Kinetics, Zymographic Analysis, and Industrial Evaluation of Alkaline Protease from Bacillus tropicus Isolated from Environmental Water Samples

Abstract

This study investigated the isolation, molecular identification, and biotechnological potential of alkaline protease-producing bacteria from fish pond (FW) and poultry farm (PW) water samples. Serial dilutions 10⁻² and 10⁻³ were cultured on skim milk agar at 37 °C and pH 9 for 72 h to screen for proteolytic activity. Among the isolates, PW10⁻²A2 exhibited the highest protease activity with a zone of hydrolysis (ZOH) of 13 mm, compared to PW10⁻²A1 (4 mm) and PW10⁻²B2 (2 mm), despite the latter having >300 colonies. Molecular identification via 16S rRNA sequencing revealed 99.84% homology with Bacillus tropicus (GenBank accession MW971701.1). Zymogram analysis indicated two protease isoforms with molecular weights ranging between 36 and 116 kDa. Batch fermentation studies showed peak protease activity (142.02 U/mL) at 96 h, with a productivity of 1.48 U/h and specific growth rate of 0.0303 h⁻¹. Functional assays demonstrated complete blood stain removal when the enzyme was combined with detergent, outperforming detergent alone. Additionally, the enzyme exhibited efficient dehairing capability, with treated cow skin showing complete hair removal within 24 h compared to control. These findings highlight Bacillus tropicus PW10⁻²A2 as a promising candidate for industrial applications in detergent formulation and leather processing.