青霉素酰化酶交联酶聚体的制备及热失活动力学

以0.53 g/mL硫酸铵为沉淀剂, 0.35%(体积分数)戊二醛为交联剂制得青霉素酰化酶交联酶聚体(CLEAs), 酶活收率30.1%, 其最适温度(57 ℃)比游离酶提高10 ℃, 最适pH(10.0)向碱性偏移1.7个单位. 对比游离酶及其CLEAs的热稳定性和热失活动力学模型发现, 游离青霉素酰化酶制成CLEAs后, 其热失活动力学模型由一步失活转变为连串失活, 失活反应活化能由248.8 kJ/mol增加至549.2 kJ/mol, 对CLEAs热稳定性大幅提高的原因进行了解释. CLEAs重复利用7次后, 酶活保留56%以上, 具有良好的重复利用性.

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% ammo-nium 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.