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

doi:10.6023/A21080385

Abstract

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

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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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Fig. & Tab. 10

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

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

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

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

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

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

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

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

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)

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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Al-MOF基多级孔Al2O3固定化酶反应器的构筑及构效关系研究