Enhanced Production and Statistical Evaluation of Alkaline Protease from EMS-Mutagenized Bacillus sp. Isolated from Poultry Farm Water

Abstract

This study evaluated the isolation, mutagenesis, molecular characterization, and biotechnological potential of alkaline protease-producing bacteria from fish pond (FW) and poultry farm (PW) water samples. Serial dilutions from 10⁻² and 10⁻³ were cultured on skim milk agar at 37 °C and pH 9 for 72 h. Among the isolates, PW10⁻²A2 exhibited the highest proteolytic activity with a zone of hydrolysis (ZOH) of 13 mm, compared to PW10⁻²A1 (4 mm) and PW10⁻²B2 (2 mm). Morphological and biochemical characterization revealed a Gram-positive, motile, endospore-forming rod consistent with Bacillus spp. Ethyl methane sulfonate (EMS)-induced mutagenesis (1 – 100 mg/mL) generated 117 mutants, with optimal enhancement observed at 20 mg/mL, producing a ZOH of 9.70 mm compared to 2.70 mm in the parental strain. High lethality rates were observed at 80 mg/mL (75%) and 100 mg/mL (77.78 %). The mutant strain (PW10-2A2) demonstrated improved biomass (maximum OD₆₀₀ = 2.010 at 120 h) compared to the wild strain (1.7413 at 96 h), and enhanced protease activity (0.142 U/mL), with 5.6807 µmol tyrosine released at 96 h. Spectrophotometric analysis showed peak enzyme activity at 96 h (OD₆₆₀ = 3.00), while mean protease activities were 2.377 (wild) and 2.1783 (mutant). These findings demonstrate that EMS mutagenesis significantly enhances protease yield and biomass production. The improved strain shows strong potential for possible industrial biotechnological applications.