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

The recovery of a recombinant !-amylase expressed in the periplasm of E. coli using packed bed, expanded bed and batch adsorption is compared. Recovery of recombinant protein using a packed bed requires complete clarification using microfiltration, resulting in loss of yield, or high speed centrifugation which reduces efficiency for viscous periplasmic feedstreams at large scale. Expanded beds ease these problems, however, low levels of cell debris and dilution of the feedstream to 5% sucrose are required to prevent particle aggregation and blockage of adaptors. Batch adsorption provides an alternative option by allowing a rapid capture step (~20 min) which removes the target protein from the feedstream thereby allowing it to be purified by further purification stages, however, the mixing of adsorbent during batch adsorption poses problems.