Abstract
Horseradish peroxidase is an enzyme commonly used in wastewater treatment due to its ability to oxidize a wide range of organic compounds, including phenols. The use of peroxidases immobilized onto magnetite nanoparticles improves the enzyme's stability and catalytic activity, but also facilitates their simply separation and reuse. In the present study, a covalent immobilization of horseradish peroxidase onto polyethylene glycol-modified magnetite nanoparticles via glutaraldehyde was performed in order to prepare a promising bio-catalyst for phenol removal from wastewater. The efficiency of the immobilized enzyme in phenol removal in the presence of hydrogen peroxide and polyethylene glycol as stabilizer was measured. The general toxicity of bio-treated water (Allium cepa test) was also investigated. All analyses were performed in parallel for the free enzyme. The immobilized enzyme showed the highest activity at a temperature of 50 °C and pH 7.0 and retained 50% of its activity after four washing cycles. Additionally, its storage stability was higher compared to the free form, as well as its tolerance against inactivation to heavy metals and organics-induced inhibition. Since the tested enzymatic system was very efficient in phenol removal from water (75% for 2 h), along with the substantial reduction of the toxicity of the tested water (48%), it can be considered as an environmentally acceptable bio-catalyst for phenol-containing wastewater treatment.
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References
Altinkaynak C, Özdemi̇r N, Öçsoy İ (2021) A rational synthesis of magnetic nanoparticles incorporated horseradish peroxidase nanoflower and its use for the removal of phenol through oxidative coupling reaction with great reusability. Mugla J Sci Technol 7:59–66. https://doi.org/10.22531/muglajsci.982993
Bilal M, Iqbal M, Hu H, Zhang X (2016) Mutagenicity and cytotoxicity assessment of biodegraded textile effluent by Ca-alginate encapsulated manganese peroxidase. Biochem Eng J 109:153–161. https://doi.org/10.1016/j.bej.2016.01.020
Bilal M, Iqbal HMN, Hu H et al (2017) Enhanced bio-catalytic performance and dye degradation potential of chitosan-encapsulated horseradish peroxidase in a packed bed reactor system. Sci Total Environ 575:1352–1360. https://doi.org/10.1016/j.scitotenv.2016.09.215
Cao Y, Wen L, Svec F et al (2016) Magnetic AuNP@Fe3O4 nanoparticles as reusable carriers for reversible enzyme immobilization. Chem Eng J 286:272–281. https://doi.org/10.1016/j.cej.2015.10.075
Caza N, Bewtra JK, Biswas N, Taylor KE (1999) Removal of phenolic compounds from synthetic wastewater using soybean peroxidase. Water Res 33:3012–3018. https://doi.org/10.1016/S0043-1354(98)00525-9
Chagas PMB, Torres JA, Silva MC, Corrêa AD (2015) Immobilized soybean hull peroxidase for the oxidation of phenolic compounds in coffee processing wastewater. Int J Biol Macromol 81:568–575. https://doi.org/10.1016/j.ijbiomac.2015.08.061
Cheah P, Qu J, Li Y et al (2021) The key role of reaction temperature on a polyol synthesis of water-dispersible iron oxide nanoparticles. J Magn Magn Mater 540:168481. https://doi.org/10.1016/j.jmmm.2021.168481
Darwesh OM, Matter IA, Eida MF (2019) Development of peroxidase enzyme immobilized magnetic nanoparticles for bioremediation of textile wastewater dye. J Environ Chem Eng 7:102805. https://doi.org/10.1016/j.jece.2018.11.049
Du P, Zhao H, Li H et al (2016) Transformation, products, and pathways of chlorophenols via electro-enzymatic catalysis: how to control toxic intermediate products. Chemosphere 144:1674–1681. https://doi.org/10.1016/j.chemosphere.2015.10.038
Dutta S, Kumar P, Yadav S et al (2023) Accelerating innovations in CH activation/functionalization through intricately designed magnetic nanomaterials: from genesis to applicability in liquid/regio/photo catalysis. Catal Commun 175:106615. https://doi.org/10.1016/j.catcom.2023.106615
Eş I, Vieira JDG, Amaral AC (2015) Principles, techniques, and applications of biocatalyst immobilization for industrial application. Appl Microbiol Biotechnol 99:2065–2082. https://doi.org/10.1007/s00253-015-6390-y
Gao J, Gu H, Xu B (2009) Multifunctional magnetic nanoparticles: design, synthesis, and biomedical applications. Acc Chem Res 42:1097–1107. https://doi.org/10.1021/ar9000026
Gao X, Pan H, Yang K et al (2023) Immobilization of horseradish peroxidase on hierarchically porous magnetic metal-organic frameworks for visual detection and efficient degradation of 2,4-dichlorophenol in simulated wastewater. Biochem Eng J 190:108760. https://doi.org/10.1016/j.bej.2022.108760
Greenberg AE, Clesceri LS, Eaton AD (1992) Standard methods for examinationof water and wastewater, 18th edn. Publication Office, American Public Health Association, Washington DC
Huang J, Chang Q, Ding Y et al (2014) Catalytic oxidative removal of 2,4-dichlorophenol by simultaneous use of horseradish peroxidase and graphene oxide/Fe3O4 as catalyst. Chem Eng J 254:434–442. https://doi.org/10.1016/j.cej.2014.05.136
Jonović M, Jugović B, Žuža M et al (2022) Immobilization of horseradish peroxidase on magnetite-alginate beads to enable effective strong binding and enzyme recycling during anthraquinone dyes’ degradation. Polymers 14:2614. https://doi.org/10.3390/polym14132614
Keshta BE, Gemeay AH, Khamis AA (2022) Impacts of horseradish peroxidase immobilization onto functionalized superparamagnetic iron oxide nanoparticles as a biocatalyst for dye degradation. Environ Sci Pollut Res 29:6633–6645. https://doi.org/10.1007/s11356-021-16119-z
Khalid N, Kalsoom U, Ahsan Z, Bilal M (2022) Non-magnetic and magnetically responsive support materials immobilized peroxidases for biocatalytic degradation of emerging dye pollutants—a review. Int J Biol Macromol 207:387–401. https://doi.org/10.1016/j.ijbiomac.2022.03.035
Kong J, Yu S (2007) Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin 39:549–559. https://doi.org/10.1111/j.1745-7270.2007.00320.x
Li J, Chen X, Xu D, Pan K (2019) Immobilization of horseradish peroxidase on electrospun magnetic nanofibers for phenol removal. Ecotoxicol Environ Saf 170:716–721. https://doi.org/10.1016/j.ecoenv.2018.12.043
Li A, Yang X, Yu B, Cai X (2020) Immobilization of horseradish peroxidase on polyglycerol-functionalized magnetic Fe3O4/nanodiamond nanocomposites and its application in phenol biodegradation. Res Chem Intermed 46:101–118. https://doi.org/10.1007/s11164-019-03937-7
Liu Y, Jia S, Wu Q et al (2011) Studies of Fe3O4-chitosan nanoparticles prepared by co-precipitation under the magnetic field for lipase immobilization. Catal Commun 12:717–720. https://doi.org/10.1016/j.catcom.2010.12.032
Lowell S, Shields JE, Thomas MA, Thommes M (2004) Characterization of porous solids and powders: surface area, pore size and density. Springer, Dordrecht
Marjani A, Zare MH, Sadeghi MH, Shirazian S, Ghadiri M (2021) Synthesis of alginate-coated magnetic nanocatalyst containing high-performance integrated enzyme for phenol removal. J Environ Chem Eng 9:104884. https://doi.org/10.1016/j.jece.2020.104884
Michałowicz J, Duda W (2007) Phenols—sources and toxicity. Pol J Environ Stud 16:347–362
Mohamed SA, Al-Harbi MH, Almulaiky YQ et al (2017) Immobilization of horseradish peroxidase on Fe3O4 magnetic nanoparticles. Electron J Biotechnol 27:84–90. https://doi.org/10.1016/j.ejbt.2017.03.010
Oh AH, Park H-Y, Jung Y-G et al (2020) Synthesis of Fe3O4 nanoparticles of various size via the polyol method. Ceram Int 46:10723–10728. https://doi.org/10.1016/j.ceramint.2020.01.080
Petronijević M, Panić S, Savić S et al (2021) Characterization and application of biochar-immobilized crude horseradish peroxidase for removal of phenol from water. Colloids Surf B Biointerfaces 208:112038. https://doi.org/10.1016/j.colsurfb.2021.112038
Rank J (2003) The method of Allium anaphase-telophase chromosome aberration assay. Ekol Vilnius 49:38–42
Rizk HE, El-Hefny NE (2020) Synthesis and characterization of magnetite nanoparticles from polyol medium for sorption and selective separation of Pd(II) from aqueous solution. J Alloys Compd 812:152041. https://doi.org/10.1016/j.jallcom.2019.152041
Safarik I, Prochazkova J, Pospiskova K (2021) Rapid magnetic modification of diamagnetic particulate and high aspect ratio materials. J Magn Magn Mater 518:167430–167436. https://doi.org/10.1016/j.jmmm.2020.167430
Sahin S, Ozmen I (2020) Covalent immobilization of trypsin on polyvinyl alcohol-coated magnetic nanoparticles activated with glutaraldehyde. J Pharm Biomed Anal 184:113195. https://doi.org/10.1016/j.jpba.2020.113195
Samsam Shariat SZA, Movahedi M, Nazem H (2019) Immobilization of lactoperoxidase on Fe3O4 magnetic nanoparticles with improved stability. Biotechnol Lett 41:1373–1382. https://doi.org/10.1007/s10529-019-02741-y
Savić SR, Stojmenović SM, Petronijević MŽ, Petronijević ŽB (2014) Phenol removal from aqueous solutions by peroxidase extracted from horseradish. Appl Biochem Microbiol 50:214–218. https://doi.org/10.1134/S0003683814020161
Schoemaker HE, Mink D, Wubbolts MG (2003) Dispelling the myths-biocatalysis in industrial synthesis. Science 299:1694–1697. https://doi.org/10.1126/science.1079237
Shakerian F, Zhao J, Li S-P (2020) Recent development in the application of immobilized oxidative enzymes for bioremediation of hazardous micropollutants—a review. Chemosphere 239:124716. https://doi.org/10.1016/j.chemosphere.2019.124716
Sharma A, Vázquez LAB, Hernández EOM et al (2022) Green remediation potential of immobilized oxidoreductases to treat halo-organic pollutants persist in wastewater and soil matrices—a way forward. Chemosphere 290:133305. https://doi.org/10.1016/j.chemosphere.2021.133305
Torres JA, Silva MC, Lopes JH et al (2018) Development of a reusable and sustainable biocatalyst by immobilization of soybean peroxidase onto magnetic adsorbent. Int J Biol Macromol 114:1279–1287. https://doi.org/10.1016/j.ijbiomac.2018.03.136
WHO (1994) Phenol, environmental health criteria 161.
Worthington K, Worthington V (2011) Peroxidase—worthington enzyme manual | Worthington biochemical. https://www.worthington-biochem.com/products/peroxidase/manual. Accessed 29 Oct 2022
Xie X, Luo P, Han J et al (2019) Horseradish peroxidase immobilized on the magnetic composite microspheres for high catalytic ability and operational stability. Enzyme Microb Technol 122:26–35. https://doi.org/10.1016/j.enzmictec.2018.12.007
Xu L, Kim M-J, Kim K-D et al (2009) Surface modified Fe3O4 nanoparticles as a protein delivery vehicle. Colloids Surf Physicochem Eng Asp 350:8–12. https://doi.org/10.1016/j.colsurfa.2009.08.022
Xu R, Yuan J, Si Y et al (2016) Estrone removal by horseradish peroxidase immobilized on a nanofibrous support with Fe3O4 nanoparticles. RSC Adv 6:3927–3933. https://doi.org/10.1039/C5RA22805K
Yuan Z, Zhang G, Ma X et al (2021) A combined abiotic oxidation-precipitation process for rapid As removal from high-As(III)-Mn(II) acid mine drainage and low As-leaching solid products. J Hazard Mater 401:123360. https://doi.org/10.1016/j.jhazmat.2020.123360
Zdarta J, Jankowska K, Bachosz K et al (2021) Enhanced wastewater treatment by immobilized enzymes. Curr Pollut Rep 7:167–179. https://doi.org/10.1007/s40726-021-00183-7
Zdarta J, Jesionowski T, Pinelo M et al (2022) Free and immobilized biocatalysts for removing micropollutants from water and wastewater: recent progress and challenges. Bioresour Technol 344:126201. https://doi.org/10.1016/j.biortech.2021.126201
Zhang H, Hay AG (2020) Magnetic biochar derived from biosolids via hydrothermal carbonization: enzyme immobilization, immobilized-enzyme kinetics, environmental toxicity. J Hazard Mater 384:121272. https://doi.org/10.1016/j.jhazmat.2019.121272
Zhang H, Xue G, Chen H, Li X (2018) Magnetic biochar catalyst derived from biological sludge and ferric sludge using hydrothermal carbonization: preparation, characterization and its circulation in Fenton process for dyeing wastewater treatment. Chemosphere 191:64–71. https://doi.org/10.1016/j.chemosphere.2017.10.026
Zhang K, Yang W, Liu Y et al (2020) Laccase immobilized on chitosan-coated Fe3O4 nanoparticles as reusable biocatalyst for degradation of chlorophenol. J Mol Struct 1220:128769. https://doi.org/10.1016/j.molstruc.2020.128769
Acknowledgements
The authors gratefully acknowledge the support of the Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grant Numbers [OI 172059], [451-03-68/2022-14/200134], [451-03-47/2023-01/200134], [451-03-47/2023-01/200133]) and BioSense Institute in Novi Sad for the analytical support (Grant Number [739570] ANTARES).
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Petronijević, M., Panić, S., Stijepović, I. et al. Covalent immobilization of horseradish peroxidase onto PEG-coated magnetite nanoparticles: application in water treatment and toxicity assessment. Int. J. Environ. Sci. Technol. 21, 3899–3912 (2024). https://doi.org/10.1007/s13762-023-05252-6
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DOI: https://doi.org/10.1007/s13762-023-05252-6