Immobilization of Cholesterol Oxidase from Streptomyces Sp. on Magnetite Silicon Dioxide by Crosslinking Method for Cholesterol Oxidation


Enzymatic biosensor has been paid much attention to the research fields due to its advantage in medical application. As one of the application, we determined the optimum value of cholesterol oxidase against cholesterol. In this work, we studied the behavior of cholesterol oxidation by enzymatic reaction to get the optimum condition for cholesterol oxidation. The enzyme that used were in two form, free cholesterol oxidase, and immobilized cholesterol oxidase. Cholesterol oxidase was produced from Streptomyces sp. by using solid state fermentation method and identified had high enzyme activity to be 5.12 U/mL. Cholesterol oxidase was simultaneously crosslinked immobilized onto magnetite coated by silicon dioxide (M-SiO2). The support was characterized by Fourier transform infrared (FTIR) to determine the functional group of modified particle and scanning electron microscope (SEM) to observe the morphological or our prepared particle. Cholesterol oxidase sensitivity to substrate was analyzed by using HPLC with different interval time measurements. The oxidation of cholesterol by free enzyme and immobilized enzyme was also investigated. The best sensitivity of cholesterol oxidase was estimated to oxidize Cso (concentration of substrate) 1.46 mM of substrate with Ce (concentration of enzyme) 20 mg/mL for 180 min. Final oxidation value of cholesterol by immobilized enzyme was greater than 60%. The results of this study revealed that immobilized enzyme for cholesterol oxidation was stable, reproducible, and sensitive.

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

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5



Concentration of enzyme (mg/mL)


Final concentration of substrate (mM)


Initial concentration of substrate (mM)


  1. 1.

    Neuvonen, M., et al. (2014). Enzymatic oxidation of cholesterol: properties and functional effects of cholestenone in cell membranes. PLoS One, 9(8), e103743.

  2. 2.

    Nawaz, M. A. H., et al. (2018). Development of a disposable electrochemical sensor for detection of cholesterol using differential pulse voltammetry. Journal of Pharmaceutical and Biomedical Analysis, 159, 398–405.

  3. 3.

    Gao, J., et al. (2019). Simultaneous detection of glucose, uric acid and cholesterol using flexible microneedle electrode array-based biosensor and multi-channel portable electrochemical analyzer. Sensors and Actuators B: Chemical, 287, 102–110.

  4. 4.

    Wang, K., et al. (2018). Ratiometric fluorescence sensor based on cholesterol oxidase-functionalized mesoporous silica nanoparticle@ ZIF-8 core-shell nanocomposites for detection of cholesterol. Talanta, 188, 708–713.

  5. 5.

    Huang, J., et al. (2015). Immobilization of cholesterol oxidase on magnetic fluorescent core-shell-structured nanoparticles. Materials Science and Engineering: C, 57, 31–37.

  6. 6.

    Pashangeh, K., et al. (2017). Biochemical characterization and stability assessment of Rhizopus oryzae lipase covalently immobilized on amino-functionalized magnetic nanoparticles. International Journal of Biological Macromolecules, 105, 300–307.

  7. 7.

    MacLachlan, J., et al. (2000). Cholesterol oxidase: sources, physical properties and analytical applications. The Journal of Steroid Biochemistry and Molecular Biology, 72(5), 169–195.

  8. 8.

    Vrielink, A., & Ghisla, S. (2009). Cholesterol oxidase: biochemistry and structural features. The FEBS Journal, 276(23), 6826–6843.

  9. 9.

    Doukyu, N., & Nihei, S. (2015). Cholesterol oxidase with high catalytic activity from Pseudomonas aeruginosa: screening, molecular genetic analysis, expression and characterization. Journal of Bioscience and Bioengineering, 120(1), 24–30.

  10. 10.

    Chen, Y., et al. (2013). Immobilization and stabilization of cholesterol oxidase on modified sepharose particles. International Journal of Biological Macromolecules, 56, 6–13.

  11. 11.

    Gholivand, M. B., & Khodadadian, M. (2014). Amperometric cholesterol biosensor based on the direct electrochemistry of cholesterol oxidase and catalase on a graphene/ionic liquid-modified glassy carbon electrode. Biosensors and Bioelectronics, 53, 472–478.

  12. 12.

    Wisitsoraat, A., et al. (2009). High sensitivity electrochemical cholesterol sensor utilizing a vertically aligned carbon nanotube electrode with electropolymerized enzyme immobilization. Sensors, 9(11), 8658–8668.

  13. 13.

    Ghosh, S., Ahmad, R., & Khare, S. (2019). Refolding of thermally denatured cholesterol oxidases by magnetic nanoparticles. International Journal of Biological Macromolecules, 138, 958–965.

  14. 14.

    Feng, B., & Liu, Y.-N. (2015). A disposable cholesterol enzyme biosensor based on ferrocene-capped gold nanoparticle modified screen-printed carbon electrode. International Journal of Electrochemical Science, 10, 4770–4778.

  15. 15.

    Ghosh, S., et al. (2018). Cholesterol-oxidase-magnetic nanobioconjugates for the production of 4-cholesten-3-one and 4-cholesten-3, 7-dione. Bioresource Technology, 254, 91–96.

  16. 16.

    Kulkarni, S. A., Sawadh, P., & Palei, P. K. (2014). Synthesis and characterization of superparamagnetic Fe 3 O 4@ SiO 2 nanoparticles. Journal of the Korean Chemical Society, 58(1), 100–104.

  17. 17.

    Zhai, Y., et al. (2016). Magnetic, fluorescence and transition metal ion response properties of 2, 6-diaminopyridine modified silica-coated fe3o4 nanoparticles. Molecules, 21(8), 1066.

  18. 18.

    Yang, L., et al. (2018). Modification and characterization of Fe3O4 nanoparticles for use in adsorption of alkaloids. Molecules, 23(3), 562.

  19. 19.

    Šulek, F., et al. (2010). Surface functionalization of silica-coated magnetic nanoparticles for covalent attachment of cholesterol oxidase. Journal of Magnetism and Magnetic Materials, 322(2), 179–185.

  20. 20.

    Dervisevic, M., et al. (2016). Amperometric cholesterol biosensor based on reconstituted cholesterol oxidase on boronic acid functional conducting polymers. Journal of Electroanalytical Chemistry, 776, 18–24.

  21. 21.

    Oliveira, C. L., et al. (2014). Effect of oxidation on the structure of human low-and high-density lipoproteins. Biophysical Journal, 106(12), 2595–2605.

  22. 22.

    Pinheiro, P., et al. (2013). Fluorescent magnetic bioprobes by surface modification of magnetite nanoparticles. Materials, 6(8), 3213–3225.

  23. 23.

    Li, D., et al. (2019). Screening and analysis of cyclooxygenase-2 inhibitors from the complex matrix: a case study to illustrate the important effect of immobilized enzyme activity in magnetic ligand fishing. Journal of Pharmaceutical and Biomedical Analysis, 175, 112,795.

  24. 24.

    Kim, M. G., & Lee, S. B. (1996). Enhanced catalytic efficiency of enzymes in organic media with the addition of the solid support: effect of silica gel on reaction rates and enzyme agglomeration. Journal of Molecular Catalysis B: Enzymatic, 2(2–3), 127–140.

  25. 25.

    Temani, M., Baccar, Z. M., & Mansour, H. B. (2014). Activity of cholesterol oxidase immobilized on layered double hydroxide nanomaterials for biosensor application: Acacia salicina scavenging power of hypercholesterolemia therapy. Microelectronic Engineering, 126, 165–168.

Download references

Author information

Correspondence to Heri Hermansyah.

Ethics declarations

This chapter does not contain any studies with human participants or animals performed by any of the authors. For this type of study formal consent is not required.

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The presenting author of this manuscript in ACB2019 is Heri Hermansyah

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Perdani, M.S., Sahlan, M., Yohda, M. et al. Immobilization of Cholesterol Oxidase from Streptomyces Sp. on Magnetite Silicon Dioxide by Crosslinking Method for Cholesterol Oxidation. Appl Biochem Biotechnol (2020).

Download citation


  • Biosensor
  • Cholesterol
  • Cholesterol oxidase
  • Enzymatic
  • Magnetite
  • Oxidation