Skip to main content
SpringerLink
Log in

Impacts of Magnetic Immobilization on the Growth and Metabolic Status of Recombinant Pichia pastoris

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Downstream processing is an expensive step for industrial production of recombinant proteins. Cell immobilization is known as one of the ideal solutions in regard to process intensification. In recent years, magnetic immobilization was introduced as a new technique for cell immobilization. This technique was successfully employed to harvest many bacterial and eukaryotic cells. But there are no data about the influence of magnetic immobilization on the eukaryotic inducted recombinant cells. In this study, impacts of magnetic immobilization on the growth and metabolic status of induced recombinant Pichia pastoris as a valuable eukaryotic model cells were investigated. Results based on colony-forming unit, OD600, and trypan blue assay indicated that magnetic immobilization had no adverse effect on the growth and viability of P. pastoris cells. Also, about 20–40% increase in metabolic activity was recorded in immobilized cells that were decorated with 0.5–2 mg/mL nanoparticles. Total protein and carbohydrate of the cells were also measured as main indicatives for cell function and no significant changes were observed in the immobilized cells. Current data show magnetic immobilization as a biocompatible technique for application in eukaryotic expression systems. Results can be considered for further developments in P. pastoris-based expression systems.

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

Similar content being viewed by others

References

  1. Daly, R., & Hearn, M. T. (2005). Expression of heterologous proteins in Pichia pastoris: A useful experimental tool in protein engineering and production. Journal of Molecular Recognition, 18, 119–138.

    Article  CAS  Google Scholar 

  2. Song, C. P., Liew, P. E., Teh, Z., Lim, S. P., Show, P. L., & Ooi, C. W. (2018). Purification of the recombinant green fluorescent protein using aqueous two-phase system composed of recyclable CO2-based alkyl carbamate ionic liquid. Frontiers in Chemistry, 6, 529.

    Article  CAS  Google Scholar 

  3. Wang, Y., Ling, C., Chen, Y., Jiang, X., & Chen, G.-Q. (2019). Microbial engineering for easy downstream processing. Biotechnology Advances, 37, 107365.

    Article  CAS  Google Scholar 

  4. Ebrahiminezhad, A., Varma, V., Yang, S., & Berenjian, A. (2016). Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation. Applied Microbiology and Biotechnology, 100, 173–180.

    Article  CAS  Google Scholar 

  5. Taghizadeh, S.-M., Berenjian, A., Chew, K. W., Show, P. L., Mohd Zaid, H. F., Ramezani, H., et al. (2020). Impact of magnetic immobilization on the cell physiology of green unicellular algae Chlorella vulgaris. Bioengineered, 11, 141–153.

    Article  CAS  Google Scholar 

  6. Raee, M. J., Ebrahiminezhad, A., Gholami, A., Ghoshoon, M. B., & Ghasemi, Y. (2018). Magnetic immobilization of recombinant E. coli producing extracellular asparaginase: An effective way to intensify downstream process. Separation Science and Technology, 53, 1397–1404.

    Article  CAS  Google Scholar 

  7. Safarik, I., Maderova, Z., Pospiskova, K., Baldikova, E., Horska, K., & Safarikova, M. (2015). Magnetically responsive yeast cells: Methods of preparation and applications. Yeast, 32, 227–237.

    CAS  PubMed  Google Scholar 

  8. Safarik, I., Pospiskova, K., Maderova, Z., Baldikova, E., Horska, K., & Safarikova, M. (2015). Microwave-synthesized magnetic chitosan microparticles for the immobilization of yeast cells. Yeast, 32, 239–243.

    CAS  PubMed  Google Scholar 

  9. Firoozi, F. R., Raee, M. J., Lal, N., Ebrahiminezhad, A., Teshnizi, S. H., Berenjian, A., et al. (2021). Application of magnetic immboilization for ethanol biosynthesis using Saccharomyces cerevisiae. Separation Science and Technology. https://doi.org/10.1080/01496395.2021.19393761-11

    Article  Google Scholar 

  10. Taghizadeh, S.-M., Ebrahiminezhad, A., Ghoshoon, M. B., Dehshahri, A., Berenjian, A., & Ghasemi, Y. (2020). Magnetic Immobilization of Pichia pastoris cells for the production of recombinant human serum albumin. Nanomaterials, 10, 111.

    Article  CAS  Google Scholar 

  11. Peng, Q., Huo, D., Li, H., Zhang, B., Li, Y., Liang, A., et al. (2018). ROS-independent toxicity of Fe3O4 nanoparticles to yeast cells: Involvement of mitochondrial dysfunction. Chemico-Biological Interactions, 287, 20–26.

    Article  CAS  Google Scholar 

  12. Otero-González, L., García-Saucedo, C., Field, J. A., & Sierra-Álvarez, R. (2013). Toxicity of TiO2, ZrO2, Fe0, Fe2O3, and Mn2O3 nanoparticles to the yeast, Saccharomyces cerevisiae. Chemosphere, 93, 1201–1206.

    Article  Google Scholar 

  13. Luo, F., Zhu, S., Hu, Y., Yang, K.-C., He, M.-S., Zhu, B., et al. (2020). Biocompatibility assessment of Fe3O4 nanoparticles using Saccharomyces cerevisiae as a model organism. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 227, 108645.

    CAS  Google Scholar 

  14. Bhargava, P., & Collins, J. J. (2015). Boosting bacterial metabolism to combat antibiotic resistance. Cell Metabolism, 21, 154–155.

    Article  CAS  Google Scholar 

  15. Cabral, D. J., Penumutchu, S., Reinhart, E. M., Zhang, C., Korry, B. J., Wurster, J. I., et al. (2019). Microbial metabolism modulates antibiotic susceptibility within the murine gut microbiome. Cell Metabolism, 30, 800–823.

    Article  CAS  Google Scholar 

  16. Stokes, J. M., Lopatkin, A. J., Lobritz, M. A., & Collins, J. J. (2019). Bacterial metabolism and antibiotic efficacy. Cell Metabolism, 30, 251–259.

    Article  CAS  Google Scholar 

  17. Ebrahiminezhad, A., Ghasemi, Y., Rasoul-Amini, S., Barar, J., & Davaran, S. (2013). Preparation of novel magnetic fluorescent nanoparticles using amino acids. Colloids and Surfaces B, 102, 534–539.

    Article  CAS  Google Scholar 

  18. Liu, P., Wang, X., Hiltunen, K., & Chen, Z. (2015). Controllable drug release system in living cells triggered by enzyme–substrate recognition. ACS Applied Materials & Interfaces, 7, 26811–26818.

    Article  CAS  Google Scholar 

  19. Olson, B. J., & Markwell, J. (2007). Assays for determination of protein concentration. Current Protocols in Protein Science. https://doi.org/10.1002/0471140864.ps0471140304s0471140848

    Article  PubMed  Google Scholar 

  20. Ebrahiminezhad, A., Varma, V., Yang, S., Ghasemi, Y., & Berenjian, A. (2015). Synthesis and application of amine functionalized iron oxide nanoparticles on menaquinone-7 fermentation: A step towards process intensification. Nanomaterials, 6, 1. https://doi.org/10.3390/nano6010001

    Article  CAS  PubMed Central  Google Scholar 

  21. Li, Y. G., Gao, H. S., Li, W. L., Xing, J. M., & Liu, H. Z. (2009). In situ magnetic separation and immobilization of dibenzothiophene-desulfurizing bacteria. Bioresource Technology, 100, 5092–5096.

    Article  CAS  Google Scholar 

  22. Vakili-Ghartavol, R., Momtazi-Borojeni, A. A., Vakili-Ghartavol, Z., Aiyelabegan, H. T., Jaafari, M. R., Rezayat, S. M., et al. (2020). Toxicity assessment of superparamagnetic iron oxide nanoparticles in different tissues. Artificial Cells, Nanomedicine, and Biotechnology, 48, 443–451.

    Article  CAS  Google Scholar 

  23. Lin, L., Wu, W., Huang, J., Sun, D., Zhou, Y., Wang, H., et al. (2013). Catalytic gold nanoparticles immobilized on yeast: From biosorption to bioreduction. Chemical Engineering Journal, 225, 857–864.

    Article  CAS  Google Scholar 

  24. Grumezescu, A. M., Mihaiescu, D. E., Mogosanu, D. E., Chifiriuc, M. C., Lazar, V., Calugarescu, I., et al. (2010). In vitro assay of the antimicrobial activity of Fe3O4 and CoFe2O4/oleic acid—Core/shell on clinical isolates of bacterial and fungal strains. Journal of Optoelectronics and Advanced Materials, 4, 1798–1801.

    CAS  Google Scholar 

  25. Ramteke, C., Ketan Sarangi, B., Chakrabarti, T., Mudliar, S., Satpute, D., & Avatar Pandey, R. (2010). Synthesis and broad spectrum antibacterial activity of magnetite ferrofluid. Current Nanoscience, 6, 587–591.

    Article  CAS  Google Scholar 

  26. Gholami, A., Rasoul-Amini, S., Ebrahiminezhad, A., Abootalebi, N., Niroumand, U., Ebrahimi, N., et al. (2016). Magnetic properties and antimicrobial effect of amino and lipoamino acid coated iron oxide nanoparticles. Minerva Biotecnologica, 28, 177–186.

    Google Scholar 

  27. Villanueva-Flores, F., Castro-Lugo, A., Ramírez, O. T., & Palomares, L. A. (2020). Understanding cellular interactions with nanomaterials: Towards a rational design of medical nanodevices. Nanotechnology, 31, 132002.

    Article  Google Scholar 

  28. Helmlinger, J., Sengstock, C., Groß-Heitfeld, C., Mayer, C., Schildhauer, T., Köller, M., et al. (2016). Silver nanoparticles with different size and shape: Equal cytotoxicity, but different antibacterial effects. RSC Advances, 6, 18490–18501.

    Article  CAS  Google Scholar 

  29. Zhang, B., Lung, P. S., Zhao, S., Chu, Z., Chrzanowski, W., & Li, Q. (2017). Shape dependent cytotoxicity of PLGA-PEG nanoparticles on human cells. Science and Reports, 7, 7315.

    Article  Google Scholar 

  30. Steckiewicz, K. P., Barcinska, E., Malankowska, A., Zauszkiewicz-Pawlak, A., Nowaczyk, G., Zaleska-Medynska, A., et al. (2019). Impact of gold nanoparticles shape on their cytotoxicity against human osteoblast and osteosarcoma in in vitro model. Evaluation of the safety of use and anti-cancer potential. Journal of Materials Science: Materials in Medicine, 30, 22. https://doi.org/10.1007/s10856-10019-16221-10852

    Article  PubMed  Google Scholar 

  31. Pichia expression kit, Protein expression, A manual of methods for expression of recombinant proteins in Pichia pastoris. Corporation, I., Ed. Invitrogen Corporation: San Diego, CA, USA, Vol. version F.

  32. Matsuo, M., Oogai, Y., Kato, F., Sugai, M., & Komatsuzawa, H. (2011). Growth-phase dependence of susceptibility to antimicrobial peptides in Staphylococcus aureus. Microbiology (Russ. Acad. Sci.), 157, 1786–1797.

    CAS  Google Scholar 

  33. Sinclair, P., Carballo-Pacheco, M., & Allen, R. J. (2019). Growth-dependent drug susceptibility can prevent or enhance spatial expansion of a bacterial population. Physical Biology, 16, 046001.

    Article  CAS  Google Scholar 

  34. Patel, J. B., Cockerill, F., & Bradford, P. A. (2015). Performance standards for antimicrobial susceptibility testing: Twenty-fifth informational supplement (pp. 29–50). Clinical and Laboratory Standard Institute.

  35. Nazemidashtarjandi, S., & Farnoud, A. M. (2019). Membrane outer leaflet is the primary regulator of membrane damage induced by silica nanoparticles in vesicles and erythrocytes. Environmental Science. Nano, 6, 1219–1232.

    Article  CAS  Google Scholar 

  36. Karlsson, H. L., Gustafsson, J., Cronholm, P., & Möller, L. (2009). Size-dependent toxicity of metal oxide particles—A comparison between nano- and micrometer size. Toxicology Letters, 188, 112–118.

    Article  CAS  Google Scholar 

  37. Ansari, F., Grigoriev, P., Libor, S., Tothill, I. E., & Ramsden, J. J. (2009). DBT degradation enhancement by decorating Rhodococcus erythropolis IGST8 with magnetic Fe3O4 nanoparticles. Biotechnology and Bioengineering, 102, 1505–1512.

    Article  CAS  Google Scholar 

  38. Berovic, M., Berlot, M., Kralj, S., & Makovec, D. (2014). A new method for the rapid separation of magnetized yeast in sparkling wine. Biochemical Engineering Journal, 88, 77–84.

    Article  CAS  Google Scholar 

  39. Nocon, J., Steiger, M. G., Pfeffer, M., Sohn, S. B., Kim, T. Y., Maurer, M., et al. (2014). Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production. Metabolic Engineering, 24, 129–138.

    Article  CAS  Google Scholar 

  40. Heyland, J., Fu, J., Blank, L. M., & Schmid, A. (2011). Carbon metabolism limits recombinant protein production in Pichia pastoris. Biotechnology and Bioengineering, 108, 1942–1953.

    Article  CAS  Google Scholar 

  41. Zahrl, R. J., Peña, D. A., Mattanovich, D., & Gasser, B. (2017). Systems biotechnology for protein production in Pichia pastoris. FEMS Yeast Research, 17, fox068. https://doi.org/10.1093/femsyr/fox1068

    Article  Google Scholar 

Download references

Acknowledgements

This experiment was funded by Shiraz University of Medical Sciences, under a PhD thesis proposal submitted at No. 18588 in the School of Pharmacy. Authors are grateful to the support provided by the University of Waikato, New Zealand.

Author information

Authors and Affiliations

  1. Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Seyedeh-Masoumeh Tagizadeh, Alireza Ebrahiminezhad, Mohammad Bagher Ghoshoon & Younes Ghasemi

  2. Department of Pharmaceutical Biotechnology, School of Pharmacy, Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Seyedeh-Masoumeh Tagizadeh, Ali Dehshahri & Younes Ghasemi

  3. Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

    Mohammad Bagher Ghoshoon

  4. School of Engineering, Faculty of Science and Engineering, The University of Waikato, Hamilton, 3240, New Zealand

    Aydin Berenjian

  5. Department of Agricultural and Biological Engineering, Pennsylvania State University, 221 Agricultural Engineering Building, University Park, PA, 16802, USA

    Aydin Berenjian

Authors
  1. Seyedeh-Masoumeh Tagizadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alireza Ebrahiminezhad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohammad Bagher Ghoshoon
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ali Dehshahri
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Aydin Berenjian
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Younes Ghasemi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Aydin Berenjian.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest which influence this work.

Additional information

Publisher's Note

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

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tagizadeh, SM., Ebrahiminezhad, A., Ghoshoon, M.B. et al. Impacts of Magnetic Immobilization on the Growth and Metabolic Status of Recombinant Pichia pastoris. Mol Biotechnol 64, 320–329 (2022). https://doi.org/10.1007/s12033-021-00420-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-021-00420-w

Keywords

Search

Navigation