Production of recombinant thermostable alpha and beta amylase enzymes via dual expression suitable for industrial usage

Thermostable α and β amylase enzymes have been used widely in starch industry and other many fields such as feed, food, paper, textile, leather and detergent manufacturing. These enzymes are produced in little amounts for industrial usage by many hyperthermophilic microorganisms. In this study, thermostable α and β amylase genes were transferred to pET-Duet-1 expression and resulting recombinant plasmids were transformed into Escherichia coli BL21 competent cells by electroporation. Genomic DNA of Bacillus stearothermophilus DSM 22 strain for α amylase and, Thermoanaerobacterium (Clostridium) thermosulfurogenes DSM 2229 strain for β amylase were used as gene sources. Enzymatic activities of recombinant BL21/αAmy and BL21/βAmy bacteria were detected on LB agar with ampicilin and starch and also with staining with iodide steam. The both enzymes were more produced by IPTG induction in recombinant BL21/αAmy and BL21/βAmy bacteria and the recombinant α and β amylase proteins were purified by Ni-NTA agarose column. Histidine labelled both proteins were analyzed by SDS-PAGE and western blot methods. Concentration of the purified proteins were calculated as 4,59 µg/ml for α amylase and 3,17 µg/ml for β amylase. For dual expression of both enzymes, recombinant BL21/αAmy were prepared as competent cells and purified pETDuet/β plasmids were transformed into this cells. Transformed bacteria containing two recombinant plasmids were tested by colony PCR and few colonies were determined comprising both recombinant plasmids. Dual expressed enzymes were analyzed by SDS-PAGE and the molecular mass of the enzymes were confirmed as 60 kDa for α amylase and 55kDa for β amylase.