Isolation, Purification, and Characterization of Protease from a Local Bacillus Strain Adapted to Extreme Temperatures in Southern Iraq

Bacteria, with their amenability to genetic manipulation and cultivation, represent a favored source of protease enzymes. This study aims to isolate and purify protease from a local Bacillus strain in southern Iraq, known for its resilience to extreme heat. In August 2022, soil samples were collected from Wasit Province, Iraq, and plated on a casein-hydrolyzed medium. Bacillus strains were selected based on microscopic and colony characteristics, with the most active isolate identified as Bacillus subtilis through morphological assessment and 16S rRNA sequencing. Protease production occurred through submerged fermentation, yielding a crude enzyme with enzymatic and specific activities of 241.3 U/mL and 613.3 U/mg, respectively. Partial purification with chilled acetone resulted in an enzyme with enzymatic and specific activities of 935.0 U/mL and 745.0 U/mg, respectively. Further DEAE chromatographic purification showed a single peak of protease activity with increasing sodium chloride ionic strength. The enzyme's specific activity reached 2491.8 U/mg, with a recovery rate of nearly 90%. Subsequent gel column filtration via 1.5 × 60 cm Sephadex G-100 displayed a single peak of protease activity and increased specific activity (4264.10 U/mg) with a 73% recovery rate. Molecular weight determination using Sephadex G-100 column indicated a size of 29.3 kDa for the B. subtilis protease. Regarding thermal stability, the enzyme demonstrated initial stability at 60°C, but prolonged exposure reduced activity. In contrast, exposure to 70°C and 80°C resulted in rapid declines in enzymatic activity. This study highlights the isolation and purification of a robust protease enzyme from a local Bacillus strain, underscoring its potential significance in various industrial applications.