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

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 degrees C) than free penicillin acylase (47 degrees C) 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.