Applied Biochemistry and Biotechnology

, Volume 186, Issue 1, pp 174–185 | Cite as

Prominent Study on Surface Properties and Diffusion Coefficient of Urease-Conjugated Magnetite Nanoparticles

  • Carlin Geor Malar
  • Muthulingam SeenuvasanEmail author
  • Kannaiyan Sathish KumarEmail author


Herein, the magnetite nanoparticles (MNs) were prepared by facile solvothermal method and its porous nature was modified using 3-(2-aminoethyl)-3-aminopropyl trimethoxysilane (AEAPS). Magnetite formation, successful amino tagging, and urease conjugation on the surface were confirmed from the presence of certain functional groups in Fourier transform infrared (FT-IR) spectra. Also, nanosize (13.2 nm) and spherical morphology of MNs were evaluated from diffraction patterns and electron micrographs respectively. Lower retentivity and coercivities in magnetization curve revealed the superparamagnetic behavior, and nitrogen adsorption/desorption curves exhibited decrease in its surface porosity. Conductivity measurements showed lower diffusion coefficient (De = 1.9 × 10−17 cm2/min) and higher diffusion with limited hydrolytic reaction in native urease and improved activity of conjugated urease with higher De (12.62 × 10−16 cm2/min). Hence, this study revealed that the surface porous nature of MNs can be altered effectively by amino tagging in order to overcome diffusional limitations thereby enhancing enzyme activity.


Magnetite Urease Activity Porosity Diffusion coefficient Diffusional limitations 


Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Mateo, C., Palomo, J. M., Fernandez-Lorente, G., Guisan, J. M., & Fernandez-Lafuente, R. (2007). Improvement of enzyme activity, stability and selectivity via immobilization techniques. Enzyme and Microbial Technology, 40(6), 1451–1463.CrossRefGoogle Scholar
  2. 2.
    Gur, S. D., Idil, N., & Aksoz, N. (2018). Optimization of enzyme co-immobilization with sodium alginate and glutaraldehyde-activated chitosan beads. Applied Biochemistry and Biotechnology, 184(2), 538–552.CrossRefPubMedGoogle Scholar
  3. 3.
    Gabrielczyk, J., Duensing, T., Buchholz, S., Schwinges, A., Jordening, H.J. (2018). A comparative study on immobilization of fructosyl transferase in biodegradable polymers by electrospinning. Applied Biochemistry and Biotechnology, 1–16.
  4. 4.
    Zou, B., Chu, Y., Xia, J., Chen, X., Huo, S. (2017). Immobilization of lipase by ionic liquid-modified mesoporous SiO2 adsorption and calcium alginate-embedding method. Applied Biochemistry and Biotechnology, 1–13.
  5. 5.
    Sheldon, R. A. (2007). Enzyme immobilization: the quest for optimum performance. Advanced Synthesis and Catalysis, 349(8-9), 1289–1307.CrossRefGoogle Scholar
  6. 6.
    Brady, D., & Jordaan, J. (2009). Advances in enzyme immobilization. Biotechnology Letters, 31(11), 1639–1650.CrossRefPubMedGoogle Scholar
  7. 7.
    Talbert, J. N., & Goddard, J. M. (2013). Influence of nanoparticle diameter on conjugated enzyme activity. Food and Bioproducts Processing, 91(4), 693–699.CrossRefGoogle Scholar
  8. 8.
    Eldin, M. S. M., & Mita, D. G. (2014). Immobilized enzymes: strategies for overcoming the substrate diffusion limitation problem. Current Biotechnology, 3(3), 207–217.CrossRefGoogle Scholar
  9. 9.
    Jia, H., Zhu, G., & Wang, P. (2003). Catalytic behaviours of enzymes attached to nanoparticles: the effect of particle mobility. Biotechnology and Bioengineering, 84(4), 406–414.CrossRefPubMedGoogle Scholar
  10. 10.
    Wang, P., Sergeeva, M. V., Lim, L. K., & Dordick, J. S. (1997). Biocatalytic plastics as active and stable materials for biotransformations. Nature Biotechnology, 15(8), 789–793.CrossRefPubMedGoogle Scholar
  11. 11.
    Rodrigues, R. C., Ortiz, C., Berenguer-Murcia, A., Torres, R., & Fernandez-Lafuente, R. (2013). Modifying enzyme activity and selectivity by immobilization. Chemical Society Reviews, 42(15), 6290–6307.CrossRefPubMedGoogle Scholar
  12. 12.
    Caldas, E. M., Novatzky, D., Deon, M., de Menezes, E. W., Hertz, P. F., Costa, T. M. H., Arenas, L. T., & Benvenutti, E. V. (2017). Pore size effect in the amount of immobilized enzyme for manufacturing carbon ceramic biosensor. Microporous and Mesoporous Materials, 247, 95–102.CrossRefGoogle Scholar
  13. 13.
    Prati, L., Bergna, D., Villa, A., Spontoni, P., Bianchi, C. L., Hu, T., Romar, H., & Lassi, U. (2017). Carbons from second generation biomass as sustainable supports for catalytic systems. Catalysis Today, 301, 239–243. Scholar
  14. 14.
    Janssen, M. H., van Langen, L. M., Pereira, S. R., van Rantwijk, F., & Sheldon, R. A. (2002). Evaluation of the performance of immobilized penicillin G acylase using active-site titration. Biotechnology and Bioengineering, 78(4), 425–432.CrossRefPubMedGoogle Scholar
  15. 15.
    Hudson, S., Cooney, J., & Magner, E. (2008). Proteins in mesoporous silicates. Angewandte Chemie, International Edition, 47(45), 8582–8594.CrossRefGoogle Scholar
  16. 16.
    Garcia, P. R. A. F., Bicev, R. N., Oliveira, C. L. P., Sant’Anna, O. A., & Fantini, M. C. A. (2016). Protein encapsulation in SBA-15 with expanded pores. Microporous and Mesoporous Materials, 235, 59–68.CrossRefGoogle Scholar
  17. 17.
    Magario, I., Ma, X., Neumann, A., Syldatk, C., & Hausmann, R. (2008). Non-porous magnetic micro-particles: comparison to porous enzyme carriers for a diffusion rate-controlled enzymatic conversion. Journal of Biotechnology, 134(1-2), 72–78.CrossRefPubMedGoogle Scholar
  18. 18.
    Seenuvasan, M., Kumar, K. S., Malar, C. G., Preethi, S., Kumar, M. A., & Balaji, N. (2014). Characterization, analysis, and application of fabricated Fe3O4-chitosan-pectinase nanobiocatalyst. Applied Biochemistry and Biotechnology, 172(5), 2706–2719.CrossRefPubMedGoogle Scholar
  19. 19.
    Keyhanian, F., Shariati, S., Faraji, M., & Hesabi, M. (2016). Magnetite nanoparticles with surface modification for removal of methyl violet from aqueous solutions. Arabian Journal of Chemistry, 9, S348–S354.CrossRefGoogle Scholar
  20. 20.
    Li, C., Wei, Y., Liivat, A., Zhu, Y., & Zhu, J. (2013). Microwave-solvothermal synthesis of Fe3O4 magnetic nanoparticles. Materials Letters, 107, 23–26.CrossRefGoogle Scholar
  21. 21.
    Seenuvasan, M., Vinodhini, G., Malar, C. G., Balaji, N., & Kumar, K. S. (2017). Magnetic nanoparticles: a versatile carrier for enzymes in bio-processing sectors. IET Nanobiotechnology.
  22. 22.
    Yanez-Vilar, S., Sanchez-Andujar, M., Gomez-Aguirre, C., Mira, J., Sennaris-Rodríguez, M. A., & Castro-Garcia, S. (2009). A simple solvothermal synthesis of MFe2O4 (M=Mn, Co and Ni) nanoparticles. Journal of Solid State Chemistry, 182, 2585–2690.CrossRefGoogle Scholar
  23. 23.
    Sahoo, B., Sahu, S. K., Nayak, S., Dhara, D., & Pramanik, P. (2012). Fabrication of magnetic mesoporous manganese ferrite nanocomposites as efficient catalyst for degradation of dye pollutants. Catalysis Science & Technology, 2(7), 1367–1374.CrossRefGoogle Scholar
  24. 24.
    Zhang, Z. L., Wang, Y. H., Tan, Q. Q., Zhong, Z. Y., & Su, F. B. (2013). Facile solvothermal synthesis of mesoporous manganese ferrite (MnFe2O4) microspheres as anode materials for lithium-ion batteries. Journal of Colloid and Interface Science, 398, 185–192.CrossRefPubMedGoogle Scholar
  25. 25.
    Wang, T., Zhang, L. Y., Wang, H. Y., Yang, W. C., Fu, Y. C., Zhou, W. L., Yu, W. T., Xiang, K. S., Sun, Z., Dai, S., & Chai, L. Y. (2013). Controllable synthesis of hierarchical porous Fe3O4 particles mediated by poly(diallyldimethylammonium chloride) and their application in arsenic removal. ACS Applied Materials & Interfaces, 5(23), 12449–12459.CrossRefGoogle Scholar
  26. 26.
    Li, S., Zhang, T., Tang, R., Qiu, H., & Wang, C. (2015). Solvothermal synthesis and characterization of monodisperse superparamagnetic iron oxide nanoparticles. Journal of Magnetism and Magnetic Materials, 379, 226–231.CrossRefGoogle Scholar
  27. 27.
    Seenuvasan, M., Malar, C. G., Preethi, S., Balaji, N., Iyyappan, J., Kumar, M. A., & Kumar, K. S. (2013). Immobilization of pectinase on co-precipitated magnetic nanoparticles for enhanced stability and activity. Research Journal of Biotechnology, 8, 24–30.Google Scholar
  28. 28.
    Seenuvasan, M., Malar, C. G., Preethi, S., Balaji, N., Iyyappan, J., Kumar, M. A., & Kumar, K. S. (2013). Fabrication, characterization and application of pectin degrading Fe3O4-SiO2 nanobiocatalyst. Materials Science & Engineering. C, Materials for Biological Applications, 33(4), 2273–2279.CrossRefGoogle Scholar
  29. 29.
    Hwang, S., Lee, K., Park, J., Min, B., Haam, S., Ahn, I., & Jung, J. (2004). Stability analysis of Bacillus stearothermophilus L1 lipase immobilized on surface-modified silica gels. Biochemical Engineering Journal, 17(2), 85–90.CrossRefGoogle Scholar
  30. 30.
    Fernandez-Lafuente, R., Rossell, C. M., Rodrigeuez, V., & Guisan, J. M. (1995). Strategies for enzyme stabilization by intramolecular crosslinking with bifunctional reagents. Enzyme and Microbial Technology, 17(6), 517–523.CrossRefGoogle Scholar
  31. 31.
    Visuri, K., Pastinen, O., Wu, X., Makinen, K., & Leisola, M. (1999). Stability of native and cross-linked crystalline glucose isomerase. Biotechnology and Bioengineering, 64(3), 377–380.CrossRefPubMedGoogle Scholar
  32. 32.
    Govardhan, C. P. (1999). Crosslinking of enzymes for improved stability and performance. Current Opinion in Biotechnology, 10(4), 331–335.CrossRefPubMedGoogle Scholar
  33. 33.
    Schoevaart, R., Wolbers, M. W., Golubovic, M., Ottens, M., Kieboom, A. P. G., van Rantwijk, F., van der Wielen, L. A., & Sheldon, R. A. (2004). Preparation, optimization, and structures of crosslinked enzyme aggregates (CLEAs). Biotechnology and Bioengineering, 87(6), 754–762.CrossRefPubMedGoogle Scholar
  34. 34.
    Karimzadeh, I., Dizaji, H. R., & Aghazadeh, M. (2016). Development of a facile and effective electrochemical strategy for preparation of iron oxides (Fe3O4 and γ-Fe2O3) nanoparticles from aqueous and ethanol mediums and in situ PVC coating of Fe3O4 superparamagnetic nanoparticles for biomedical applications. Journal of Magnetism and Magnetic Materials, 416, 81–88.CrossRefGoogle Scholar
  35. 35.
    Atacan, K., Cakiroglu, B., & Ozacar, M. (2017). Efficient protein digestion using immobilized trypsin onto tannin modified Fe3O4 magnetic nanoparticles. Colloids and Surfaces B, 156, 9–18. Scholar
  36. 36.
    Karimzadeh, I., Aghazadeh, M., Doroudi, T., Ganjali, M. R., & Kolivand, P. H. (2017). Superparamagnetic iron oxide (Fe3O4) nanoparticles coated with PEG/PEI for biomedical applications: a facile and scalable preparation route based on the cathodic electrochemical deposition method. Advances in Physical Chemistry, 2017, 1–7. Scholar
  37. 37.
    Asgari, S., Fakhari, Z., & Berijani, S. (2014). Synthesis and characterization of Fe3O4 magnetic nanoparticles coated with carboxymethyl chitosan grafted sodium methacrylate. Journal Nanostructured, 4, 55–63.Google Scholar
  38. 38.
    Akbari, B., Tavadashti, M. P., & Zandrahimi, M. (2011). Particle size characterization of nanoparticles—a practical approach. Iranian Journal of Materials Science and Engineering, 8, 48–56.Google Scholar
  39. 39.
    Grunwald, P. (1989). Determination of effective diffusion coefficients an important parameter for the efficiency of immobilized biocatalysts. Biochemical Education, 17(2), 99–102.CrossRefGoogle Scholar
  40. 40.
    Tanaka, H., Matsumura, M., & Veliky, I.A. (1984). Diffusion characteristics of substrates in Ca-alginate gel beads, Biotechnology and Bioengineering 26, 053–058, 1, 53.Google Scholar
  41. 41.
    Sant, S., Nadeau, V., & Hildgen, P. (2005). Effect of porosity on the release kinetics of propafenone-loaded PEG-g-PLA nanoparticles. Journal of Controlled Release, 107(2), 203–214.CrossRefPubMedGoogle Scholar
  42. 42.
    Oh, J., & Kim, J. (2000). Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres. Enzyme and Microbial Technology, 27(6), 356–361.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Chemical EngineeringSSN College of EngineeringKalavakkamIndia
  2. 2.Department of Petrochemical EngineeringSVS College of EngineeringCoimbatoreIndia

Personalised recommendations