A novel cold-active alkaline protease from Bacillus mobilis TK31: random mutagenesis, characterization, and its...

This study introduces a novel cold-active alkaline protease-producing Bacillus mobilis TK31 strain, first isolated from Türkiye and identified through biochemical and molecular analyses. Combined mutagenesis using UV irradiation and ethyl methanesulfonate (EMS) enhanced protease production of the strain under optimized conditions. Proteases from wild-type and mutant strains were partially purified by acetone precipitation and subjected to preliminary comparative characterization. Both enzymes exhibited optimal activity at a pH of 9.0 and a temperature of 15 °C. The mutant 18C-179 enzyme showed a 3.75-fold activity increase over the wild-type post-optimization. The enzymes' pH and temperature stability, chemical resistance, and substrate specificity were evaluated, with the mutant displaying superior stability at extreme pH values, higher temperatures, and enhanced resistance to specific chemicals. Casein hydrolysis products were identified chromatographically. The wild-type and mutant enzymes demonstrated promising potential in detergent, photography, and pharmaceutical applications. This work presents a cold-active alkaline protease and highlights its potential for further industrial biotechnology studies through strain improvement.