Simultaneous Production of Alpha and Beta Amylase Enzymes Using Separate Gene Bearing Recombinant Vectors in the Same Escherichia Coli Cells

Abstract

The present study describes the simultaneous expression of thermostable industrial alpha (alpha) and beta (beta) amylase enzymes that have been used widely in starch industry. Genomic DNA of Bacillus stearothermophilus DSM 22 strain for alpha amylase and, Thermoanaerobacterium (Clostridium) thermosulfurogenes DSM 2229 strain for beta amylase were used as gene sources. Both genes were ligated into pETDuet-1 expression vector separately and resulting recombinant vectors were transformed into Escherichia coli BL21 competent cells by electroporation. The cells were first transformed by pETDuet-1/ alpha Amy recombinant plasmid, then the competent cells carrying this plasmid were prepared for the transformation of pETDuet-1/beta Amy plasmid. Enzymatic activities of bacterial colonies were detected on LB agar staining with iodide. Both enzymes were more produced by IPTG induction in BL21 cells and were purified using Ni-NTA agarose column. SDS-PAGE and western blot analyses showed that the molecular weight of purified alpha and beta amylase to be approximately 60 kDa and 55kDa, respectively. The concentration of the purified alpha and beta amylase were calculated as 4.59 mu g/mL and 3.17 mu g/mL with IPTG as an inducer in LB medium. The present study proposes a novel and efficient method for the production of thermostable alpha and beta amylases at the same E coli cells containing separate engineered plasmid vectors.