Immobilization of Laccase from Trametes hirsuta onto CMC Coated Magnetic Nanoparticles

Document Type : Original Article


1 Chemical & Petroleum Engineering Department, Sharif University of Technology, Tehran, Iran

2 Biochemical & Bioenvironmental Research Center, Sharif University of Technology, Azadi Avenue, Tehran, Iran


In this study, Fe3O4/CMC magnetic nanoparticles were synthesized through co-precipitation method. Afterward, laccase from Trametes hirsuta was immobilized onto Carboxymethyl cellulose (CMC)-coated magnetic Fe3O4 nanoparticles by covalent bonding between carboxyl groups of carboxymethyl cellulose and amine group of laccases. Also, the resulted magnetic nanoparticles and immobilized laccase were characterized by Fourier-transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and dynamic light scattering (DLS) analysis. Moreover, the vital factors in enzyme immobilization, such as contact time, amount of N-hydroxysuccinimide (NHS), and the amount of nanoparticles were optimized, which successively 48 h, 0.01 g, and 0.0125 g were achieved for 0.01g of N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC). Activity recovery of 51 ± 0.8% was achieved by optimizing the immobilization process. The results also indicated that the loading of laccase onto carboxymethyl cellulose-coated Fe3O4 nanoparticles was approximately 120 (mg/g). Finally, the immobilized laccases on magnetic support could save nearly 50% of their initial activity after five consecutive cycles.  


1. Sheldon, R.A., "Enzyme immobilization: The quest for optimum
performance", Advanced Synthesis & Catalysis,  Vol. 349, No.
8‐9, (2007), 1289-1307. 
2. Sheldon, R.A., "Cleas, combi-cleas and ‘smart’magnetic cleas:
Biocatalysis in a bio-based economy", Catalysts,  Vol. 9, No. 3,
(2019), 261. 
3. Heydarzadeh Darzi, H., Gilani, S., Farrokhi, M., Nouri, S. and
Karimi, G., "Textural and structural characterizations of
mesoporous chitosan beads for immobilization of alphaamylase:
Diffusivity and sustainability of biocatalyst",
International Journal of Engineering, Transactions B.
Applications,  Vol. 32, No. 2, (2019), 207-216. 
4. Arca-Ramos, A., Kumar, V., Eibes, G., Moreira, M. and Cabana,
H., "Recyclable cross-linked laccase aggregates coupled to
magnetic silica microbeads for elimination of pharmaceuticals
from municipal wastewater", Environmental Science and
Pollution Research,  Vol. 23, No. 9, (2016), 8929-8939. 
5. Cabana, H., Alexandre, C., Agathos, S.N. and Jones, J.P.,
"Immobilization of laccase from the white rot fungus coriolopsis
polyzona and use of the immobilized biocatalyst for the
continuous elimination of endocrine disrupting chemicals",
Bioresource Technology,  Vol. 100, No. 14, (2009), 3447-3458. 
6. Yang, J., Lin, Y., Yang, X., Ng, T.B., Ye, X. and Lin, J.,
"Degradation of tetracycline by immobilized laccase and the
proposed transformation pathway", Journal of Hazardous
Materials,  Vol. 322, (2017), 525-531. 
7. D'Annibale, A., Ricci, M., Quaratino, D., Federici, F. and
Fenice, M., "Panus tigrinus efficiently removes phenols, color
and organic load from olive-mill wastewater", Research
Microbiology,  Vol. 155, No. 7, (2004), 596-603. 
8. Chea, V., Paolucci-Jeanjean, D., Sanchez, J. and Belleville,
M.P., "Potentialities of a membrane reactor with laccase grafted
membranes for the enzymatic degradation of phenolic
compounds in water", Membranes (Basel),  Vol. 4, No. 4,
(2014), 678-691. 
9. Durán, N., Rosa, M.A., D’Annibale, A. and Gianfreda, L.,
"Applications of laccases and tyrosinases (phenoloxidases)
immobilized on different supports: A review", Enzyme and
Microbial Technology,  Vol. 31, No. 7, (2002), 907-931. 
10. JA, N., "Enzymatic treatment of waters and wastes. In: Tarr ma,
editor. Chemical degradation methods for wastes and pollutants:
Environmental and industrial applications, chapter 10, New
york,  (2003). 
11. Gilani, S., Najafpour, G., Moghadamnia, A. and Kamaruddin,
A., "Kinetics and isotherm studies of the immobilized lipase on
chitosan support", International Journal of EngineeringTransactions
12. Kavardi, S.S., Alemzadeh, I. and Kazemi, A., "Optimization of
lipase immobilization", International Journal of Engineering, 
Transactions C: Aspects, Vol. 25, No. 1, (2012), 1-9. 
13. Tavares, A.P., Silva, C.G., Dražić, G., Silva, A.M., Loureiro,
J.M. and Faria, J.L., "Laccase immobilization over multi-walled
carbon nanotubes: Kinetic, thermodynamic and stability
studies", Journal of Colloid and Interface Science,  Vol. 454,
(2015), 52-60. 
14. Mohammadi, M., As’habi, M.A., Salehi, P., Yousefi, M.,
Nazari, M. and Brask, J., "Immobilization of laccase on epoxyfunctionalized
silica and its application in biodegradation of
phenolic compounds", International Journal of Biological
Macromolecules,  Vol. 109, (2018), 443-447. 
15. Chen, W.-H., Vázquez-González, M., Zoabi, A., Abu-Reziq, R.
and Willner, I., "Biocatalytic cascades driven by enzymes
encapsulated in metal–organic framework nanoparticles",
Nature Catalysis,  Vol. 1, No. 9, (2018), 689-695. 
16. Lassouane, F., Aït-Amar, H., Amrani, S. and Rodriguez-Couto,
S., "A promising laccase immobilization approach for bisphenol
a removal from aqueous solutions", Bioresource Technology, 
Vol. 271, (2019), 360-367. 
17. Yang, J., Wang, Z., Lin, Y., Ng, T.B., Ye, X. and Lin, J.,
"Immobilized cerrena sp. Laccase: Preparation, thermal
inactivation, and operational stability in malachite green
decolorization", Scientific Reports,  Vol. 7, No. 1, (2017),
18. Datta, S., Christena, L.R. and Rajaram, Y.R., "Enzyme
immobilization: An overview on techniques and support
materials", 3 Biotech,  Vol. 3, No. 1, (2013), 1-9. 
19. Wang, Y., Chen, X., Liu, J., He, F. and Wang, R.,
"Immobilization of laccase by Cu(2+) chelate affinity interaction
on surface-modified magnetic silica particles and its use for the
removal of 2,4-dichlorophenol", Environmental Science
Pollution Research,  Vol. 20, No. 9, (2013), 6222-6231.  
20. Sheldon, R.A. and Woodley, J.M., "Role of biocatalysis in
sustainable chemistry", Chemical Reviews,  Vol. 118, No. 2,
(2017), 801-838. 
21. Shaarani, S.M., Jahim, J.M., Rahman, R.A., Idris, A., Murad,
A.M.A. and Illias, R.M., "Silanized maghemite for cross-linked
enzyme aggregates of recombinant xylanase from trichoderma
reesei", Journal of Molecular Catalysis B: Enzymatic,  Vol.
133, (2016), 65-76. 
22. Bayramoglu, G., Yilmaz, M. and Arica, M.Y., "Reversible
immobilization of laccase to poly(4-vinylpyridine) grafted and
Cu(ii) chelated magnetic beads: Biodegradation of reactive
dyes", Bioresource Technology,  Vol. 101, No. 17, (2010),
23. Xu, J., Sun, J., Wang, Y., Sheng, J., Wang, F. and Sun, M.,
"Application of iron magnetic nanoparticles in protein
immobilization", Molecules,  Vol. 19, No. 8, (2014), 1146511486.
24. Reza, R., Pérez, C.M., González, C.R., Romero, H. and Casillas,
P.G., "Effect of the polymeric coating over fe3o4 particles used
for magnetic separation", Open Chemistry,  Vol. 8, No. 5,
(2010), 1041-1046. 
25. Golizadeh, M., Karimi, A., Gandomi-Ravandi, S., Vossoughi,
M., Khafaji, M., Joghataei, M.T. and Faghihi, F., "Evaluation of
cellular attachment and proliferation on different surface
charged functional cellulose electrospun nanofibers",
Carbohydrate Polymers,  Vol. 207, (2019), 796-805. 
26. Orelma, H., Teerinen, T., Johansson, L.-S., Holappa, S. and
Laine, J., "Cmc-modified cellulose biointerface for antibody
conjugation", Biomacromolecules,  Vol. 13, No. 4, (2012),
27. Sitthichai, S., Pilapong, C., Thongtem, T. and Thongtem, S.,
"Cmc-coated fe3o4 nanoparticles as new mri probes for
hepatocellular carcinoma", Applied Surface Science,  Vol. 356,
(2015), 972-977. 
28. Nejad, Z.G., Borghei, S. and Yaghmaei, S., "Kinetic studies of
bisphenol a in aqueous solutions by enzymatic treatment",
International Journal of Environmental Science and
Technology,  Vol. 16, No. 2, (2019), 821-832. 
29. Ghobadi Nejad, Z., Borghei, S. and Yaghmaei, S., "Effect of
polyethylene glycol and triton x-100 on the enzymatic treatment
of bisphenol a", International Journal of Engineering,
Transactions B: Applications,  Vol. 31, No. 11, (2018), 18161823.
30. Sheldon, R.A. and van Pelt, S., "Enzyme immobilisation in
biocatalysis: Why, what and how", Chemical Society Reviews, 
Vol. 42, No. 15, (2013), 6223-6235. 
31. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J.,
"Protein measurement with the folin phenol reagent", Journal of
Biological Chemistry,  Vol. 193, (1951), 265-275. 
32. Chen, H., Zhou, J. and Deng, J., "Helical polymer/fe 3 o 4 nps
constructing optically active, magnetic core/shell microspheres:
Preparation by emulsion polymerization and recycling
application in enantioselective crystallization", Polymer
Chemistry,  Vol. 7, No. 1, (2016), 125-134. 
33. Cai, Y., Yuan, F., Wang, X., Sun, Z., Chen, Y., Liu, Z., Wang,
X., Yang, S. and Wang, S., "Synthesis of core–shell structured
@ carboxymethyl cellulose magnetic composite for highly
efficient removal of eu (iii)", Cellulose,  Vol. 24, No. 1, (2017),
34. Kumar, V.V. and Cabana, H., "Towards high potential magnetic
biocatalysts for on-demand elimination of pharmaceuticals",
Bioresource Technology,  Vol. 200, (2016), 81-89. 
35. Patel, S.K., Choi, S.H., Kang, Y.C. and Lee, J.-K., "Large-scale
aerosol-assisted synthesis of biofriendly fe 2 o 3 yolk–shell
particles: A promising support for enzyme immobilization",
Nanoscale,  Vol. 8, No. 12, (2016), 6728-6738. 
36. Wissink, M., Beernink, R., Pieper, J., Poot, A., Engbers, G.,
Beugeling, T., Van Aken, W. and Feijen, J., "Immobilization of
heparin to edc/nhs-crosslinked collagen. Characterization and in
vitro evaluation", Biomaterials,  Vol. 22, No. 2, (2001), 151163.
37. Sam, S., Touahir, L., Salvador Andresa, J., Allongue, P.,
Chazalviel, J.-N., Gouget-Laemmel, A., Henry de Villeneuve,
C., Moraillon, A., Ozanam, F. and Gabouze, N.,
"Semiquantitative study of the edc/nhs activation of acid
terminal groups at modified porous silicon surfaces", Langmuir, 
Vol. 26, No. 2, (2009), 809-814. 
38. Zhang, Y., Xu, J.-L., Yuan, Z.-H., Qi, W., Liu, Y.-Y. and He,
M.-C., "Artificial intelligence techniques to optimize the
edc/nhs-mediated immobilization of cellulase on eudragit l-100",
International Journal of Molecular Sciences,  Vol. 13, No. 7,
(2012), 7952-7962. 
39. Hou, J., Dong, G., Ye, Y. and Chen, V., "Laccase
immobilization on titania nanoparticles and titaniafunctionalized
membranes", Journal of Membrane Science, 
Vol. 452, (2014), 229-240. 
40. Park, J.-M., Kim, M., Park, J.-Y., Lee, D.-H., Lee, K.-H., Min, J.
and Kim, Y.-H., "Immobilization of the cross-linked paranitrobenzyl
esterase of bacillus subtilis aggregates on to magnetic beads",ProcessBiochemistry,Vol.45,No.2,(2010),259-263.