青霉素酰化酶交联酶聚体的制备及热失活动力学

doi:10.7503/cjcu20131009

摘要/Abstract

摘要：

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.

关键词: 交联酶聚体, 青霉素酰化酶, 固定化, 稳定性, 热失活动力学

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% 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.

Key words: Cross-linked enzyme aggregate(CLEA), Penicillin acylase, Immobilization, Enzyme stability, Thermal deactivation kinetics

中图分类号:

TrendMD:

慕洋洋, 甄倩楠, 王梦凡, 齐崴, 苏荣欣, 何志敏. 青霉素酰化酶交联酶聚体的制备及热失活动力学. 高等学校化学学报, 2014, 35(6): 1212.

MU Yangyang, ZHEN Qiannan, WANG Mengfan, QI Wei, SU Rongxin, HE Zhimin. Preparation and Thermal Kinetic Deactivation of Cross-linked Enzyme Aggregates of Penicillin Acylase^†. Chem. J. Chinese Universities, 2014, 35(6): 1212.

图/表 9

Fig.1 Effects of ammonium sulfate(A) and glutaraldehyde(B) concentration on the activity yieldActivity yield(%)=Ai/A0×100%; Ai is the activity of enzyme aggregates or CLEAs; A0 is the activity of free enzyme.

Fig.2 Optimal temperatures(A) and pH(B) of free penicillin acylase(a) and its CLEAs(b) Relative activity(%) was calculated by assuming the activity at optimal temperature or pH as 100%.

Fig.3 Thermal stability of free penicillin acylase(a—c) and its CLEAs(d—f)Residual activity(%) was calculated by assuming the initial activity of enzyme samples as 100%. Temperature/K: a, d: 329.15; b, e: 331.15; c, f: 333.15.

Fig.4 Thermal deactivation kinetic curves of free enzyme(A—C) and its CLEAs(A'—C')(A, A') One step model; (B, B') parallel model; (C, C') serial model. ● 329.15 K; ▲ 331.15 K; ■ 333.15 K.

Table 1 One-step deactivation parameters of free penicillin acylase and its CLEAs

Free enzyme			CLEAs
T/K	k_d/min^-1	R²	k_d/min^-1	R²
329.15	0.0083	0.9985	0.0012	0.6480
331.15	0.0264	0.9904	0.0018	0.7587
333.15	0.0343	0.9966	0.0044	0.6548

Table 2 Parallel deactivation parameters of free penicillin acylase and its CLEAs

Free enzyme					CLEAs
T/K	α	k_d1/min^-1	k_d2/min^-1	R²	α	k_d1/min^-1	k_d2/min^-1	R²
329.15	-28.55	0.0109	0.0110	0.9985	0.0372	0.0012	0.0012	0.5308
331.15	12.45	0.0265	0.0265	0.9808	1.320×10^-5	0.0019	0.0019	0.6897
333.15	-33.91	0.0270	0.0267	0.9976	0.6542	0.0372	0.0016	0.9919

Table 3 Serial deactivation parameters of free penicillin acylase and its CLEAs

Free enzyme						CLEAs
T/K	α	β	k_d1/min^-1	k_d2/min^-1	R²	α	β	k_d1/min^-1	k_d2/min^-1	R²
329.15	0.5017	0.5017	0.0083	0.0083	0.9972	0.6531	0.3446	4.024×10^-4	0.0132	0.9935
331.15	0.1048	0.8952	1.1743	0.0240	0.9653	0.7156	0.2837	9.871×10^-4	0.0205	0.9887
333.15						0.6542	0.3461	0.0016	0.0372	0.9892

Fig.5 Activation energy of thermal inactivation reaction

Fig.6 Reusability of CLEAs

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