Extracellular Release of Recombinant (alpha)-Amylase from Escherichia coli Using Pulsed Electric Field

It is difficult for Escherichia coli to secrete products such as recombinant enzymes, because the Gram-negative bacterium has a double membrane structure and so some of the products are accumulated in a periplasmic space. In this study, we demonstrated that recombinant (alpha)-amylase can be released from recombinant E. coli HB101/pHI301A during cultivation by applying a pulsed electric field (PEF). When a PEF (12 kV, 2 Hz) was applied for 30 min with an interval of 30 min from the point of OD660 = 0.7, the amount of released (alpha)-amylase was about 30% of the total amount of (alpha)-amylase produced in the cells. As a result of SDS-PAGE and activity staining analyses, it was confirmed that the released proteins were not all of the intracellular proteins, and the (alpha)-amylase, which was identical with intracellular (alpha)-amylase, was released by applied PEF cultivation. PEF treatment could be useful for easy release of periplasmic protein with selectivity.