羧基功能化Fe3O4固定化酶反应器的构筑及性能研究

doi:10.6023/A23010015

摘要/Abstract

对固定化酶的载体进行功能化修饰, 通过改善载体和酶的界面连接可使酶分子在载体表面形成高度有序的二维排列, 从而提高酶的催化活性和操作稳定性. 用柠檬酸修饰的Fe₃O₄磁性纳米粒子(CA-Fe₃O₄)易于磁性分离且表面富含羧基, 可作为一种优良载体通过吸附法固定化氯过氧化物酶(CPO)构筑CPO@CA-Fe₃O₄酶反应器、共固定化CPO和葡萄糖氧化酶(GOx)构筑GOx&CPO@CA-Fe₃O₄级联酶反应器. 将酶反应器应用于催化氧化结晶紫染料的脱色时, 两种酶反应器均显示出良好的催化活性、对底物的亲和性与专一性、热稳定性, 在实际水样中也有良好的应用效果. 与CPO@CA-Fe₃O₄相比, GOx&CPO@CA-Fe₃O₄酶反应器因级联反应中H₂O₂的原位产生而表现出更优异的催化性能.

关键词: 羧基功能化四氧化三铁(CA-Fe₃O₄), 氯过氧化物酶, 固定化酶, 级联酶反应器, 酶催化, 稳定性

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

引用此文

高丰琴, 刘洋, 张引莉, 蒋育澄. 羧基功能化Fe₃O₄固定化酶反应器的构筑及性能研究[J]. 化学学报, 2023, 81(4): 338-344.

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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图/表 10

图1. 固定化级联酶催化氧化结晶紫脱色示意图

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

图2. Fe3O4和CA-Fe3O4的XRD (a)、红外光谱(b)和透射电镜(c)图

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

图3. CA-Fe3O4和固定化酶的激光共聚焦图(a、b、c)和热重曲线图(d)

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

表1. 固定化方法与结晶紫脱色效果

Table 1. Immobilization method and decoloration effect of crystal violet

图4. CA-Fe3O4吸附法固定化酶示意图

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

图5. GOx&CPO@CA-Fe3O4催化结晶紫脱色反应条件

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

表2. 游离CPO和固定化CPO的酶动力学参数

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

图6. 游离酶和固定化酶在(a) 20~70 ℃和(b) 50 ℃下的热稳定性

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

图7. 固定化CPO的重复使用性

Figure 7. Reusability of immobilized CPO

图8. 在自来水和自然水源中使用固定化酶催化氧化结晶紫脱色效果(从左到右分别为缓冲溶液和自来水、黑河水、沣峪口水、人工湖水源)

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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