介孔金属有机骨架固定化漆酶及其活性艳蓝KN-R降解性能

doi:10.16085/j.issn.1000-6613.2024-0349

摘要/Abstract

摘要：

为了克服游离漆酶不稳定且难以回收利用的问题，以具有高比表面积且孔径与漆酶尺寸相近的介孔金属有机骨架PCN-333(Al)作为固定漆酶载体，通过物理吸附法制备了PCN-333(Al)固定化漆酶［Lac/PCN-333(Al)］，并将其用于活性艳蓝KN-R（RBBR）的降解。结果表明，Lac/PCN-333(Al)具有超高漆酶负载量（688.9mg/g），酶活性回收率达70.9%。同时，PCN-333(Al)的孔道为漆酶提供了良好的保护作用，Lac/PCN-333(Al)的pH、温度、储存稳定性均高于游离漆酶。此外，Lac/PCN-333(Al)的米氏常数（K_m）值为97.6μmol/L，远低于游离漆酶，表明固定后漆酶与底物的亲和力增加。最后，在加入0.2mL介体ABTS（0.2mmol/L）条件下Lac/PCN-333(Al) 200min内对RBBR的降解率高达89.0%，降解速率是游离漆酶的4.5倍，且经过5次重复使用后降解率仍可达78.5%，具有良好的可重复使用性。本研究为新型固定化漆酶的研发及其在染料污水处理中的应用提供了参考。

关键词: 金属有机骨架, 固定化, 酶, 生物催化, 废水

Abstract:

In order to overcome the unstable and unrecyclable problem of free laccase, mesoporous metal-organic framework PCN-333(Al) with high specific surface area and pore size similar to laccase was selected as the carrier of immobilized laccase, and PCN-333(Al) immobilized laccase [Lac/PCN-333(Al)] was prepared through physical adsorption for reactive brilliant blue KN-R (RBBR) degradation. The results showed that Lac/PCN-333(Al) had extremely high laccase loading (688.9mg/g), and its activity recovery was 70.9%. The pores of PCN-333(Al) provided good protection for laccase, and the pH stability, thermal stability and storage stability of Lac/PCN-333(Al) were improved compared with free laccase. Besides, the Michaelis Menten constant (K_m) value of Lac/PCN-333(Al) was 97.6μmol/L, which was much lower than that of free laccase, indicating the affinity of laccase for the substrate became higher after being immobilized. Finally, the degradation efficiency of RBBR by Lac/PCN-333(Al) reached 89.0% within 200min when 0.2mL of ABTS (0.2mmol/L) was added, the degradation rate was 4.5 times higher than that of free laccase, and the degradation efficiency maintained 78.5% after repeated use for 5 times, indicating it had good reusability. This study provided a reference for the development of new immobilized laccase and its application in the treatment of dye wastewater.

Key words: metal-organic framework, immobilization, enzyme, biocatalysis, waste water

中图分类号:

X703.1

张新宇, 陶梦滢, 于小婷, 赵钟兴, 赵祯霞. 介孔金属有机骨架固定化漆酶及其活性艳蓝KN-R降解性能[J]. 化工进展, 2025, 44(3): 1758-1767.

ZHANG Xinyu, TAO Mengying, YU Xiaoting, ZHAO Zhongxing, ZHAO Zhenxia. Laccase immobilized on mesoporous metal-organic framework and its performance of reactive brilliant blue KN-R degradation[J]. Chemical Industry and Engineering Progress, 2025, 44(3): 1758-1767.

图/表 14

图1 PCN-333(Al)和Lac/PCN-333(Al)的SEM图像及FITC-Lac/PCN-333(Al)的CLSM图像

图2 PCN-333(Al)和Lac/PCN-333(Al)的XRD谱图和N2吸附-脱附等温线

图3 漆酶、Lac/PCN-333(Al)和PCN-333(Al)的FTIR谱图

图4 PCN-333(Al)和Lac/PCN-333(Al)的热重曲线

图5 漆酶初始浓度对Lac/PCN-333(Al)上漆酶负载量和酶活性回收率的影响

表1 PCN-333(Al)对漆酶的负载量和酶活性回收率与文献报道其他载体对比

漆酶载体	漆酶负载量 /mg·g^-1	酶活性回收率 /%	参考文献
HS NMIL-88(Fe)	213.2	75.8	[20]
H-COF-OMe	567.0	85.0	[23]
dPCN-224	80.0	90.0	[31]
ILs-NH2-MIL-101	61.3	83.3	[32]
Fe₃O₄-NH₂@MIL-100(Fe)	61.6	72.1	[33]
Fe₃O₄@TA/PEI	39.9	53.1	[34]
Fe-Cu-MPC	189.0	63.1	[35]
Fe₃O₄@SiO₂-chitosan	158.1	79.1	[36]
3D printed PLA scaffolds	198.0	89.2	[37]
PCN-333(Al)	688.9	70.9	本工作

图6 pH和温度对游离漆酶和Lac/PCN-333(Al)活性的影响

图7 游离漆酶和Lac/PCN-333(Al)的热稳定性和储存稳定性

图8 Lac/PCN-333(Al)的循环使用稳定性

图9 游离漆酶和Lac/PCN-333(Al)的Lineweaver-Burk谱图（游离或固定化的漆酶含量均为0.006μmol/L）

表2 游离漆酶和Lac/PCN-333(Al)的动力学参数

样品	V_max/μmol·L^-1·min^-1	K_m/μmol·L^-1	K_cat/min^-1	(K_cat/K_m)/L·μmol^-1·min^-1
游离漆酶	173.0	235.3	2.9×10⁴	123.3
Lac/PCN-333(Al)	112.2	97.6	1.9×10⁴	194.7

图10 加入不同浓度ABTS条件下游离漆酶对RBBR的降解效果

图11 加入不同浓度ABTS条件下Lac/PCN-333(Al)对RBBR的降解效果

图12 Lac/PCN-333(Al)重复降解RBBR

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