Study on Construction and Performance of Immobilized Enzyme Reactors by Carboxyl-functionalized Fe3O4

doi:10.6023/A23010015

Abstract

The interface connection between carrier and enzyme can be improved by functionalizing the carrier of immobilized enzyme. This facilitates the formation of highly ordered two-dimensional arrangement of enzyme molecules on the surface of the carrier, thus improving the catalytic activity and operational stability of the enzymes. The surface of Fe₃O₄ modified with citric acid (CA-Fe₃O₄) is rich in carboxyl groups, which can be used as an excellent carrier due to its magnetic and easy separation characteristics. In this work, the adsorption method was more suitable for constructing enzyme reactors on the carrier of CA-Fe₃O₄, compared with covalent method. The CPO@CA-Fe₃O₄ reactor was constructed to immobilize chloroperoxidase (CPO) and the cascade enzyme reactor of GOx&CPO@CA-Fe₃O₄ was constructed by co-immobilization of CPO and glucose oxidase (GOx). In catalytic reaction, H₂O₂ was used as oxidant for CPO@CA-Fe₃O₄, while for GOx&CPO@CA-Fe₃O₄, the in-situ formation of H₂O₂ was caused by the oxidation of β-D-glucose by GOx. When the molar ratio of CPO to GOx was 3∶4 for immobilization, the effect of the cascade reaction can reach the best. When the enzyme reactors were applied to catalyze the oxidation of the decolorization of crystal violet, both enzyme reactors showed good catalytic activity, affinity and specificity for substrate. The thermal stabilities of the immobilized enzyme reactors were significantly improved. Both the enzyme reactors retained more than 55% of the activity at 70 ℃ for 1 h and more than 60% of the activity at 50 ℃ for 8 h, while the free enzyme was almost completely inactivated under the same reaction conditions. In contrast with CPO@CA-Fe₃O₄, the GOx&CPO@CA-Fe₃O₄ reactor showed better catalytic performance due to the in-situ generation of H₂O₂ in the cascade reaction. And this advantage was particularly reflected in the application of thermal stability and actual water samples. The important application potential has been shown about the immobilization of CPO enzyme reactors on CA-Fe₃O₄ carrier as a catalyst.

Key words: carboxyl-functionalized ferroferric oxide (CA-Fe₃O₄), chloroperoxidase, immobilized enzyme, cascade enzyme reactor, enzyme catalysis, stability

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Fengqin Gao, Yang Liu, Yinli Zhang, Yucheng Jiang. Study on Construction and Performance of Immobilized Enzyme Reactors by Carboxyl-functionalized Fe₃O₄[J]. Acta Chimica Sinica, 2023, 81(4): 338-344.

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Fig. & Tab. 10

Figure 1. Schematic diagram of decolorization of crystal violet catalyzed by immobilized cascaded enzymes

Figure 2. XRD pattern (a), FT-IR spectra (b) and TEM (c) images of Fe3O4 and CA-Fe3O4

Figure 3. Confocal microscopy images (a, b, c) and TGA curves (d) of CA-Fe3O4 and enzymes@CA-Fe3O4

方法	pH	时间/h	结晶紫脱色率/%
方法	pH	时间/h	第1次	第2次	第3次	第4次
共价法	3.5	1.0	76.9	55.1	39.5	32.1
吸附法	3.5	1.0	87.1	64.2	55.3	48.3

Table 1. Immobilization method and decoloration effect of crystal violet

Figure 4. Schematic diagram of immobilization of enzymes on the carrier of CA-Fe3O4 by adsorption

Figure 5. Reaction conditions for decolorization of crystal violet catalyzed by GOx&CPO@CA-Fe3O4

Enzyme	V_max/(mmol•L^－1•s^－1)	K_m/(mmol•L^－1)	k_cat/s^－1	(k_cat/K_m)/(mmol^－1•L•s^－1)
Free CPO	0.1003	0.03009	310.5	1.0×10⁴
CPO@CA-Fe₃O₄	0.07746	0.01781	239.8	1.3×10⁴
GOx&CPO@CA-Fe₃O₄	0.07179	0.01651	222.3	1.3×10⁴

Table 2. Enzymatic kinetic parameters of free CPO and immobilized CPO

Figure 6. Thermal stability of the free and immobilized enzymes: (a) at different temperatures from 20 to 70 ℃ and (b) at 50 ℃

Figure 7. Reusability of immobilized CPO

Figure 8. Decolorization effects of crystal violet catalyzed by immobilized enzymes in tap water and actual wastewater (From left to right, samples from buffer and tap water, Hei river, Fengyu river, a man-made lake)

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羧基功能化Fe₃O₄固定化酶反应器的构筑及性能研究

Study on Construction and Performance of Immobilized Enzyme Reactors by Carboxyl-functionalized Fe₃O₄

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羧基功能化Fe3O4固定化酶反应器的构筑及性能研究