Abstract: Proteases, as one of the most important groups of industrial enzymes, account for about 60% of the global enzyme market. Their ability to hydrolyze peptide bonds has enabled wide applications in detergent, pharmaceutical, food, leather, an‎d textile industries. However, the limited stability of many natural proteases under harsh processing conditions such as high temperatures, extreme pH values, an‎d the presence of chemical agents remains a major challenge for their industrial utilization. This study aimed to improve the functional properties an‎d stability of a bacterial protease through chemical mutagenesis of the producing strain. In this research, Bacillus halotolerans was used as the parental strain an‎d cultivated in liquid LB medium. To induce mutations, cells were treated with 100 µg/mL ethidium bromide (EtBr) for 120 minutes. After mutagenic treatment, the resulting colonies were cultured on skim milk agar, an‎d preliminary screening was performed based on the ratio of hydrolysis halo diameter to colony diameter (H/C index). Forty-six colonies showing the highest proteolytic activity were selec‎ted an‎d grouped into three categories: A (largest halo), B (moderate), an‎d T (smallest). Quantitative enzyme assays were performed using the stan‎dard casein hydrolysis method, an‎d absorbance was measured at 660 nm. Seven mutant colonies (A1, A4, A13, T1, A14, A20, T26, an‎d T19) exhibited protease activity 2–3 times higher than that of the parent strain. Enzyme stability tests under harsh conditions (95 °C an‎d pH 8.5) revealed that the protease from colony A4, with an activity of 83,422 U/g, showed significantly greater thermal an‎d alkaline stability than the parental enzyme (33,733 U/g). To identify molecular alterations, genomic DNA of selec‎ted colonies was extracted, an‎d the protease-encoding gene (aprE) was amplified by PCR an‎d sequenced using the Sanger method. Sequence comparison confirmed point mutations leading to amino acid substitutions. In colony A4, cysteine was replaced by serine at position 168 (C168S) an‎d arginine by lysine at position 324 (R324K). Additional substitutions, such as S142C an‎d H349S, were identified in colonies T19 an‎d A20. These amino acid changes, particularly those involving cysteine, appear to enhance structural stability via disulfide bond formation, thereby improving enzyme thermostability. Overall, the findings indicate that chemical mutagenesis using ethidium bromide is an effective an‎d economical approach to generate genetic diversity an‎d enhance the performance of bacterial proteases. The results provide valuable insights for developing robust industrial strains an‎d producing stable, high-performance proteases for harsh processing environments.

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بدرالدين ابراهيم سيدطباطبايي , محمد صدقي