Preparation and characterization of oxidized Multi-walled Carbon Nanotubes-Immobilized Aspergillus sp. Laccase Hybrid Materials

Haroun A.A.
Chemical Industries Research Division, National Research Centre, 12622 Dokki, Giza, Egypt
Masoud R.A.
Chemical Industries Research Division, National Research Centre, 12622 Dokki, Giza, Egypt


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This work deals with preparation and characterization of immobilized laccase (Aspergillus sp.) over oxidized multi-walled carbon nanotubes (ox-MWCNTs) via simple mixing technique. The resulting materials were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), transmission electron microscope (TEM) and particle size distribution analysis using dynamic light scattering technique (DLS). The results showed that the TEM images exhibited more separate individual carbon bundles with particle size around of 396 nm after enzyme immobilization rather than the spaghetti-like tubes with size about 180 nm in the case of ox-MWCNTs. Also, the lowering in the zeta potential negative value (-5 mv) proved that the free carboxyl groups at ox-MWCNT surface were decreased after enzyme immobilization. Moreover, the thermal stability was decreased after enzyme immobilization using TGA. These results confirmed that the laccase could be reacted at the side walls of the ox-MWCNTs without structure damage. The biocatalytic effect of the immobilized laccase was investigated after its incubation with silver nitrate solution for 1 and 24 h. It can be concluded that the biocatalytic efficiency of the immobilized laccase could be enhanced after its incubation with silver nitrate solution for 24 h at room temperature relative to the free form. On the other hand, the enzyme stability was improved after immobilization up to 50ºC and at pH 3.0, while no remarkable differences on the activity values were observed for immobilized and free laccases at acidic pH range (4-6).


  • Multi-walled carbon nanotubes,
  • Functionalization,
  • Biocatalysts,
  • Enzyme immobilization,
  • Laccase enzyme,
  • Enzyme stability
  • ...More


  1. TA Hilder, JM. Hill, Theoretical comparison of nanotube materials for drug delivery, IET Digital Library, 3 (2008) 18-24.
  2. A.A. Haroun, H.A.A. Taie, Cytotoxicity and antioxidant activity of beta vulgaris extract released from grafted carbon nanotubes-based nanocomposites, Macromolecular Symposia, 337 (2014) 25-33.
  3. E. Khaled, MS. Kamel, HNA. Hassan, A.A. Haroun, AM. Youssef, Hassan, Y. Aboul-Enein, Novel multi walled carbon nanotubes/β-cyclodextrin based carbon paste electrode for flow injection potentiometric determination of piroxicam, Talanta, 97 (2012) 96-102.
  4. S. Prakash, M. Malhotra, W. Shao, C. Tomaro-Duchesneau, S. Abbasi, Polymeric nanohybrids and functionalized carbon nanotubes as drug delivery carriers for cancer therapy, Advanced Drug Delivery Reviews, 63 (2011) 1340-1351.
  5. C.G. Silva, A.P.M. Tavares, G. Dražić, A.M.T. Silva, J.M. Loureiro, J.L. Faria, Controlling the surface chemistry of multiwalled carbon nanotubes for the production of highly efficient and stable laccase-based biocatalysts, Chem Plus Chem, 79 (2014) 1116-1122.
  6. Y. Amano, A. Koto, S. Matsuzaki, H. Sakamoto, T. Satomura, S.I. Suye, Construction of a biointerface on a carbon nanotube surface for efficient electron transfer, Materials Letters, 174 (2016) 184-187.
  7. Y. Fan, G. Wu, F. Su, K. Li, L. Xu, X. Han, Y. Yan, Lipase oriented-immobilized on dendrimer-coated magnetic multi-walled carbon nanotubes toward catalyzing biodiesel production from waste vegetable oil, Fuel, 178 (2016) 172-178.
  8. S. Gupta, C.R. Prabha, C.N. Murthy, Functionalized multi-walled carbon nanotubes/ polyvinyl alcohol membrane coated glassy carbon electrode for efficient enzyme immobilization and glucose sensing, Journal of environmental chemical engineering, 4 (2016) 3734-3740.
  9. C. Ji, J. Hou, V. Chen, Cross-linked carbon nanotubes-based biocatalytic membranes for micro-pollutants degradation: performance, stability, and regeneration, Journal of Membrane Science, 520 (2016) 869–880.
  10. A.A. Haroun, H.A. Taie, Preparation and rational biological evaluation of functionalized carbon nanotube with plant extracts. Proceeding at 2nd Int. Symposium on Materials and Sustainable Development, 9-10 November, Algeria, (2015).
  11. A.A. Haroun, F. Ayoob, E. Nashy, O. Mohamed, A. Rabi, Sol-gel preparation and in vitro kinetic release study of albendazole-immobilized MWCNTs, Egyptian Journal of Chemistry, 62 (2019) 645 – 654.
  12. F. Ayoob, A.A. Haroun, E. Nashy, O. Mohamed, S. Abdelshafy, A. Rabi, Preparation, characterization and in vitro toxicity study of antiparasitic drugs loaded onto functionalized MWCNTs, Egyptian Journal of Chemistry, 63 (2020) 3829-3836.
  13. A.A.Haroun, H.A. Amin, S.H. Abd El-Alim, Preparation, characterization and In vitro biological activity of soyasapogenol B loaded onto functionalized multi-walled carbon nanotubes, Current Bioactive Compounds, 14 (2018) 364-372.
  14. A.A. Haroun, H.A. Amin, S.H. Abd El-Alim, Imobilization and in vitro evaluation of soyasapogenol B onto functionalized multi-walled carbon nanotubes, IRBM 39 (2018) 35-42.
  15. A.A. Haroun, A.M. Elnahrawy, H.I. Abd-Alla, Sol-gel preparation and in vitro cytotoxic activity of nanohybrid structures based on multi-walled carbon nanotubes and silicate, Inorganic and Nano-Metal Chemistry, 47 (2017) 1023-1027.
  16. A.A. Haroun, B.M. Zaki, M. Shalash, R.A. Morsy, Preparation and Histological Study of Multi-Walled Carbon Nanotubes Bone Graft in Management of Class II Furcation Defects in Dogs, Open Access Macedonian Journal of Medical Sciences, 7 (2019) 3634-3641.
  17. A.A. Haroun, A.H. Mossa, S.M. Mohafrash, Preparation and biochemical evaluation of functionalized multi-walled carbon nanotubes with P. granatum extract, Current Bioactive Compounds, 5 (2019) 138-144.
  18. A.A. Haroun, Carbon Nanotubes as Innovative Materials for Bone Grafting Applications, Crimson PublishersWings to the Research, 2 (2019) 1-3.
  19. H.A. Amin, A.A. Haroun, Comparative studies of free and immobilized Aspergillus flavus onto functionalized multiwalled carbon nanotubes for soyasapogenol B production, Egyptian Pharmaceutical Journal, 16 (2017) 138-143.
  20. A.A. Haroun, E.F. Ahmed, M. Esawy, Immobilization and characterization of levansucrase enzyme onto functionalized multi-walled carbon nanotubes, Egyptian Journal of Chemistry, 61 (2018) 667-678.
  21. AA. Haroun, HM. Ahmed, EF. Ahmed, (2019) Functionalized multi-walled carbon nanotubes as emerging carrier for biological applications, Proceedings of the 5th World Congress on New Technologies.
  22. A.A. Haroun, H.M. Ahmed, A.H. Mossa, S.M. Mohafrash, E.F. Ahmed, Production, characterization and immobilization of Aspergillus versicolor L-asparaginase onto multi-walled carbon nanotubes, Biointerface Research in Applied Chemistry, 10 (2020) 5733-740.
  23. AM. Abdel Naby, MA. Hashem, MA. Esawy, Immobilisation of Bacillus subtilis œ-amylase.and characterization of its enzyme properties, Microbiological Research, 153 (1998) 319-325.
  24. A.L Cordeiro, T. Lenk, C. Werner, Immobilization of Bacillus licheniformis α-amylase onto reactive polymer films, Journal of Biotechnology, 154 (2011) 216-221.
  25. EA. Karam, WAA. Wahab, SAA. Saleh, ME. Hassan, AL. Kansoh, MA. Esawy, Production, immobilization and thermodynamic studies of free and immobilized Aspergillus awamori amylase, International Journal of Biological Macromolecules, 102 (2017) 694-703.
  26. H. Bisswanger, Enzyme assays review, Perspectives in Science, 1 (2014) 41–55.
  27. S. Sadeghzadeha, Z.G. Nejadb, S. Ghasemic, M. Khafajid, S.M. Borghei, Removal of bisphenol A in aqueous solution using magnetic crosslinked laccase aggregates from Trametes hirsute, Bioresource Technology, 306 (2020) 123-169.
  28. M. Masjoudi, M. Golgoli, Z.G. Nejad, S. Sadeghzadeh, S.M. Borghei, Pharmaceuticals removal by immobilized laccase on polyvinylidene fluoride nanocomposite with multi-walled carbon nanotubes, Chemosphere, 263 (2021) 128043.
  29. K.S. Muthuvelu, R. Rajarathinama, R.N. Selvaraj, V.B. Rajendren, A novel method for improving laccase activity by immobilization onto copper ferrite nanoparticles for lignin degradation, International Journal of Biological Macromolecules, 152 (2020) 1098–1107.
  30. X. Chen, B. He, M. Feng, D. Zhao, J. Sun, Immobilized laccase on magnetic nanoparticles for enhanced lignin model compounds degradation, Chinese Journal of Chemical Engineering, 28 (2020) 2152–2159.
  31. X. Qiua, S. Wanga, S. Miaoa, H. Suob, H. Xua, Y. Hu, Co-immobilization of laccase and ABTS onto amino-functionalized ionic liquid-modified magnetic chitosan nanoparticles for pollutants removal, Journal of Hazardous Materials, 401 (2021) 123353.
  32. C. Zhang, S. You, Y. Liu, C. Wang, Q. Yan, W. Qi, R. Su, Z. He, Construction of luffa sponge-based magnetic carbon nanocarriers for laccase immobilization and its application in the removal of bisphenol A, Bioresource Technology, 305 (2020) 123085.
  33. M. Bilal, Z. Jing, Y. Zhao, H.M.N. Iqbal, Immobilization of fungal laccase on glutaraldehyde crosslinked chitosan beads and its bio-catalytic potential to degrade bisphenol A, Biocatalysis and Agricultural Biotechnology, 19 (2019) 101174.
  34. C. Zhang, S. You, J. Zhang, W. Qi, R. Su, Z. He, An effective in-situ method for laccase immobilization: Excellent activity, effective antibiotic removal rate and low potential ecological risk for degradation products, Bioresource Technology, 308 (2020) 123271.
  35. J. Liu, X. Shen, Z. Zheng, M. Li, X. Zhuc, H. Cao, C. Cui, Immobilization of laccase by 3D bioprinting and its application in the biodegradation of phenolic compounds, International Journal of Biological Macromolecules, 164 (2020) 518–525.
  36. D. Wang, J. Lou, J. Yuan, J. Xu, R. Zhu, Q. Wang, X. Fan, Laccase immobilization on core-shell magnetic metal-organic framework microspheres for alkylphenol ethoxylate compound removal, Journal of Environmental Chemical Engineering, 9 (2021) 105000.
  37. W. Zhoua, W. Zhanga, Y. Cai, Laccase immobilization for water purification: A comprehensive review, Chemical Engineering Journal, 403 (2021) 126272.
  38. P. Cao, H. Liu, D. Wu, X. Wang, Immobilization of laccase on phase-change microcapsules as self-thermoregulatory enzyme carrier for biocatalytic enhancement, Chemical Engineering Journal, 405 (2021) 126695.
  39. Z. Lia, Z. Chena, Q. Zhua, J. Songa, S. Lia, X. Liu, Improved performance of immobilized laccase on Fe3O4@C-Cu2+ nanoparticles and its application for biodegradation of dyes, Journal of Hazardous Materials, 399 (2020) 123088.
  40. M. Primo, G. Kravanja, Z. Knez, A. Crnjac, M. Leitgeb, Immobilized laccase in the form of (magnetic) cross-linked enzyme aggregates for sustainable diclofenac (bio) degradation, Journal of Cleaner Production, 275 (2020) 124121.
  41. H. Yamaguchi, M. Miyazaki, Laccase aggregates via poly-lysine-supported immobilization onto PEGA resin, with efficient activity and high operational stability and can be used to degrade endocrine-disrupting chemicals, Catalysis Science and Technology, 11 (2020) 934-942.
  42. Y. Zhang, M. Piao, L. He, L. Yao, T. Piao, Z. Liua, Y. Piao, Immobilization of laccase on magnetically separable biochar for highly efficient removal of bisphenol A in water, RSC Advances, 10 (2020) 4795-4804.
  43. W. Feng, P. Ji, Enzymes immobilized on carbon nanotubes, Biotechnology Advances, 29 (2011) 889-895.
  44. G. Diamantidis, A. Effosse, P. Potier, R. Bally, Purification and characterization of the first bacterial laccase in rhizopheric bacteria Azospirillium lipoferum, Soil Biology and Biochemistry, s32 (2000) 919-927.
  45. O.V. Morozova, G.P. Shumakovich, M.A. Gorbacheva, S.V. Shleev, A.I.Yaropolov, Blue laccases, Biochemistry, 72 (2007) 1136-1150.
  46. M.W. Marshall, S. Popa-Nita, J.G. Shapter, Measurement of functionalized carbon nanotubes carboxylic acid groups using a simple chemical process, Carbon, 44 (2006) 1137-1141.
  47. P. habimana, J. Gao, J.P. Mwizerwa, J.B. Ndayambaje, H. Liu, P. Luan, L. Ma, Y. Jiang, Improvement of laccase activity via covalent immobilization over mesoporous silica coated magnetic multiwalled carbon nanotubes for the discoloration of synthetic dyes, ACS Omega 6 (2021) 2777–2789.
  48. H.J. Lee, S.J. Oh, J.Y. Choi, J. Kim, L.S. Han, J.B. Back, In situ synthesis of poly(ethylene terephthalate) (PET) in ethylene glycol containing terephthalic acid and functionalized multiwalled carbon nanotubes (MWNTs) as an approach to MWNT/PET nanocomposites, Chemistry of Materials, 17 (2005) 5057-5064.
  49. P. Baldrian, Fungal laccases - occurrence and properties, FEMS Microbiology Reviews, 30 (2006) 215-245.
  50. S.F. Zofair, A. Arsalan, M.A. Khan, F.A. Alhumaydhi, H. Younus, Immobilization of laccase on sepharose-liked antibody support for decolourization of phenol red, International Journal of Biological Macromolecules, 161 (2020) 78-87.
  51. N.A. Daronch, M. Kelbert, C.S. Pereira, D. de Olivera, Elucidating the choice of precise matrix for laccase immobilization, A Review, Chemical Engineering Journal, 397 (2020) 12506.
  52. F. Patrick, G. Mtui, A. M. Mshandete, G. Johansson, A. Kivaisi, Purification and characterization of a laccase from the basidiomycete Funalia trogii (Berk.) isolated in Tanzania, African Journal of Biochemistry Research, 3 (2009) 250-258.
  53. A. Daâssi, A. Prieto, H. Zouari-Mechichi, M. J. Martínez, M. Nasri, T. Mechichi, Degradation of bisphenol A by different fungal laccases and identification of its degradation products, International Biodeterioration & Biodegradation, 110 (2016) 181-188.
  54. D. Tuncay, H. Yagar, Decolorization of reactive blue-19 textile dye by Boletus edulis laccase immobilized onto rice husks, International Journal of Environmental Science and Technology, 17 (2020) 3177-3188.
  55. K.S. Muthuvelu, R. rajarathinam, R.N. Searaj, V.B.A. Rajendren, A novel method for improving laccase activity by immobilization onto copper ferrite nanoparticles for linin degradation, International Journal of Biological Macromolecules, 152 (2020) 1098-1107.
  56. S. Rouhani, S. Azizi, R.W. Kibechu, B.B. Mamba, T.A.M. Msagati, Laccase immobilized Fe3O4-grahene oxide nano-biocatalyst improve stability and immobilization efficiency in the green preparation of sulfa drugs, Catalysts, 10 (2020) 1-15.



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Volume 3, Issue 4, Year 2021

Published 2021-07-12


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