Production, Purification and Partial Characterization of Organo-Solvent Tolerant Protease from Newly Isolated Bacillus sp. BBXS-2

Proteolytic enzymes are applied in various industries such as detergent, leather, food, textile, cosmetics, pharmaceutical, and synthetic biotechnology. To meet commercial needs, microbial strains of high value in terms of cost-effective production have been focused. In this study, thermophilic strain, Bacillus sp. BBXS-2 was activated on simple growth medium and then transferred to Luria Bertani (LB) medium. Maximum protease concentration of 6723 U/mL under optimized fermentation conditions (molasses, corn steep liquor, pH 9.0, and 45°C). The results showed, there was no difference observed in bacterial growth and protease titer while replacing yeast extract with corn steep liquor so, in this way, about 90% cost of nitrogen source can be saved. The extracellular enzyme was purified to homogeneity from cell-free supernatant by ammonium sulphate precipitation followed by dialysis and ion exchange chromatography, recovery yield reduced from 100 to 22% and purification fold increased from 1 to 9.82. The enzyme was active in broad pH and temperature range 8-12 pH and 30-60°C, with maximum activity at pH 10.0 and 60°C, respectively. Protease retained more than 90% activity after incubation at 40°C for 2 weeks in the presence of (40% v/v) organic solvents including ethanol, methanol, and isopropanol. Overall, research suggests that this strain is a more promising candidate and possess practical ability to use in industries.