Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

Abstract

AbstractThe release of Penicillin acylase from Escherichia coli cells through mechanical cell disruption using high‐pressure homogenization and sonication was studied. From these cell disruption processes, the enzyme activity was totally released although with low specific activities, 0.1–0.3 IU(mg prot)−1. Intracellular total soluble protein release was quantified and modelled by a first order kinetic model. The effect of the driving force for each mechanical method, namely acoustic power input and homogenization pressure, on the respective kinetic disruption constants was also analysed. The release of Penicillin acylase by cell permeabilization using osmotic shock was also evaluated. The effects of cell concentration, penicillin acylase activity in E coli cells, type of buffer, pH, hypertonic solution composition, temperature and time used for osmotic shock were evaluated. Using cold osmotic shock, highly selective penicillin acylase release was attained with specific enzyme activities of about 4 IU(mg prot)−1 and enzyme activity release yields higher than 90%. The high purity of the penicillin acylase was a consequence of the optimized differential enzyme release method which was validated by SDS gel electrophoresis.© 2002 Society of Chemical Industry