Strain Improvement of Bacillus Subtilis for Enhanced α-Amylase Production Using UV and Ethidium Bromide Mutagenesis
DOI:
https://doi.org/10.68050/JAMS.2026.359Keywords:
α-Amylase; Bacillus subtilis; strain improvement; UV mutagenesis; ethidium bromide; starch hydrolysis; DNS assay; submerged fermentation; partial purification; industrial biotechnology.Abstract
Microbial α-amylase is an industrially important starch-hydrolyzing enzyme with applications spanning food processing, starch conversion, textiles, detergents, paper manufacture, fermentation and other biotechnological processes. The present study evaluated strain improvement of an amylolytic bacterial isolate, designated PSSR201801 and identified in the supplied study records as Bacillus subtilis, using physical and chemical mutagenesis. A rhizospheric soil sample collected near Meerut College, Meerut was processed by serial dilution and spread plating. Amylolytic colonies were screened on starch-containing minimal agar and visualized by iodine flooding, after which the selected isolate was purified by quadrant streaking. UV irradiation and ethidium bromide (EtBr) treatment were subsequently used to generate mutant populations, and colonies were re-screened for starch hydrolysis. The experimental record indicates that UV exposure progressively reduced recoverable colony numbers from approximately 220 at 5 min to 45 at 20 min, while EtBr treatment yielded 76 colonies at 6 µL and 32 colonies at 8 µL, with lower treatments reported as lawn growth. The 20-min UV-derived culture and a positive EtBr-derived culture were selected for subsequent enzyme production studies. Growth-curve measurements showed that PSSR201801 increased from an OD620 of 0 at inoculation to 0.33 at 48 h before declining to 0.25 at 72 h. Amylase production was assessed using the dinitrosalicylic acid (DNS) reducing-sugar assay, followed by partial purification through ammonium sulfate precipitation and dialysis. The supplied records report enhanced amylase activity following mutagenic treatment, including a stated doubling of enzyme activity after 20 min of UV exposure; however, replicate-level numerical enzyme-activity values were not available in the source files and therefore are not fabricated here. Overall, the study supports random mutagenesis as a practical route for generating candidate high-producing Bacillus variants and provides a foundation for subsequent molecular characterization, statistical optimization and scale-up.
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