Production, Purification and Characterization of Thermostable Amylase from Thermophilic Bacteria Isolated from Water and Sediments of Wanzaye Hot Spring, Ethiopia

Thermostable α-amylases are among the most important industrial biocatalysts because of their ability to hydrolyze starch under elevated temperatures. Hot springs are promising reservoirs of thermophilic microorganisms producing robust enzymes; however, Ethiopian geothermal ecosystems remain unexplored. This study investigated thermophilic bacteria isolated from the water and sediments of Wanzaye Hot Spring, Ethiopia, for the production, purification, and biochemical characterization of thermostable α-amylases. In this study, twenty bacterial isolates were recovered, of which four highly active thermophilic isolates (WA7, WA10, WA11, and WA12) belonging to the genus Bacillus were selected for detailed investigation. During the study, maximum enzyme production was achieved after 72 h of solid-state fermentation using wheat bran at 55°C and pH 7. Thin-layer chromatography identified maltose as the principal hydrolysis product, confirming α-amylase activity, while SDS-PAGE revealed an approximate molecular mass of 50 KDa. The enzymes exhibited maximum catalytic activity at 65°C (WA7, WA10, and WA12) and 70°C (WA11), retained more than 76.8% of their initial activity after incubation at 40–80°C for 60 min, and remained active over a broad pH range (6.0–8.5). Furthermore, the purified enzymes hydrolyzed raw enset ( Ensete ventricosum ) starch with conversion efficiencies exceeding 70%, demonstrating their potential for starch processing and other high-temperature industrial bioprocesses. These findings identify Wanzaye Hot Spring as a valuable source of industrially relevant thermostable α-amylase-producing bacteria.