Preparation and Thermal Kinetic Deactivation of Cross-linked Enzyme Aggregates of Penicillin Acylase†

Abstract

Cross-linked enzyme aggregates( CLEAs) is an efficient approach to obtain immobilized enzymes without the use of any pre-existing carriers. The preparation of CLEAs usually consisted of two simple steps: precipitation and cross-linking. In this work,CLEAs of penicillin acylase was preparaed using 50% ammonium sulphate solution as precipitant and 0. 35% glutaraldehyde as cross-linking agent. The resulting CLEAs obtained an activity yield of 30. 1% and higher optimal temperature( 57 ℃) than free penicillin acylase( 47 ℃) as well as an obvious shift of optimal pH from 8. 3 to 10. 0. Moreover,compared with free enzyme, the thermal stability of CLEAs was largely enhanced which facilitated the application of penicillin acylase in high-temperature reaction systems. Based on the thermal deactivation kinetics study,it was found that the deactivation model of penicillin acylase changed from one-step model to serial model through immobilizing as CLEAs. The higher deactivation energy of CLEAs( 549. 2 kJ / mol) than free enzyme( 248. 8 kJ / mol) also explained the excellent stability of CLEAs under high temperature environment. In addition,CLEAs exhibited favorable reusability after using 7 times.