Daily accumulation of feather waste due to the development of the poultry industry is a critical issue, considering the environmental and health hazards. Among feather waste disposal methods, biodegradation is a practical, cheap, and environment-friendly method. This study aimed to enhance the keratinolytic activity of FUM-2 isolate, a native keratinolytic bacterium, using mutations and culture improvement. The isolate was identified as Bacillus paralicheniformis based on biochemical, morphological, and molecular analysis. Using the one factor at a time (OFAT) method, the isolate produced more keratinase at 45 °C, pH 11, 2% carbon substrate concentration, 75% aeration rate, and 48 hours incubation. Also, it was able to degrade both α and β keratin. Culture improvement increased enzyme production from 507.6 U/mL to 1706.4 U/mL. In random mutation using ultraviolet radiation, three mutants were isolated, and a 10-minute UV-treated mutant of UVF2-D showed a 36.3% increase in keratinolytic activity compared to its parent. In the next round of mutagenesis by ethidium bromide, seven double mutants were isolated from the UVF2-D mutant, among which the EUF2-D2 double mutant showed a 79% increase in keratinolytic activity. No viable mutants were isolated from the wild strain using ethidium bromide. Our results show that many organisms can potentially produce beneficial products that could be improved using random mutagenesis and optimization methods, which are more useful for strain improvement than complex and costly molecular techniques.

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