磁性固定化漆酶的制备及其对染料的高效降解

doi:10.13475/j.fzxb.20250401301

摘要/Abstract

摘要： 为解决生物酶生产成本高、稳定性差以及无法重复使用等问题,通过将生物酶固定于纳米粒子表面,可提升其稳定性与重复使用性。基于醛基与氨基形成席夫碱的反应原理,将漆酶共价固定在Fe₃O₄纳米粒子表面,成功制备出磁性固定化漆酶,并考察了其耐热性、耐酸性、金属离子抗污染性、有机物抗污染性、抑制剂抗污染性及重复使用性。结果表明:磁性固定化漆酶的稳定性与抗污染性均得到提升,且重复使用性能表现优异,在重复使用10次后仍可保持59.3%的初始活性;此外,该磁性固定化漆酶在染料降解领域展现出优异性能,可高效降解高浓度的孔雀石绿、结晶紫、灿烂绿、活性红、亮蓝及偶氮荧光桃红,初次降解率可达81.6%~98.8%;经过10个循环的重复降解,其降解率仍能维持在62.2%~90.5%。该结果证实,磁性固定化漆酶在染料降解方面具有一定的优势。

关键词: 固定化漆酶, 重复性, 稳定性, 抗污染性, 染料, 生物降解, 降解率, 废水处理

Abstract:

Objective The discharge of dyeing effluents presents a serious environmental challenge. The release of dye wastewater generated by the textile industry is considered to be the major sources of water pollution. Dyes pose significant obstacles for conventional treatment methods due to their remarkable photolytic stability and resistance to microbial degradation. Moreover, the majority of dyes, along with their degradation byproducts, exhibit toxic, carcinogenic, mutagenic, and allergenic properties that pose significant risks to human health. Consequently, there exists an urgent demand for effective strategies aiming at the removal of dyes from wastewater.

Method Amine-functionalized Fe₃O₄ nanoparticles were synthesized for the immobilization of laccase, aiming at enhancing the efficient degradation of dyes. The morphology, structure, magnetic properties, and surface characteristics of the Fe₃O₄ nanoparticles were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), magnetic measurements, and Fourier transform infrared spectroscopy (FT-IR). The activity assays of laccase, including optimal temperature and pH conditions, thermostability, pH stability, tolerance to metal ions and organic solvents, resistance to inhibitors, and recyclability of the immobilized laccase, were systematically investigated. Additionally, the degradation kinetics and reusability concerning dyes were also evaluated.

Results TEM images revealed that the synthesized Fe₃O₄ nanoparticles exhibited a spherical morphology, with a size range of 110-130 nm. XRD analysis confirmed that the resultant product displayed a cubic crystalline structure characteristic of Fe₃O₄. FT-IR spectra illustrated the presence of abundant amide groups on the surface of these nanoparticles. Magnetic measurements demonstrated that the prepared material possessed ferromagnetic properties, exhibiting a saturation magnetization of 42.3 emu/g. The Bradford protein assay indicated that approximately 3.1 mg of laccase was loaded per gram onto the amino-functionalized Fe₃O₄ nanoparticles. The optimal temperature for both immobilized laccase and free laccase was 50 ℃, the former however exhibited a broader temperature distribution range compared to its free counterpart. The optimal pH for immobilized laccase was 3.0, representing a shift of 1.5 towards acidity when contrasted with the free laccase. After undergoing treatment at 60 ℃ for 10 h, the immobilized laccase retained 71.3% of its initial activity, demonstrating an enhancement of 14.4% over that of the free laccase. Moreover, the pH stability of immobilized laccase showed increased resistance to organic solvents, metal ions, and inhibitors in comparison to its free enzyme counterpart. The immobilized laccase maintained remarkable reusability, preserving 59.3% of its initial activity even after 10 cycles of reuse. Furthermore, the immobilized laccase showcased exceptional efficacy in treating dye wastewater, achieving degradation rates between 81.6% and 98.8% for triphenylmethane, azo, and anthraquinone dyes. The degradation of dyes by the immobilized laccase was a relatively swift process. The initial 30 min interval performed 79.1% to 97.9% of the total degradation achieved. Subsequently, within the span of 60 min, the maximum degradation rate was gradually attained. Additionally, after undergoing 10 consecutive degradation cycles, the efficiency level was kept in the range from 62.2% to 90.5%, underscoring its potential as an effective strategy for biodegrading dye effluents.

Conclusion An innovative method for the synthesis of amino-functionalized Fe₃O₄ nanoparticles has been developed, positioning them as magnetic carriers for laccase immobilization. These magnetic nanoparticles have been thoroughly characterized, demonstrating their promising applicability in studies focused on laccase immobilization. The amino-functionalized Fe₃O₄, can be easily separated using an external magnet and exhibits a straightforward immobilization process along with remarkable loading capacity and catalytic activity for the immobilized laccase. The immobilized laccase demonstrated to possess excellent reusability and enhanced thermal and pH stability, and remarkable organic compounds, inhibitors and metal ions tolerance. Additionally, the immobilized laccase showcases effective and sustainable degradation capabilities for high concentrations of dyes. This highlights the exceptional potential of immobilized laccase in addressing textile wastewater treatment within practical applications.

Key words: immobilized laccase, reusability, stability, anti-polluting, dye, biodegradation, degradation rate, wastewater treatment

中图分类号:

TS190

谢围围, 朱庆鹏, 宋娇娇, 陈志明. 磁性固定化漆酶的制备及其对染料的高效降解[J]. 纺织学报, 2025, 46(12): 163-170.

XIE Weiwei, ZHU Qingpeng, SONG Jiaojiao, CHEN Zhiming. Synthesis of magnetic immobilized laccase and its efficient degradation of dyes[J]. Journal of Textile Research, 2025, 46(12): 163-170.

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链接本文: http://www.fzxb.org.cn/CN/10.13475/j.fzxb.20250401301

http://www.fzxb.org.cn/CN/Y2025/V46/I12/163

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参考文献 33

[1]	MOGHIMI Dehkordi M, POURNUROZ Nodeh Z, SOLEIMANI Dehkordi K, et al. Soil, air, and water pollution from mining and industrial activities: sources of pollution, environmental impacts, and prevention and control methods[J]. Results in Engineering, 2024, 23: 102729. doi: 10.1016/j.rineng.2024.102729
[2]	RAJENDRAN S, KALAIRAJ A, SENTHILVELAN T. A comprehensive review on enzymatic decolorization of various azo dyes using laccase for the abatement of industrial pollution[J]. Biomass Conversion and Biorefinery, 2025, 15(9): 13079-13101. doi: 10.1007/s13399-024-06104-0
[3]	RAYAROTH M P, ARAVIND U K, ARAVINDAKUMAR C T. Effect of inorganic ions on the ultrasound initiated degradation and product formation of triphenylmethane dyes[J]. Ultrasonics Sonochemistry, 2018, 48: 482-491. doi: S1350-4177(18)30351-1 pmid: 30080575
[4]	BELLO-GIL D, ROIG-MOLINA E, FONSECA J, et al. An enzymatic system for decolorization of wastewater dyes using immobilized CueO laccase-like multicopper oxidase on poly-3-hydroxybutyrate[J]. Microbial Biotechnology, 2018, 11(5): 881-892. doi: 10.1111/mbt2.2018.11.issue-5
[5]	BILAL M, ASGHER M, IQBAL M, et al. Chitosan beads immobilized manganese peroxidase catalytic potential for detoxification and decolorization of textile effluent[J]. International Journal of Biological Macromolecules, 2016, 89: 181-189. doi: 10.1016/j.ijbiomac.2016.04.075 pmid: 27130652
[6]	NOUREN S, BHATTI H N, IQBAL M, et al. By-product identification and phytotoxicity of biodegraded Direct Yellow 4 dye[J]. Chemosphere, 2017, 169: 474-484. doi: S0045-6535(16)31615-0 pmid: 27889513
[7]	XU H M, SUN X F, WANG S Y, et al. Development of laccase/graphene oxide membrane for enhanced synthetic dyes separation and degradation[J]. Separation and Purification Technology, 2018, 204: 255-260. doi: 10.1016/j.seppur.2018.04.036
[8]	李万新, 舒大武, 安芳芳, 等. 碳化钛与三价铁离子协同过硫酸钠对活性染料废水的降解[J]. 纺织学报, 2025, 46(1): 138-147.
	LI Wanxin, SHU Dawu, AN Fangfang, et al. Degradation of reactive dye wastewater by titanium carbide and Fe³⁺ activated sodium persulfate[J]. Journal of Textile Research, 2025, 46(1): 138-147.
[9]	ABBAS M, ADIL M, EHTISHAM-UL-HAQUE S, et al. Vibrio fischeri bioluminescence inhibition assay for ecotoxicity assessment: a review[J]. Science of the Total Environment, 2018, 626: 1295-1309. doi: 10.1016/j.scitotenv.2018.01.066
[10]	CHEN J G, LI Y B, YANG Y, et al. How cushion communities are maintained in alpine ecosystems: a review and case study on alpine cushion plant reproduction[J]. Plant Diversity, 2017, 39(4): 221-228. doi: 10.1016/j.pld.2017.07.002
[11]	SABER-SAMANDARI S, SABER-SAMANDARI S, JONEIDI-YEKTA H, et al. Adsorption of anionic and cationic dyes from aqueous solution using gelatin-based magnetic nanocomposite beads comprising carboxylic acid functionalized carbon nanotube[J]. Chemical Engineering Journal, 2017, 308: 1133-1144. doi: 10.1016/j.cej.2016.10.017
[12]	NAVARRO P, GABALDÓN J A, GÓMEZ-LÓPEZ V M. Degradation of an azo dye by a fast and innovative pulsed light/H₂O₂ advanced oxidation process[J]. Dyes and Pigments, 2017, 136: 887-892. doi: 10.1016/j.dyepig.2016.09.053
[13]	KADAM A A, JANG J, LEE D S. Supermagnetically tuned halloysite nanotubes functionalized with aminosilane for covalent laccase immobilization[J]. ACS Applied Materials & Interfaces, 2017, 9(18): 15492-15501.
[14]	RAN F P, ZOU Y L, XU Y X, et al. Fe₃O₄@MoS₂@PEI-facilitated enzyme tethering for efficient removal of persistent organic pollutants in water[J]. Chemical Engineering Journal, 2019, 375: 121947. doi: 10.1016/j.cej.2019.121947
[15]	DARVISHI F, MORADI M, JOLIVALT C, et al. Laccase production from sucrose by recombinant Yarrowia lipolytica and its application to decolorization of environmental pollutant dyes[J]. Ecotoxicology and Environmental Safety, 2018, 165: 278-283. doi: S0147-6513(18)30882-0 pmid: 30205329
[16]	WANG F, GUO C, LIU H Z, et al. Immobilization of Pycnoporus sanguineus laccase by metal affinity adsorption on magnetic chelator particles[J]. Journal of Chemical Technology & Biotechnology, 2008, 83(1): 97-104. doi: 10.1002/jctb.v83:1
[17]	WANG X Y, LIU L, JIANG B B, et al. Synthesis of the immobilized laccase on N-doped carbon nanonets for photothermal detection of hydroquinone[J]. Microchemical Journal, 2025, 208: 112432. doi: 10.1016/j.microc.2024.112432
[18]	PATEL S K S, ANWAR M Z, KUMAR A, et al. Fe₂O₃ yolk-shell particle-based laccase biosensor for efficient detection of 2, 6-dimethoxyphenol[J]. Biochemical Engineering Journal, 2018, 132: 1-8. doi: 10.1016/j.bej.2017.12.013
[19]	ORABY K R M, VILLALONGA A, HASSAN F S M, et al. Immobilization of laccase on Fe₃O₄@SiO₂ core@shell magnetic nanoparticles for methylene blue biodegradation[J]. Process Biochemistry, 2025, 148: 10-16. doi: 10.1016/j.procbio.2024.11.012
[20]	TULLY J, YENDLURI R, LVOV Y. Halloysite clay nanotubes for enzyme immobilization[J]. Biomacromolecules, 2016, 17(2): 615-621. doi: 10.1021/acs.biomac.5b01542 pmid: 26699154
[21]	ZHANG H, ZHANG X, WANG L, et al. Functionalized chitosan and alginate composite hydrogel-immobilized laccase with sustainable biocatalysts for the effective removal of organic pollutant bisphenol A[J]. Catalysts, 2024, 14(5): 304. doi: 10.3390/catal14050304
[22]	XIA T T, LIU C Z, HU J H, et al. Improved performance of immobilized laccase on amine-functioned magnetic Fe₃O₄ nanoparticles modified with polyethylenimine[J]. Chemical Engineering Journal, 2016, 295: 201-206. doi: 10.1016/j.cej.2016.03.044
[23]	ZHANG W, WANG Q R, SONG J F, et al. Ionic liquid interfacial modification of magnetic metal-organic framework enhances laccase stability and catalytic performance in degrading phenolic pollutants[J]. Process Safety and Environmental Protection, 2025, 194: 1081-1091. doi: 10.1016/j.psep.2024.12.083
[24]	朱脉勇, 陈齐, 童文杰, 等. 四氧化三铁纳米材料的制备与应用[J]. 化学进展, 2017, 29(11): 1366-1394. doi: 10.7536/PC170559
	ZHU Maiyong, CHEN Qi, TONG Wenjie, et al. Preparation and application of Fe₃O₄ nanomaterials[J]. Progress in Chemistry, 2017, 29(11): 1366-1394. doi: 10.7536/PC170559
[25]	FARAJI M, YAMINI Y, REZAEE M. Magnetic nanoparticles: synthesis, stabilization, functionalization, characterization, and applica-tions[J]. Journal of the Iranian Chemical Society, 2010, 7(1): 1-37. doi: 10.1007/BF03245856
[26]	FAROKHIAN P, MAMAGHANI M, MAHMOODI N O, et al. An expeditious one-pot synthesis of pyrido [2, 3-d] pyrimidines using Fe₃O₄-ZnO-NH₂-PW₁₂O₄₀ nanocatalyst[J]. Journal of Chemical Research, 2019, 43(3/4): 135-139. doi: 10.1177/1747519819856978
[27]	HUAN W W, YANG Y X, WU B, et al. Degradation of 2, 4-DCP by the immobilized laccase on the carrier of Fe₃O₄@SiO₂-NH2[J]. Chinese Journal of Chemistry, 2012, 30(12): 2849-2860. doi: 10.1002/cjoc.v30.12
[28]	YANG L, GUO Y L, ZHAN W C, et al. One-pot synthesis of aldehyde-functionalized mesoporous silica-Fe₃O₄ nanocomposites for immobilization of penicillin G acylase[J]. Microporous and Mesoporous Materials, 2014, 197: 1-7. doi: 10.1016/j.micromeso.2014.05.044
[29]	TAKEUCHI R, SATO N. Hydroformylation of alkenes having organosilicon substituents[J]. Journal of Organometallic Chemistry, 1990, 393(1): 1-10. doi: 10.1016/0022-328X(90)87193-H
[30]	YONG Y, BAI Y X, LI Y F, et al. Characterization of Candida rugosa lipase immobilized onto magnetic microspheres with hydrophilicity[J]. Process Biochemistry, 2008, 43(11): 1179-1185. doi: 10.1016/j.procbio.2008.05.019
[31]	CHEN Z M, SONG J J, ZHU Q P, et al. Synthesis of Fe₃O₄@PVBC-TMT nanoparticles for the efficient removal of heavy metals ions[J]. RSC Advances, 2019, 9(69): 40546-40552. doi: 10.1039/C9RA08037F
[32]	HIDAKA H, KOBAYASHI R. Pharmacology of protein kinase inhibitors[J]. Annual Review of Pharmacology and Toxicology, 1992, 32: 377-397. pmid: 1605572
[33]	QIAO W C, LIU H. Enhanced decolorization of malachite green by a magnetic graphene oxide-CotA laccase composite[J]. International Journal of Biological Macromolecules, 2019, 138: 1-12. doi: S0141-8130(19)32813-2 pmid: 31302127

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