Optimisation of penicillin acylase extraction by AOT/isooctane reversed micellar systems

Escherichia coli penicillin acylase was successfully extracted from a crude extract obtained by osmotic shock into the reversed micelle phase of the anionic surfactant AOT in isooctane and back-extracted, almost pure, to a final aqueous phase by simple salt concentration change (1 mol 1(-1) KCI). This work describes the effects of pH, ionic strength and surfactant concentration on the enzyme transfer process from the aqueous to the organic phase by the direct contact between the two phases. The best results were obtained after 3 min of contact between the two phases (with an agitation of 100 rpm), 0.10 mol 1(-1) NaCl, pH 5.5 and 0.05 mol 1(-1) AOT. Under the conditions described the equilibrium distribution, corresponding to ca. 60% of enzyme activity transfer and enzyme inactivation lower than 10% were obtained. NaCl concentration shows to be a critical factor since its increase to 0.125 mol 1(-1) reduces the extracted activity to only 10% of the loaded activity. The optimum pH for extraction ties within a narrow interval (5.5+/-0.5). Since this value is lower than the enzyme pI, the transfer process is dominated by enzyme-surfactant electrostatic interactions. The AOT concentration of 0.05 mol I-I was established as a compromise between extraction yield and enzyme precipitation at the interface. (C) 2002 Elsevier Science B.V All rights reserved.