Skip to main content
SpringerLink
Log in

Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe3O4) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of Km and Vmax. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The Vmax values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg−1 min−1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13

Similar content being viewed by others

References

  1. Bornscheuer, U. T., & Angew. (2003). Chemie International Edition, 423, 336–337.

    Google Scholar 

  2. Sheldon, R. A. (1993). Chirotechnology: industrial synthesis of optically active compounds. New York: Marcel Dekker.

    Google Scholar 

  3. Mozhaev, V. V., Khmelnitsky, Y. L., Sergeeva, M. V., Belova, A. B., Klyachko, N. L., Levashov, A. V., & Martinek, K. (1989). Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures. European Journal of Biochemistry, 184, 597–602.

    Article  CAS  Google Scholar 

  4. Ozyilmaz, G., & Yağız, E. (2012). Isoamylacetate production by entrapped and covalently bound Candida rugosa and porcine pancreatic lipases. Food Chemistry, 135, 2326–2332.

    Article  CAS  Google Scholar 

  5. Berry, C. C., & Curtis, A. S. G. (2003). Functionalisation of magnetic nanoparticles for applications in biomedicine. Journal of Physics D: Applied Physics, 36, 198–206.

    Article  Google Scholar 

  6. Konerack_a, M., Kopčanský, P., Antalík, M., Ramchand, C. N., Lobo, D., Mehta, R. V., & Upadhyay, R. V. (1999). Immobilization of proteins and enzymes to fine magnetic particles. Journal of Magnetism and Magnetic Materials, 201, 427–430.

    Article  CAS  Google Scholar 

  7. Lee, J., Lee, Y., Youn, J., Na, H., Yu, T., Kim, H., Lee, S. M., Koo, Y. M., Kwak, J. H., Park, H. G., Chang, H. N., Hwang, M., Park, J. G., Kim, J., & Hyeon, T. (2008). Simple synthesis of functionalized superparamagnetic magnetite/silica core/shell nanoparticles and their application as magnetically separable high performance biocatalysts. Small, 4, 143–152.

    Article  CAS  Google Scholar 

  8. Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K., & Chauhan, B. (2003). Microbial α-amylases: a biotechnological perspective. Process Biochemistry, 38, 1599–1616.

    Article  CAS  Google Scholar 

  9. Kandra, L. (2003). a-Amylases of medical and industrial importance. Journal of Molecular Structure, 666, 487–498.

    Article  Google Scholar 

  10. Pandey, A., Nigam, P., Soccol, C. R., Soccol, V. T., Singh, D., & Mohan, R. (2000). Advances in microbial amylases. Applied Biochemistry and Biotechnology, 31, 135–152.

    Article  CAS  Google Scholar 

  11. Reddy, N. S., Nimmagadda, A., & Sambasiva Rao, K. R. S. (2003). An overview of the microbial a-amylase family. African Journal of Biotechnology, 2, 645–648.

    Article  CAS  Google Scholar 

  12. Kahraman, M. V., Bayramoglu, G., Kayaman, N. A., & Gungor, A. (2007). Alpha-amylase immobilization on functionalized glass beads by covalent attachment. Food Chemistry, 104, 1385–1392.

    Article  CAS  Google Scholar 

  13. Bellino, M. G., & Regazzoni, A. E. (2010). Amylase-functionalized mesoporous silica thin films as robust biocatalyst platforms. Applied Materials & Interfaces, 2, 360–365.

    Article  CAS  Google Scholar 

  14. Tripathi, P., Kumari, A., Rath, P., & Kayastha, A. M. (2007). Immobilization of α-amylase from mung beans on amberlite MB 150 and chitosan beads: a comparative study. Journal of Molecular Catalysis B: Enzymatic, 49, 69–74.

    Article  CAS  Google Scholar 

  15. Jaiswal, N., Prakash, O., Talat, M., Hasan, S. H., & Pandey, R. K. (2012). α-Amylase immobilizati on on gelatin: optimization of process variables. Journal Genetic Engineering and Biotechnology, 10, 161–167.

    Article  CAS  Google Scholar 

  16. Tumturk, H., Aksoy, S., & Hasirci, N. (2000). Covalent immobilization of alpha-amylase onto poly(2-hydroxyethyl methacrylate) poly(styerene-2-hydroxyethyl methacrylate) microspheres and the effect of Ca2+ ions on the enzyme activity. Food Chemistry, 68, 259–266.

    Article  CAS  Google Scholar 

  17. Pascoal, A. M., Mitidieri, S., & Fernandes, K. F. (2011). Immobilization of α-amylase from Aspergillus niger onto polyaniline. Food and Bioproducts Processing, 89, 300–306.

    Article  CAS  Google Scholar 

  18. Singh, V., & Ahmed, S. (2012). Silver nanoparticles (AgNPs) doped gum acacia–gelatin–silica nanohybrid: an effective support for diastase immobilization. International Journal of Biological Macromolecules, 50, 353–361.

    Article  CAS  Google Scholar 

  19. Eslamipour, F., & Hejazi, P. (2016). Evaluating effective factors on the activity and loading of immobilized a-amylase onto magnetic nanoparticles using a response surface-desirability approach. RSC Advances, 6, 20187–20197.

    Article  CAS  Google Scholar 

  20. Guo, H., Tang, Y., Yu, Y., Xue, L., & Qian, J. Q. (2016). Covalent immobilization of α-amylase on magnetic particles ascatalyst for hydrolysis of high-amylose starch. International Journal of Biological Macromolecules, 87, 537–544.

    Article  CAS  Google Scholar 

  21. Tuzmen, N., Kalburcu, T., & Denizli, A. (2012). α-Amylase immobilization onto dye attached magnetic beads: optimization and characterization. Journal of Molecular Catalysis B, 78, 16–23.

    Article  CAS  Google Scholar 

  22. Baskar, G., Afrin Banu, N., Helan Leuca, G., Gayathri, V., & Jeyashree, N. (2015). Magnetic immobilization and characterization of α-amylase as nanobiocatalyst for hydrolysis of sweet potato starch. Chemical Engineering Journal, 102, 18–23.

    CAS  Google Scholar 

  23. Motevalizadeh, S. F., Khoobi, M., Sadighi, A., Khalilvand-Sedagheh, M., Pazhouhandeh, M., Ramazani, A., Faramarzi, M. A., & Abbas Shafiee, A. (2015). Lipase immobilization onto polyethylenimine coated magnetic nanoparticles assisted by divalent metal chelated ions. Journal of Molecular Catalysis B, 120, 75–83.

    Article  CAS  Google Scholar 

  24. Chen, X., Lam, K. F., Zhang, Q., Pan, B., Arruebo, M., & Yeung, K. L. (2009). Synthesis of highly selective magnetic mesoporous adsorbent. Journal of Physical Chemistry, 113, 9804–9813.

    Article  CAS  Google Scholar 

  25. Jang, J., & Lim, H. (2010). Characterization analytical application of surface modified magnetic nanoparticles. Microchemical Journal, 94, 148–158.

    Article  CAS  Google Scholar 

  26. Abareshi, M., Goharshadi, E. K., Zebarjad, S. M., Fadafan, H. K., & Youssefi, A. (2010). Fabrication, characterization and measurement of thermal conductivity of Fe3O4 nanofluids. Journal of Magnetism and Magnetic Materials, 322, 3895–3901.

    Article  CAS  Google Scholar 

  27. Stober, W., Fink, A., & Bohn, E. J. (1968). Controlled growth of monodisperse silica spheres in the micron size range. Journal of Colloid and Interface Science, 26, 62–69.

    Article  Google Scholar 

  28. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  29. Bernfeld, P. (1955). α and β amylases. Methods in Enzymology, 1, 149–158.

    Article  CAS  Google Scholar 

  30. Sohrabi, N., Rasouli, N., & Torkzadeh, M. (2014). Chemical Engineering Journal, 240, 426–433.

    Article  CAS  Google Scholar 

  31. Ranjbakhsh, E., Bordbar, A. K., Abbasi, M., Khosropour, A. R., & Shams, E. (2012). Chemical Engineering Journal, 179, 272–276.

    Article  CAS  Google Scholar 

  32. Swarnalatha, V., Esther, R. A., & Dhamodharan, R. (2013). Journal of Molecular Catalysis B: Enzymatic, 96, 6–13.

    Article  CAS  Google Scholar 

  33. Singh, V., & Kumar, P. (2011). Carboxy methyl tamarind gum–silica nanohybrids for effective immobilization of amylase. Journal of Molecular Catalysis B: Enzymatic, 70, 67–73.

    Article  CAS  Google Scholar 

  34. Jahir Khan, A., Husain, Q., & Azam, A. (2012). Biotechnology and Bioprocess Engineering, 17, 377–384.

    Article  Google Scholar 

  35. Ramesh, M. V., & Lonsane, B. K. (1990). Effect of metal salts and protein modifying agents on activity of thermostable α-amylase produced by Bacillus licheniformis M27 under solid state fermentation. Chemie Mikrobiologie Technologie der Lebensmittel, 12, 129–136.

    CAS  Google Scholar 

  36. Lo, H., Lin, L., Chen, H., Hsu, W., & Chang, C. (2001). Enzymatic properties of a SDS-resistant Bacillus sp. TS-23 α-amylase produced by recombinant Escherichia coli. Process Biochemistry, 36, 743–750.

    Article  CAS  Google Scholar 

  37. Ai, Z., Jiang, Z., Li, L., Deng, W., Kusakabe, I., & Li, H. (2005). Process Biochemistry, 40, 2707–2714.

    Article  CAS  Google Scholar 

  38. Turunc, O., Kahraman, M. V., Akdemir, Z. S., Kayaman-Apohan, N., & Gungor, A. (2009). Immobilization of alpha-amylase onto cyclic carbonate bearing hybrid material. Food Chemistry, 112(4), 992–997.

    Article  CAS  Google Scholar 

  39. Dey, G., Nagpal, V., & Banerjee, R. (2002). Immobilization of alpha-amylase from Bacillus circulans grs 313 on coconut fiber. Applied Biochemistry and Microbiology, 102, 303–313.

    Google Scholar 

  40. Mukherjee, A., Kumar, T., Rai, S., & Roy, J. (2010). Biotechnology and Bioprocess Engineering, 15, 984–992.

    Article  CAS  Google Scholar 

  41. Sedaghat, M. E., Ghiaci, M., Aghaei, H., & Soleimanian-Zad, S. (2009). Immobilization of alpha-amylase on Na-bentonite and modified bentonite. Applied Clay Science, 46, 125–130.

    Article  CAS  Google Scholar 

  42. Swarnalatha, V., Esther, R. A., & Dhamodharan, R. (2013). Immobilized of a-amylase on gum acacia stabilized magnetite nanoparticles, an easily recoverable and reusable support. Journal \of Molecular Catalysis B: Enzymatic, 96, 6–13.

Download references

Acknowledgments

The authors wish to acknowledge the support of this work by Shiraz University Research Council.

Author information

Authors and Affiliations

  1. Professor Massoumi Laboratory, Department of Chemistry, Faculty of Sciences, Shiraz University, Shiraz, 71454, Iran

    M. Akhond, Kh. Pashangeh & G. Absalan

  2. Department of Biology, Faculty of Sciences, Shiraz University, Shiraz, 71454, Iran

    H. R. Karbalaei-Heidari

Authors
  1. M. Akhond
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kh. Pashangeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. R. Karbalaei-Heidari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Absalan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. Absalan.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Akhond, M., Pashangeh, K., Karbalaei-Heidari, H.R. et al. Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme. Appl Biochem Biotechnol 180, 954–968 (2016). https://doi.org/10.1007/s12010-016-2145-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-016-2145-1

Keywords

Search

Navigation