Fitsum Tigu (PhD)Asnake Desalegn (PhD)Misgana Anbessa2025-08-172025-08-172025-02https://etd.aau.edu.et/handle/123456789/6875Alpha amylase is a biological catalyst which breakdown large molecules of starch to smaller unit. It has many functions in industries like food, fermentation, detergent, textile, and pharmaceuticals. This study aimed to isolate, characterize, and evaluate alpha-amylase-producing mesophilic bacteria from Bishoftu lakes for potential application in detergent formulation. The samples were isolated using serial dilution and nutrient agar media. Hydrolysis method was employed to screen all isolates for their potential of amylase production by starch plate agar method. Overall, 110 isolates were tested as positive for amylase activity among 169 isolates. Based on amylolytic index (AI), 60 isolates were used for submerged state fermentation and 9 most potent amylase producing isolates were subjected for further analysis. The impact of temperature, pH, incubation time, carbon source, organic and inorganic nitrogen sources, metal ions, inoculum size, and various concentrations of starch, and sodium chloride on the isolates were performed. The maximum alpha-amylase production of all the 9 isolates was observed at 40ºC in 24 h of incubation time, pH 7, glucose, tryptone, beef extract, ammonium chloride, zinc sulphate, potassium phosphate, 1.5% sodium chloride concentration using basal media. The biochemical tests like methyl red, Voges Proskaur, citrate, urease and catalase tests were conducted. A total of 9 isolates were identified. 7 isolates were grouped as B. cereus and the rest as, A. rivipollensis and K. pasteurii based on 16S rRNA, gyrB and elongation factor Tu (tuf) gene sequencing. A. rivipollensis (G2W1) recorded 4.5 ± 0.1 U/mL after applying all optimum growth conditions, which was the highest one. The second was K. pasteurii (G3W1) (4.3 ± 0.4 U/mL) and the third was B. cereus (B1W1) (4.1 ± 0.1 U/mL). The optimum conditions for the crude enzyme activity were 45ºC, pH 7, 15 min of reaction time, Ca2+, and in which 5 ± 0.04 U/mL of enzyme activity was recorded by A. rivipollensis. Ca2+ was the best enhancer among metal ion as cofactors. Partially purified enzyme has a molecular weight of 50 kDa, with specific activity of 10.1 U/mg. The effectiveness of alpha amylase was checked by preparing a detergent and the soap made by this enzyme has successfully cleaned the pieces of cloth that were purposively contaminated by potato waste.en-USAmylaseBacteriaBishoftu LakesEnzyme ActivityOptimizationThesis