Alpha-amylase is one of the most important industrial enzymes, widely used in sectors such as food, detergents, textiles, an‎d animal feed. Given the industryʹs need for enzymes with higher efficiency an‎d stability, this research was conducted to improve the activity an‎d thermal stability of alpha-amylase through chemical mutagenesis using ethidium bromide on the bacterial strain Bacillus spizizenii S1. For this purpose, bacteria were treated with 1 mg/mL of ethidium bromide, an‎d mutant colonies were screened based on superior enzyme production an‎d the formation of clear halos on starch agar medium. Enzyme activity was assessed using the DNS assay method an‎d measuring optical density at 540 nm, while its thermal stability was eva‎luated at 90°C over different time intervals. Additionally, to identify molecular changes, the amyE gene in the superior mutants was amplified an‎d sequenced. The results indicated that mutagenesis led to a significant increase in enzymatic activity in several colonies, with strains R1, R3, an‎d R6 showing the highest levels of activity. Strain R1 was identified as the top performer, exhibiting a 120% increase in activity compared to the wild-type. Furthermore, the thermal stability test revealed a remarkable improvement in the mutant strains; strain R3 demonstrated the highest thermal resistance with a 251% increase in stability at 90°C. Sequence analysis of the amyE gene confirmed several nucleotide substitutions an‎d amino acid changes in regions affecting the enzymeʹs structure an‎d function. In conclusion, this study demonstrates that chemical mutagenesis with ethidium bromide is an efficient method for obtaining bacterial strains capable of producing alpha-amylase with higher activity an‎d stability for various industrial applications.

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مجيد طالبي , صادق زارعي