Produção e caracterização da α-amilase produzida por Priestia koreensis em Escherichia coli

Abstract

α-Amylases are enzymes widely used in the food, textile, pharmaceutical, and biofuel industries due to their ability to hydrolyze starch α-1,4-glycosidic bonds. To meet industrial demands, heterologous expression has emerged as an alternative for increasing enzyme production, and recombinant enzymes are gaining prominence with the aid of recombinant DNA technology. This study aimed to produce and characterize an α-amylase from Priestia koreensis cloned into Escherichia coli JM110, using the pDMU 02 expression vector under the control of the *lac* operon. α-Amylase production was confirmed by the formation of starch degradation halos, and enzyme activity assays were performed using Fuwa’s iodometric method. An IPTG concentration of 0.3 mM resulted in the highest enzyme production after 10 hours of cultivation. Characterization revealed an optimal temperature of 50°C, an optimal pH of 6, and good thermostability, with the enzyme retaining high amylolytic activity for up to 60 minutes between 50°C and 80°C, though activity progressively declined after prolonged incubation at 100°C. Ion-exchange chromatography using a HiTrap Q FF column failed to purify the enzyme, indicating a weak interaction between the α-amylase and the resin. Kinetic analysis revealed an apparent Km of 1 mg/mL and an apparent Vmax of 434.78 U/mL, indicating high substrate affinity. The α-amylase exhibits properties suitable for biotechnological and industrial applications.