Al-MOF基多级孔Al2O3固定化酶反应器的构筑及构效关系研究

doi:10.6023/A21080385

摘要/Abstract

针对生物酶在固相载体负载后存在的催化活性与稳定性之间“此消彼长”的问题, 本工作采用“自牺牲模板”策略以铝基金属有机骨架材料(Al-MOF)为前驱体设计制备多级孔Al₂O₃ (MHAl₂O₃)材料, 再以“聚多巴胺(PDA)”仿生膜对材料表面进行功能化修饰后用以固载辣根过氧化物酶(HRP). 通过调节前驱体的煅烧温度来实现载体孔径大小的调控, 探讨了载体的孔道限域效应对固定化酶反应器催化活性的影响, 所得固定化酶反应器的热稳定性和重复使用性显著提高. 为了解析固定化酶反应器的构效关系, 采用酶动力学和热动力学参数研究了固定化酶反应器催化过程中酶与底物的相互作用, 结果表明固载后酶分子对底物的亲和性和专一性得到提升. 将固定化酶反应器用于模拟废水中苯胺黑药的催化降解时, 表现出非常高效的催化效率.

关键词: 金属有机骨架, 辣根过氧化物酶, 固定化酶反应器, 酶催化, 构效关系

Although enzyme catalysis has many advantages such as green, high efficiency and so on, the industrial application of enzyme is often hampered by a lack of long-term operational stability and difficult to re-use. These drawbacks can generally be overcome by immobilization of the enzyme on a solid carrier. However, the immobilized enzyme reactor often can not remain all the initial catalytic activity due to the enzyme shedding during reuse, change of conformation and the difficulty for substrates to access the active site of enzyme in the carrier, etc. The contradiction between catalytic activity and stability of biological enzyme molecules supported on solid phase carriers has become a bottleneck in the design and preparation of immobilized enzyme reactors. Therefore, it is a challenging task to construct an immobilized enzyme reactor with both high catalytic activity and high stability. In this work, aluminum-based metal-organic framework (Al-MOF)-derived hierarchical porous Al₂O₃ (MHAl₂O₃) was prepared by the self-sacrificial template strategy and functionally modified with “polydopamine (PDA)” bionic membrane for entrapping horseradate peroxidase (HRP). The pore size was regulated by changing the calcination temperature of precursor. The influence of channel confinement effect of the carrier on the catalytic activity of the enzyme reactor was discussed, and the thermal stability and reusability of the HRP@PDA@MHAl₂O₃ was significantly improved. After 1 h at 70 ℃, 98.5% of the catalytic activity of HRP@PDA@MHAl₂O₃ could be maintained. After 10 times of reuse, 90.9% of the catalytic activity was still maintained. In order to analyze the structure-activity relationship of the enzyme reactor, the interaction between enzyme and substrate in the reaction catalyzed by HRP@PDA@MHAl₂O₃ was studied by means of enzyme kinetics and thermodynamic parameters, which showed that the affinity and specificity of the enzyme to the substrate were improved. When the enzyme reactor was applied to the catalytic degradation of aniline aerofloat in simulated wastewater, the degradation rate of 40 mg•L^-1 substrate in 30 min reached 82.7% under the best reaction conditions.

Key words: metal-organic framework (MOF), horseradish peroxidase, immobilized enzyme reactor, enzyme catalysis, structure-activity relationship

引用此文

高霞, 潘会宾, 贺曾贤, 杨柯, 乔成芳, 刘永亮, 周春生. Al-MOF基多级孔Al₂O₃固定化酶反应器的构筑及构效关系研究[J]. 化学学报, 2021, 79(12): 1502-1510.

Xia Gao, Huibin Pan, Zengxian He, Ke Yang, Chengfang Qiao, Yongliang Liu, Chunsheng Zhou. Construction and Structure-Activity Relationship of Immobilized Enzyme Reactor Based on Al-MOF-Derived Al₂O₃ with Hierarchical Structure[J]. Acta Chimica Sinica, 2021, 79(12): 1502-1510.

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图/表 10

图1. HRP@PDA@MHAl2O3的制备示意图

Figure 1. Schematic diagram of preparation of HRP@PDA@MHAl2O3

图2. (a) Al-MOF和MHAl2O3的XRD图谱, (b) Al2O3和MHAl2O3的XRD图谱

Figure 2. (a) XRD patterns of Al-MOF and MHAl2O3, (b) XRD patterns of Al2O3 and MHAl2O3

图3. Al-MOF (a), MHAl2O3-400 (b), MHAl2O3-600 (c)和 MHAl2O3-800 (d)的场发射透射电镜图

Figure 3. FETEM images of Al-MOF (a), MHAl2O3-400 (b), MHAl2O3-600 (c) and MHAl2O3-800 (d)

图4. PDA@MHAl2O3 (a)和FITC-HRP@PDA@MHAl2O3 (b)的激光共聚焦显微镜图

Figure 4. Laser confocal microscopy images of the PDA@MHAl2O3 (a) and FITC-HRP@PDA@MHAl2O3 (b)

图5. 游离HRP和固定化酶反应器的热稳定性曲线

Figure 5. Thermal stability curves of free HRP and immobilized enzyme reactors

图6. 固定化酶反应器的重复使用次数

Figure 6. Operational reusability of immobilized enzyme reactors

Enzyme	v_max/(mmol•L^-1•s^-1)	K_m/(mmol•L^-1)	k_cat/s^-1	(k_cat/K_m)/(L•mmol^-1•s^-1)
Free HRP	0.5355	0.4093	21.4200	52.3333
HRP@MHAl₂O₃	1.1254	0.1125	45.0160	400.1422
HRP@PDA@MHAl₂O₃	1.3456	0.0807	53.8240	666.9641

表1. 游离HRP和固定化酶反应器的酶动力学参数

Table 1. Enzymatic kinetic parameters of free HRP and immobilized enzyme reactors

Enzyme	K_a×10^-3/(L•mol^-1)	ΔH/(kJ•mol^-1)	ΔS/(J•mol^-1•K^-1)	n	TΔS/(kJ•mol^-1)	ΔG/(kJ•mol^-1)
Free HRP	0.75±0.01	1088.0±0.01	3703.0±0.2	1.23±0.1	1104.0±0.1	–16.41±0.04
HRP@MHAl₂O₃	0.88±0.01	996.9±0.02	3409.0±0.1	1.36±0.1	1016.0±0.2	–16.79±0.06
HRP@PDA@MHAl₂O₃	1.03±0.01	142.2±0.01	534.4±0.1	6.91±0.1	159.3±0.2	–17.19±0.06

表2. 游离HRP和固定化酶反应器的热动力学参数a

Table 2. Thermodynamic binding parameters for free HRP and immobilized enzyme reactorsa

图7. (a)体系pH值, (b)降解时间, (c)底物浓度对苯胺黑药降解效果的影响和(d)固定化酶反应器催化降解苯胺黑药的重复使用情况

Figure 7. Effect of pH value (a), time (b) and substrate concentration (c) on degradation of aniline aerofloat, and reusability of immobilized enzyme reactors for catalytic degradation of aniline aerofloat (d)

图8. HRP催化ABTS生成ABTS•+的过氧化反应

Figure 8. Peroxidation of ABTS to ABTS•+ catalyzed by HRP

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Al-MOF基多级孔Al₂O₃固定化酶反应器的构筑及构效关系研究

Construction and Structure-Activity Relationship of Immobilized Enzyme Reactor Based on Al-MOF-Derived Al₂O₃ with Hierarchical Structure

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