An integrated downstream processing strategy for the recovery and partial purification of penicillin acylase from crude media

An integrated process strategy for the recovery of penicillin acylase was developed, based on precipitation of non‐enzymatic proteins directly from Escherichia coli homogenates or crude extracts using Rolquat (quaternary ammonium salt) and adsorption of the enzyme on Amp‐Seph (3.8 µmole ampicillin cm^?3) under pseudo‐affinity conditions. The effect of pH, concentrations of ammonium sulfate and Rolquat, and also concentrations of protein and cell debris on the precipitation of non‐enzymatic proteins from homogenates and crude extracts of penicillin acylase were analysed. The method of addition of Rolquat to homogenates and crude extracts significantly influenced the size of the precipitated particles. Improved results on the specific activity of penicillin acylase were obtained for 22% and 1% (w/v) of ammonium sulfate and Rolquat, respectively, added sequentially to enzyme solutions and at room temperature. Under these experimental conditions, the specific activity of penicillin acylase in homogenates and crude extracts was enhanced 2.5–3.0‐fold. Finally, the integrated process strategy was implemented first by precipitation of non‐enzymatic proteins and recovery of penicillin acylase directly from the enzyme solution treated with Rolquat using an adsorption/filtration system with an overall yield of 86%. This system allows simultaneously the filtration of cell debris and fine precipitated particles, in situ recovery of penicillin acylase by its adsorption on Amp‐Seph, and selective desorption of the enzyme with a specific activity of 11 IU (mg prot)^?1 and a desorption yield of 95%. © 2002 Society of Chemical Industry