase production from Bacillus velezensis sp. By utilizing different media types and optimizing key process parameters, the study achieved a notable increase in enzyme yield. The systematic use of LB broth for initial growth, followed by tailored seed media and controlled submerged fermentation, set a solid foundation for optimization. The application of RSM, leveraging the Box-Behnken Design (BBD) model, effectively identified and optimized critical factors, leading to a substantial improvement in α-amylase activity. This optimized approach not only highlights the potential of RSM in enzyme production but also provides a framework for scaling up the process for industrial applications.