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Purification and Biochemical Characterization of Two Anionic Peroxidase Isoenzymes from Raphanus sativus L. var niger Roots

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Abstract

Peroxidase is a commonly used enzyme with a wide range of applications. Horseradish (Armoracia rusticana) is the most well-known source of peroxidase enzyme. Peroxidases extracted from other plant sources have also been proved as useful, sometimes even superior, comparing to traditional horseradish peroxidase (HRP). In the present study, two novel peroxidase isoenzymes were purified and characterized from Raphanus sativus L. var niger roots. Two anionic peroxidase isoenzymes were purified using diafiltration, ammonium sulfate precipitation, DEAE anion-exchange chromatography, and concanavalin A affinity chromatography. The heaviest anionic isoenzyme (isoenzyme A) has a MW of about 110 KD, and the other anionic isoenzyme (isoenzyme B) has a MW of 97 KD. Both isoenzymes have an optimum temperature of 40 °C, but the activity of isoenzyme B is much more dependent on temperature with a Q10 of 3.5, while isoenzyme A has a Q10 of 1.7. These isoenzymes showed great thermal stability at 37 °C and 4 °C. Isoenzyme A showed the highest activity at pH 5 and it was found to be more stable at pH 6, whereas isoenzyme B showed the highest activity at pH 6 and is more stable at pH 7. Isoenzyme A has a Km value of 10.63 mM and 0.043 mM, and isoenzyme B has a Km of 15.38 mM and 0.067 mM for 4-aminoantipyrine and H2O2, respectively. The isoenzymes purified from Raphanus sativus L. var niger offer excellent chemical and thermal stability, which encourages further studies on their suitability for biotechnological applications.

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References

  1. El-Khonezy, M. I., Abd-Elaziz, A. M., Dondeti, M. F., Fahmy, A. S., & Mohamed, S. A. (2020). Purification and characterization of cationic peroxidase from ginger (Zingiber officinale). Bulletin of the National Research Centre, 44(1), 11.

    Article  Google Scholar 

  2. Regalado, C., García-Almendárez, B. E., & Duarte-Vázquez, M. A. (2004). Biotechnological applications of peroxidases. Phytochemistry Reviews, 3(1), 243–256.

    Article  CAS  Google Scholar 

  3. Pandey, V. P., Awasthi, M., Singh, S., Tiwari, S., & Dwivedi, U. N. (2017). A comprehensive review on function and application of plant peroxidases. Biochemistry and Analytical Biochemistry, 6(1), 1009–2161.

    Article  Google Scholar 

  4. Prokopowicz, Z., Marcinkiewicz, J., Katz, D. R., & Chain, B. M. (2012). Neutrophil myeloperoxidase: Soldier and statesman. Archivum immunolgiae et therapiae experimentalis, 60(1), 43–54.

    Article  CAS  Google Scholar 

  5. Ursini, F., Maiorino, M. (2013). Glutathione Peroxidases. In: Lennarz, W. J., Lane MDBT-E of BC (Second E, editors. Waltham: Academic Press. p. 399–404. Available from: https://www.sciencedirect.com/science/article/pii/B9780123786302003832.

  6. Dhruvaraj, M. R. (2017). Role of peroxidase in clinical assays: A short review. The Journal of Clinical Nutrition, 3(2), 14.

    Google Scholar 

  7. Jeong, Y.-M., Oh, M. H., Kim, S. Y., Li, H., Yun, H.-Y., Baek, K. J., et al. (2010). Indole-3-acetic acid/horseradish peroxidase induces apoptosis in TCCSUP human urinary bladder carcinoma cells. Die Pharmazie - An International Journal of Pharmaceutical Sciences, 65(2), 122–126.

    CAS  Google Scholar 

  8. Greco, O., Folkes, L. K., Wardman, P., Tozer, G. M., & Dachs, G. U. (2000). Development of a novel enzyme/prodrug combination for gene therapy of cancer: Horseradish peroxidase/indole-3-acetic acid. Cancer Gene Therapy, 7(11), 1414–1420.

    Article  CAS  Google Scholar 

  9. Karam, J., & Nicell, J. A. (1997). Potential applications of enzymes in waste treatment. Journal of Chemical Technology and Biotechnology, 69(2), 141–153. https://doi.org/10.1002/(SICI)1097-4660(199706)69:2%3C141::AID-JCTB694%3E3.0.CO.

    Article  CAS  Google Scholar 

  10. Kwang-Soo, S., & Chang-Jin, K. (1998). Decolorisation of artificial dyes by peroxidase from the white-rot fungus, Pleurotus ostreatus. Biotechnology Letters, 20(6), 569–72. https://doi.org/10.1023/A:1005301812253.

    Article  Google Scholar 

  11. Kariminiaae-Hamedaani, H.-R., Sakurai, A., & Sakakibara, M. (2007). Decolorization of synthetic dyes by a new manganese peroxidase-producing white rot fungus. Dye Pigment [Internet]. 72(2):157–62. Available from: https://www.sciencedirect.com/science/article/pii/S0143720805002676.

  12. Bhunia, A., Durani, S., & Wangikar, P. P. (2001). Horseradish peroxidase catalyzed degradation of industrially important dyes. Biotechnology and Bioengineering, 72(5), 562–7. https://doi.org/10.1002/1097-0290(20010305)72:5%3C562::AID-BIT1020%3E3.0.CO.

    Article  CAS  PubMed  Google Scholar 

  13. Klibanov, A. M., Tu, T. M., & Scott, K. P. (1983). Peroxidase-catalyzed removal of phenols from coal-conversion waste waters. Science, 221(4607), 259–261.

    Article  CAS  Google Scholar 

  14. Giannoudi L, Piletska E V, Piletsky SA. Development of biosensors for the detection of hydrogen peroxide BT - Biotechnological applications of photosynthetic proteins: Biochips, biosensors and biodevices. In: Giardi MT, Piletska E V, editors. Boston, MA: Springer US; 2006. p. 175–91. Available from: https://doi.org/10.1007/978-0-387-36672-2_16.

  15. Ambrosi, A., Airò, F., & Merkoçi, A. (2010). Enhanced gold nanoparticle based ELISA for a breast cancer biomarker. Analytical Chemistry, 82(3), 1151–6. https://doi.org/10.1021/ac902492c.

    Article  CAS  PubMed  Google Scholar 

  16. Yang, H., Bever, C. S., Zhang, H., Mari, G. M., Li, H., Zhang, X., et al. (2019). Comparison of soybean peroxidase with horseradish peroxidase and alkaline phosphatase used in immunoassays. Analytical Biochemistry, 581, 113336.

    Article  CAS  Google Scholar 

  17. Ryan, B. J., Carolan, N., & Ó’Fágáin, C. (2006). Horseradish and soybean peroxidases: Comparable tools for alternative niches? Trends in Biotechnology [Internet]. 24(8):355–63. Available from: https://www.sciencedirect.com/science/article/pii/S0167779906001557.

  18. Bisen, P. S. (2014). Laboratory protocols in applied life sciences. CRC Press.

    Book  Google Scholar 

  19. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680–685.

    Article  CAS  Google Scholar 

  20. Swain, M., & Ross, N. W. (1995). A silver stain protocol for proteins yielding high resolution and transparent background in sodium dodecyl sulfate-polyacrylamide gels. Electrophoresis, 16(6), 948–951.

    Article  CAS  Google Scholar 

  21. Shental-Bechor, D., & Levy, Y. (2008). Effect of glycosylation on protein folding: A close look at thermodynamic stabilization. Proceedings of the National Academy of Sciences, 105(24), 8256–8261.

    Article  CAS  Google Scholar 

  22. Han, Q., Li, G., & Li, J. (2000). Purification and characterization of chorion peroxidase from Aedes aegypti eggs. Archives of Biochemistry and Biophysics, 378(1), 107–115.

    Article  CAS  Google Scholar 

  23. Zeyadi, M., & Almulaiky, Y. Q. (2020). A novel peroxidase from Ziziphus jujuba fruit: Purification, thermodynamics and biochemical characterization properties. Science Reports, 10(1), 8007. https://doi.org/10.1038/s41598-020-64599-9.

    Article  CAS  Google Scholar 

  24. Sariri, R., Jafarian, V., Sajedi, R. H., & Khajeh, K. (2006). Inhibition of horseradish peroxidase by thiol type inhibitors: Mercaptoethanol and mercaptoacetic acid. Journal of Molecular Liquids, 128(1–3), 175–177.

    Article  CAS  Google Scholar 

  25. Rojas-Reyes, J. O., Robles-Olvera, V., Carvajal-Zarrabal, O., Castro Matinez, C., Waliszewski, K. N., & Aguilar-Uscanga, M. G. (2014). Purification and characterization of peroxidase from avocado (Persea americana Mill, cv. Hass). Journal of the Science of Food and Agriculture, 94(9), 1844–53.

    Article  CAS  Google Scholar 

  26. Kouakou, T. H., Dué, E. A., Kouadio, N. E. J. P., Niamké, S., Kouadio, Y. J., & Mérillon, J.-M. (2009). Purification and characterization of cell suspensions peroxidase from cotton (Gossypium hirsutum L.). Applied Biochemistry and Biotechnology, 157(3), 575–92.

    Article  CAS  Google Scholar 

  27. Feng, J.-Y., Liu, J.-Z., & Ji, L.-N. (2008). Thermostability, solvent tolerance, catalytic activity and conformation of cofactor modified horseradish peroxidase. Biochimie, 90(9), 1337–1346.

    Article  CAS  Google Scholar 

  28. Gilabert, M. A., Fenoll, L. G., García-Molina, F., Tudela, J., García-Cánovas, F., & Rodríguez-López, J. N. (2004). Kinetic characterization of phenol and aniline derivates as substrates of peroxidase.

  29. Zhao, J., Lu, C., & Franzen, S. (2015). Distinct enzyme–substrate interactions revealed by two dimensional kinetic comparison between dehaloperoxidase-hemoglobin and horseradish peroxidase. The Journal of Physical Chemistry B, 119(40), 12828–12837.

    Article  CAS  Google Scholar 

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Funding

This research was supported by Tehran University of Medical Sciences and Health Services (grant number 46714).

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Authors and Affiliations

  1. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Hooman Askari, Aliasghar Rahimian & Mahdi Aminian

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  1. Hooman Askari
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  2. Aliasghar Rahimian
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  3. Mahdi Aminian
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Contributions

All of the authors made significant contributions to the design and conception of study. Material preparation, data collection, and analysis were performed by Hooman Askari, Aliasghar Rahimian, and Mahdi Aminian. All authors read and approved the final manuscript.

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Correspondence to Mahdi Aminian.

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Askari, H., Rahimian, A. & Aminian, M. Purification and Biochemical Characterization of Two Anionic Peroxidase Isoenzymes from Raphanus sativus L. var niger Roots. Appl Biochem Biotechnol 194, 2219–2235 (2022). https://doi.org/10.1007/s12010-021-03736-0

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