Identification of a Novel Extracellular Alkaline Protease-Producing Bacillus cereus G۲ Isolated From Petroleum Sludge and Optimization of Enzyme Production

Background: Alkaline proteases are of significant utility in the field of biotechnology, with extensive applications in spanning numerous sectors, including those in the medical and health, pharmaceuticals, food, detergents, and leather industries. Methods: The bacterial strain G۲, isolated from petroleum sludge at the Homs Refinery in Syria, was evaluated for its ability to produce alkaline protease using a skim milk agar medium. Protease production was confirmed through a protease assay. The molecular identification of the selected isolate was conducted through sequencing of the ۱۶S ribosomal deoxyribonucleic acid (rDNA). Culture conditions, including variables such as inoculum size, incubation duration, temperature, pH, and various carbon and nitrogen sources, were optimized to achieve protease production. Results: Strain G۲ exhibited alkaline proteolytic activity, thereby establishing its status as an effective protease producer. A ۱۶S rDNA analysis of the intended strain revealed that the organism was identified as Bacillus cereus. The highest level of enzyme production was observed when the isolate, at an inoculum size of ۳%, was cultured in the production medium for ۴۸ hours at a pH rate of ۱۰ and ۴۵° C, using ۱% glucose and ۱% yeast extract as carbon and nitrogen sources, respectively. The alkaline protease activity was ultimately found to be ۴.۴۴-fold higher (۵۳۳.۳ U/mL) compared to the initial optimization of enzyme production (۱۲۰ U/mL). Conclusion: This constitutes the inaugural account of the alkaliphilic B. cereus G۲ strain, which was isolated from sludge petroleum and is capable of secreting alkaline protease. The findings regarding the optimal cultural conditions for the production of thermophilic alkaline protease provide a crucial foundation for future research into the potential biotechnological applications of this strain in alkaline protease production.