Enhanced Starch Hydrolysis by Induced Mutagenesis: Achieving Alpha-Amylase Over-Production in a Native Bacillus Strain

Low enzymatic titer remains a major bottleneck in deploying wild-type bacterial strains for commercial alpha-amylase production, restricting their economic viability across brewing, textile desizing, and starch-processing sectors. To overcome this production constraint, the present investigation isolated indigenous amylolytic bacteria from soil environments in Chh. Sambhajiangar, India, and evaluated comparative physical versus chemical mutagenesis protocols for targeted catalytic enhancement. Initial screening across 12 bacterial isolates on starch agar media identified three potential producers, among which a Gram-positive, rod-shaped bacterium designated as Bacillus sp. isolate 5a displayed the highest baseline starch hydrolysis halo of 20 mm. Mutational induction revealed that exposure duration directly governed amylolytic amplification for both mutagens across 5 to 20 min intervals. Physical mutagenesis via short-wave ultraviolet irradiation (254 nm) achieved incremental increases in the clearance zone, peaking at 27 mm after 20 min of exposure (a 35% improvement over the unmutated parent). In contrast, chemical mutagenesis utilizing 0.1% ethidium bromide triggered an exceptional surge in enzyme secretion, expanding the hydrolysis diameter from 20 mm to 32 mm at 20 min exposure—delivering an overall 60% boost in clearance diameter. These findings underscore the superior mutagenic efficiency of ethidium bromide intercalation over ultraviolet-induced dimerization, providing an accessible, robust, and cost-effective strain improvement route for developing high-yielding industrial Bacillus strains.