Purification of Cowpea (Vigna unguiculata) Peroxidase with Single-Step Affinity Chromatography and, Characterization Study

Yıl 2021, Cilt: 7 Sayı: 2, 28 - 34, 09.12.2021

Şeyma Taşbaşı Aykut Öztekin

Öz

In this report, cowpea (Vigna unguiculata) peroxidase (POD) enzyme was isolated and characterized. Four different amino benzohydrazide derivatives were used as ligand in affinity chromatography and with 4-amino 3-bromo 2-methylbenzohidrazide, 53.6% purification yield (91.6 fold) was achieved in a single-step. The molecular weight of the purified cowpea POD showing single band at SDS-PAGE was calculated as 39 kDa. For evaluating the cowpea PODs affinity to ligands, IC50 and Ki values were determined. The molecule that caused the highest inhibition at the lowest concentration was found to be a 4-amino 3-bromo 5-chlorobenzohydrazide with the IC50 value of 78 µM. In the characterization assays, optimum parameters were determined for purified cowpea POD as pH= 6.0, ionic strength= 0.3 M and temperature= 40 ºC. In addition, substrate specificity tests were carried out with guaiacol, H2O2, 4-methylcatechol and ABTS substrates. According to the obtained data, the KM values of these substrates were calculated as 7.21 mM, 17.03 mM, 7.28 mM and 49.21 mM, respectively.

Anahtar Kelimeler

Peroxidase, Cowpea, Purification, Affinity chromatography, Characterization

Kaynakça

• AL-SENAIDY, A. M., & ISMAEL, M. A. (2011). Purification and characterization of membrane-bound peroxidase from date palm leaves (Phoenix dactylifera L.). Saudi Journal of Biological Sciences, 18(3), 293-298.
• ALMAZ, Z., OZTEKIN, A., ABUL, N., GERNI, S., EREL, D., KOCAK, S. M., SENGUL, M. E., OZDEMIR, H. (2021). A new approach for affinity‐based purification of horseradish peroxidase. Biotechnology and applied biochemistry, 68(1), 102-113.
• BELINKY, P., LASSER, H., DOSORETZ, C. (2014). Methods of producing lignin peroxidase and its use in skin and hair lightening. In: Google Patents.
• CHEVALLET, M., LUCHE, S., RABILLOUD, T. (2006). Silver staining of proteins in polyacrylamide gels. Nature protocols, 1(4), 1852-1858.
• CRAIG, A. R., & HARTZ JR, T. P. (1989). Use of polyoxyethylene ethers to improve the performance of immunoassays employing peroxidase conjugates. In: Google Patents.
• 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), 1-9.
• HU, Y., WU, J., LUO, P., MO, Y. (2012). Purification and partial characterization of peroxidase from lettuce stems. African Journal of Biotechnology, 11(11), 2752-2756.
• JANSON, J. C. (1984). Large-scale affinity purification—state of the art and future prospects. Trends in Biotechnology, 2(2), 31-38.
• KOHLER, A. C., SALE, K. L., SIMMONS, B. A. (2020). Hybrid peroxidases with enhanced activity and stability. In: Google Patents.
• KOKSAL, E., BURSAL, E., AGGUL, A. G., GULCIN, I. (2012). Purification and characterization of peroxidase from sweet gourd (Cucurbita Moschata Lam. Poiret). International journal of food properties, 15(5), 1110-1119.
• LAVERY, C. B., MACINNIS, M. C., MACDONALD, M. J., WILLIAMS, J. B., SPENCER, C. A., BURKE, A. A., IRWIN, D. JG., D’CUNHA, G. B. (2010). Purification of peroxidase from horseradish (Armoracia rusticana) roots. Journal of agricultural and food chemistry, 58(15), 8471-8476.
• LINEWEAVER, H., & BURK, D. (1934). The determination of enzyme dissociation constants. Journal of the American chemical society, 56(3), 658-666.
• OZTEKIN, A., ALMAZ, Z., GERNI, S., EREL, D., KOCAK, S. M., SENGÜL, M. E., OZDEMIR, H. (2019). Purification of peroxidase enzyme from radish species in fast and high yield with affinity chromatography technique. Journal of Chromatography B, 1114, 86-92.
• OZTEKIN, A., ALMAZ, Z., ONLU, S. (2021). Production and purification of peroxidases from callus cultures of white and red cabbage for enzymatic decolourization of reactive blue 19 and acid blue 25 dyes. Biocatalysis and Biotransformation, 1-10.
• OZTEKIN, A., & TASBASI, S. (2020). A novel peroxidase from runner bean (Phaseolus coccineus L.): Enhanced affinity purification, characterization, and dye decolorization activity. Journal of Food Biochemistry, 44(10), e13411.
• OZTEKIN, A. (2020). Guaiakol Peroksidazın Soğan Köklerinden Afinite Kromatografisi ile Saflaştırılması. Journal of the Institute of Science and Technology, 10(2), 1163-1170.
• POKORA, A. R., & CYRUS JR, W. L. (1990). Biocatalytic process for preparing phenolic resins using peroxidase or oxidase enzyme. In: Google Patents.
• SINGH, R. S., SINGH, T., PANDEY, A. (2019). Microbial enzymes—an overview. Advances in enzyme technology, 1-40.
• TAO, Y.-M., WANG, S., LUO, H.-L., YAN, W.-W. (2018). Peroxidase from jackfruit: Purification, characterization and thermal inactivation. International journal of biological macromolecules, 114, 898-905.