Comparative biochemical characterization and industrial potential of alkaline proteases from wild-type Bacillus...

In this study, a wild-type alkaline protease-producing Bacillus strain isolated from soil was biochemically and molecularly characterized. The strain was identified as Bacillus subtilis PTK56 based on 16 S rRNA analysis. Random mutagenesis using EMS (Ethyl methanesulfonate) generated multiple variants, and the mutant with the highest protease productivity was selected. Enzyme production conditions for both strains were optimized, and the partially purified enzymes were comparatively characterized. The mutant protease exhibited a 1.34-fold higher activity than the wild type. Zymogram analysis confirmed the functional impact of the mutation through the appearance of an additional activity band. Both enzymes displayed an optimal pH of 9.0 and an optimal temperature of 55 °C. They retained ≥ 97% of their initial activities between 30 and 60 °C and maintained ≥ 95% stability within the pH range of 6.0-11.0. In the presence of metal ions (5-10 mM) and organic solvents (10%), both enzymes preserved more than 90% activity. In non-ionic detergents (1% Triton X-100, Tween-20, and Tween-80), both enzymes exhibited high stability, retaining > 90% activity with Tween-20 and Tween-80, while the wild-type retained ~ 79% and the mutant ~ 92% with Triton X-100. The mutant enzyme showed markedly higher stability, performing 39% better than the wild type in 5% Triton X-100 and 26% better in 5% H_₂O_₂. Both enzymes also maintained ≥ 80% stability across 1-15% NaCl concentrations. HPLC analysis of casein hydrolysis products revealed higher amino acid release by the mutant enzyme. Overall, both proteases demonstrate strong potential for applications in detergent, photographic, and pharmaceutical industries.