Genetic Engineering and Fermentation Optimization of Bacillus pacificus HS-Cas sol 5.2 for Enhanced Protease Activity

Amino acid fertilizers offer key advantages over conventional ones but are significantly more expensive. A more cost-effective alternative could be combining high-protein dairy industry waste with specific microbes to create a nutrient-rich product. Twelve bacteria were isolated from blood and meat meal. A Bacillus pacificus strain was selected after the comparison of the radius of action of the proteolytic activity (PAR) in the isolated bacteria. Genomic characterization after sequencing showed that the most similar genome to the isolated strain is Bacillus pacificus EB422T. This new strain was named HS-Cas sol 5.2 and a whole genome-derived proteomic analysis 72 proteases were identified. The presence of these enzymes highlights the biotechnological potential of this new strain, particularly for industry applications involving the conversion of complex proteins into fertilizers. Microbial extracts were obtained from cultures grown in various media, pH, temperatures, and for different periods of time, using both solid and liquid fermentation. These extracts were prepared from the culture medium where the microorganism was grown or from the cell pellet lysate, and their protease activity was measured via azocasein hydrolysis. In addition, the strain was transformed with four of its identified protease genes (Vpr, lytH, ysdC, and nprB) and the most active protease from Bacillus subtilis (aprE) to generate five new strains. Our main results indicate that optimizing growth conditions and heterologous gene expression are effective strategies for boosting the microorganism proteolytic capacity.