Purification of a periplasmic enzyme by a stirred adsorbent, and by expanded and packed beds

The recovery of a recombinant a-amylase expressed in the periplasm of E. co li using packed bed, expanded bed and batch adsorption is compared. Recover y of recombinant protein using a packed bed requires complete clarification using microfiltration, resulting in loss of yield, or high speed centrifug ation which reduces efficiency for viscous periplasmic feedstreams at large scale. Expanded beds ease these problems, however, low levels of cell debr is and dilution of the feedstream to 5% sucrose are required to prevent par ticle aggregation and blockage of adaptors. Batch adsorption provides an al ternative option by allowing a rapid capture step (similar to 20 min) which removes the target protein from the feedstream thereby allowing it to be p urified by further purification stages, however, the mixing of adsorbent du ring batch adsorption poses problems.