Feasibility of Combining Novel LacN with Traditional Enzyme Immobilization Techniques: Further Enhancement of Enzyme Activity and Stability

Abstract

Laccase-inorganic hybrid nanoflowers (LacN) represent a novel enzyme immobilization strategy. The integration of this approach with traditional adsorption-based immobilization offers a novel and efficient pathway for laccase immobilization. In this study, LacN was immobilized onto two types of Cu/HAP-BC (natural mineral-derived Cu/nHAP-BC and porcine bone-derived Cu/pHAP-BC) to synthesize LacN@Cu/HAP-BC. Single-factor experiments, combined with Box-Behnken response surface methodology, were used to optimize the immobilization parameters. The optimal preparation conditions were determined as 60% LacN suspension, pH 6.0, reaction time of 2 h for LacN@Cu/nHAP- BC; and 80% LacN suspension, pH 6.0, reaction time of 2 h for LacN@Cu/pHAP-BC. Multiple characterizations confirmed uniform anchoring of flower-like LacN on porous Cu/HAP-BC. Compared with free laccase, LacN@Cu/HAP-BC showed significantly improved stability: after 30 d of storage, LacN@Cu/nHAP-BC and LacN@Cu/pHAP-BC retained 81.35% and 76.70% of their initial activities, respectively (vs. 42.98% for free laccase); the thermal half-lives (t_1/2) at 65 °C were 502 min and 433 min, respectively, approximately 3.0–3.5 times that of free laccase (142 min). Additionally, the two composites retained 71.98% and 80.15% of their initial activity after 10 consecutive catalytic cycles, showing satisfactory reusability. These results verify that the composites deliver favorable storage, thermal, and cyclic stability, acting as a feasible biocatalyst candidate for lab-level water treatment and biomass conversion. Further tests on continuous reactors and real wastewater pollutants are needed to confirm its industrial applicability.

Keywords

Laccase nanoflowers / Copper based carrier / Enzyme immobilization / Enzyme activity / Stability

References

[1] Zhao H, Zhao Y, Wang Y, Xiao G, Su H. Adsorption and High-Value Transformation of Volatile Fatty Acids from Microbial Fermentation Products: A Review. Green Chem. Technol. 2025, 2, 10001. DOI:10.70322/gct.2025.10001

[2] Ghattavi S, Homaei A. Marine enzymes: Classification and application in various industries. Int. J. Biol. Macromol. 2023, 230, 123136. DOI:10.1016/j.ijbiomac.2023.123136

[3] Ge J, Lei J, Zare RN. Protein-inorganic hybrid nanoflowers. Nat. Nanotechnol. 2012, 7, 428-432. DOI:10.1038/nnano.2012.80

[4] Zhang M, Zhang Y, Yang C, Ma C, Tang J. Enzyme-inorganic hybrid nanoflowers: Classification, synthesis, functionalization and potential applications. Chem. Eng. J. 2021, 415, 129075. DOI:10.1016/j.cej.2021.129075

[5] Wang H, Tang L-X, Ye Y-F, Ma J-X, Li X, Si J, et al. Laccase immobilization and its degradation of emerging pollutants: A comprehensive review. J. Environ. Manag. 2024, 359, 120984. DOI:10.1016/j.jenvman.2024.120984

[6] Gupta RK, Patel SKS, Lee J-K. Novel cofactor regeneration-based magnetic metal-organic framework for cascade enzymatic conversion of biomass-derived bioethanol to acetoin. Bioresour. Technol. 2024, 408, 131175. DOI:10.1016/j.biortech.2024.131175

[7] Wang Z, Ren D, Jiang S, Yu H, Cheng Y, Zhang S, et al. The study of laccase immobilization optimization and stability improvement on CTAB-KOH modified biochar. BMC Biotechnol. 2021, 21, 47. DOI:10.1186/s12896-021-00709-3

[8] Wang Z, Ren D, Yu H, Zhang S, Zhang X, Chen W. Preparation optimization and stability comparison study of alkali-modified biochar immobilized laccase under multi-immobilization methods. Biochem. Eng. J. 2022, 181, 108401. DOI:10.1016/j.bej.2022.108401

[9] Wang Z, Ren D, Zhao Y, Huang C, Zhang S, Zhang X, et al. Remediation and improvement of 2,4-dichlorophenol contaminated soil by biochar-immobilized laccase. Environ. Technol. 2021, 42, 1679-1692. DOI:10.1080/09593330.2019.1677782

[10] Wang Z, Ren D, Cheng Y, Zhang X, Zhang S, Chen W. Immobilization of laccase on chitosan functionalized halloysite nanotubes for degradation of Bisphenol A in aqueous solution: degradation mechanism and mineralization pathway. Heliyon 2022, 8, e09919. DOI:10.1016/j.heliyon.2022.e09919

[11] Wang Z, Ren D, Zhang X, Zhang S, Chen W. Adsorption-degradation of malachite green using alkali-modified biochar immobilized laccase under multi-methods. Adv. Powder Technol. 2022, 33, 103821. DOI:10.1016/j.apt.2022.103821

[12] Wang Z, Ren D, Wu J, Jiang S, Yu H, Cheng Y, et al. Study on adsorption-degradation of 2,4-dichlorophenol by modified biochar immobilized laccase. Int. J. Environ. Sci. Technol. 2022, 19, 1393-1406. DOI:10.1007/s13762-021-03151-2

[13] Jiang S, Ren D, Wang Z, Zhang S, Zhang X, Chen W. Improved stability and promoted activity of laccase by One-Pot encapsulation with Cu (PABA) nanoarchitectonics and its application for removal of Azo dyes. Ecotoxicol. Environ. Saf. 2022, 234, 113366. DOI:10.1016/j.ecoenv.2022.113366

[14] Jiang S, Wang Z, Qin Y, Cao B, Chi R. Eco-Friendly Synthesis of Spherical Lanthanum-Doped Hydroxyapatite from Phosphogypsum Waste: A High-Efficiency Strategy for Fluoride Removal in Aqueous Solutions. Green Chem. Technol. 2026, 3, 10008. DOI:10.70322/gct.2026.10008

[15] Ding T, Chi R, Yu J, Yin W, Hu Z, Zhao Q. Synthesis of Hydroxyapatite Porous Microspheres for Efficient Adsorption of Copper Ion from Water. Green Chem. Technol. 2026, 3, 10011. DOI:10.70322/gct.2026.10011

[16] Liu Y, Fan C, Zhang B, Wen B, Zhang L. The Crystal Structure of Apatite and Copper-Doped Apatite. Green Chem. Technol. 2025, 2, 10005. DOI:10.70322/gct.2025.10005

[17] Wang Z, Ren D, Shang S, Zhang S, Zhang X, Chen W. Novel synthesis of Cu-HAP/SiO2@carbon nanocomposites as heterogeneous catalysts for Fenton-like oxidation of 2,4-DCP. Adv. Powder Technol. 2022, 33, 103509. DOI:10.1016/j.apt.2022.103509

[18] Wang M, Ren D, Wang Z, Li Y, Zhang S, Gong X, et al. A novel Cu/HAP@sBC composite as a persulfate activator for the removal of 2,4,6-trichlorophenol. Catal. Commun. 2023, 184, 106793. DOI:10.1016/j.catcom.2023.106793

[19] Wang Z, Ren D, Chi R. Synthesis of copper-based Fenton-like catalyst (Cu/HAP-sBC) for oxidative degradation of p-nitrophenol in aqueous solution. J. Mol. Struct. 2025, 1339, 142383. DOI:10.1016/j.molstruc.2025.142383

[20] Akram W, Garud N. Design expert as a statistical tool for optimization of 5-ASA-loaded biopolymer-based nanoparticles using Box Behnken factorial design. Futur. J. Pharm. Sci. 2021, 7, 1-17. DOI:10.1186/s43094-021-00299-z

[21] Martin E, Audonnet F, Yaacoub D, Dubessay P, Michaud P. Nernst-Michaelis-Menten framework unlocks electrochemical kinetics for laccases. Bioelectrochemistry 2025, 165, 109003. DOI:10.1016/j.bioelechem.2025.109003

[22] Costa JB, Lima MJ, Sampaio MJ, Neves MC, Faria JL, Morales-Torres S, et al. Enhanced biocatalytic sustainability of laccase by immobilization on functionalized carbon nanotubes/polysulfone membranes. Chem. Eng. J. 2019, 355, 974-985. DOI:10.1016/j.cej.2018.08.178

[23] Wu X, Ge J, Hou M. Metal-organic frameworks and inorganic nanoflowers: A type of emerging inorganic crystal nanocarrier for enzyme immobilization. Catal. Sci. Technol. 2015, 5, 5077-5085. DOI:10.1039/C5CY01181G

[24] Vo TN, Tran TD, Nguyen HK, Kong DY, Kim MI, Kim IT. In situ growth of hybrid nanoflowers on activated carbon fibers as electrodes for mediatorless enzymatic biofuel cells. Mater. Lett. 2020, 281, 128662. DOI:10.1016/j.matlet.2020.128662

[25] Dlamini ML, Lesaoana M, Kotze I, Richards HL. Zeolitic imidazolate frameworks as effective crystalline supports for aspergillus-based laccase immobilization for the biocatalytic degradation of carbamazepine. Chemosphere 2023, 311, 137142. DOI:10.1016/j.chemosphere.2022.137142

[26] Rong J, Zhang T, Qiu F, Zhu Y. Preparation of Efficient, Stable, and Reusable Laccase-Cu₃(PO₄)₂ Hybrid Microspheres Based on Copper Foil for Decoloration of Congo Red. ACS Sustain. Chem. Eng. 2017, 5, 4468-4477. DOI:10.1021/acssuschemeng.7b00820

[27] Li X, Wu Z, Tao X, Li R, Tian D, Liu X. Gentle one-step co-precipitation to synthesize bimetallic CoCu-MOF immobilized laccase for boosting enzyme stability and Congo red removal. J. Hazard. Mater. 2022, 438, 129525. DOI:10.1016/j.jhazmat.2022.129525

[28] Yuan Y, Cai W, Xu J, Cheng J, Du K-S, Recyclable laccase by coprecipitation with aciduric Cu-based MOFs for bisphenol A degradation in an aqueous environment. Colloids Surf. B Biointerfaces 2021, 204, 111792. DOI:10.1016/j.colsurfb.2021.111792

[29] Zapata-Castillo P, Villalonga-Santana L, Islas-Flores I, Rivera-Muñoz G, Ancona-Escalante W, Solís-Pereira S. Synergistic action of laccases from Trametes hirsuta Bm2 improves decolourization of indigo carmine. Lett. Appl. Microbiol. 2015, 61, 252-258. DOI:10.1111/lam.12451

[30] Han Z, Fan X, Yu S, Li X, Wang S, Lu L. Metal-organic frameworks (MOFs): A novel platform for laccase immobilization and application. J. Environ. Chem. Eng. 2022, 10, 108795. DOI:10.1016/j.jece.2022.108795

[31] Zhang K, Yang W, Liu Y, Zhang K, Chen Y, Yin X. Laccase immobilized on chitosan-coated Fe₃O₄ nanoparticles as reusable biocatalyst for degradation of chlorophenol. J. Mol. Struct. 2020, 1220, 128769. DOI:10.1016/j.molstruc.2020.128769

[32] Patel SKS, Gupta RK, Karuppanan KK, Padhi DK, Ranganathan S, Paramanantham P, et al. Trametes versicolor Laccase-Based Magnetic Inorganic-Protein Hybrid Nanobiocatalyst for Efficient Decolorization of Dyes in the Presence of Inhibitors. Materials 2024, 17, 1790. DOI:10.3390/ma17081790

[33] da Silva CKH, Polidoro AS, Ruschel PMC, Thue PS, Jacques RA, Lima ÉC, et al. Laccase covalently immobilized on avocado seed biochar: A high-performance biocatalyst for acetaminophen sorption and biotransformation. J. Environ. Chem. Eng. 2022, 10, 107731. DOI:10.1016/j.jece.2022.107731

[34] Patel SKS, Gupta RK, Kim S-Y, Kim I-W, Kalia VC, Lee J-K. Rhus vernicifera laccase immobilization on magnetic nanoparticles to improve stability and its potential application in bisphenol A degradation. Indian J. Microbiol. 2021, 61, 45-54. DOI:10.1007/s12088-020-00912-4

[35] Zdarta J, Jankowska K, Wyszowska M, Kijeńska-Gawrońska E, Zgoła-Grześkowiak A, Pinelo M, et al. Robust biodegradation of naproxen and diclofenac by laccase immobilized using electrospun nanofibers with enhanced stability and reusability. Mater. Sci. Eng. C 2019, 103, 109789. DOI:10.1016/j.msec.2019.109789

[36] Panwar V, Lzaod S, Dutta T. Thermostable Bacterial Laccase: Catalytic Properties and Its Application in Biotransformation of Emerging Pollutants. ACS Omega 2023, 8, 34710-34719. DOI:10.1021/acsomega.3c03627

[37] Taheran M, Naghdi M, Brar SK, Knystautas EJ, Verma M, Surampalli RY. Degradation of chlortetracycline using immobilized laccase on Polyacrylonitrile-biochar composite nanofibrous membrane. Sci. Total Environ. 2017, 605, 315-321. DOI:10.1016/j.scitotenv.2017.06.185

[38] Naghdi M, Taheran M, Brar SK, Kermanshahi-pour A, Verma M, Surampalli RY. Pinewood nanobiochar: A unique carrier for the immobilization of crude laccase by covalent bonding. Int. J. Biol. Macromol. 2018, 115, 563-571. DOI:10.1016/j.ijbiomac.2018.04.105

[39] Wang J, Yu S, Feng F, Lu L. Simultaneous purification and immobilization of laccase on magnetic zeolitic imidazolate frameworks: Recyclable biocatalysts with enhanced stability for dye decolorization. Biochem. Eng. J. 2019, 150, 107285. DOI:10.1016/j.bej.2019.107285

[40] Olajuyigbe FM, Adetuyi OY, Fatokun CO. Characterization of free and immobilized laccase from Cyberlindnera fabianii and application in degradation of bisphenol A. Int. J. Biol. Macromol. 2019, 125, 856-864. DOI:10.1016/j.ijbiomac.2018.12.106

[41] Masjoudi M, Golgoli M, Nejad ZG, Sadeghzadeh S, Borghei SM. Pharmaceuticals removal by immobilized laccase on polyvinylidene fluoride nanocomposite with multi-walled carbon nanotubes. Chemosphere 2021, 263, 128043. DOI:10.1016/j.chemosphere.2020.128043

[42] Yavaşer R, Karagözler AA. Laccase immobilized polyacrylamide-alginate cryogel: A candidate for treatment of effluents. Process Biochem. 2021, 101, 137-146. DOI:10.1016/j.procbio.2020.11.021

[43] Hu W, Zong J, Gong J, Xue R, Lv Y, Liu B, et al. Lipase immobilization via dynamic defect-engineered ZIF-8: A bioactivity-enhanced platform for high-efficiency screening of herbal lipase inhibitors. Int. J. Biol. Macromol. 2025, 335, 149264. DOI:10.1016/j.ijbiomac.2025.149264

[44] Zhao Q, Zhang X, Wang S, Deng L, Bai Y, Du K. Robust and Versatile OPH-Cu Phosphate Hybrid Nanoflower for Organophosphorus Compound Degradation. J. Agric. Food Chem. 2025, 73, 18210-18219. DOI:10.1021/acs.jafc.5c03908