Affinity Purification and Characterization of Polyphenol Oxidase from Helianthus tuberosus L.

Year 2009, Volume: 37 Issue: 4, 313 - 325, 01.12.2009

Mahmut Erzengin

Abstract

Polyphenol oxidase PPO of Jerusalem artichoke Helianthus Tuberosus L. tubers was purified using a Sepharose-4B-L-tyrosine-p-amino benzoic acid affinity gel. Both nativeand SDS-gel electrophoresis analyses of the purified PPO gave a single band ca. 65 kDa based on SDS-PAGE , indicating that it is a monomer. The purified PPO showed activity towards diphenolic and triphenolic substrates but not with the monophenolic substrates, suggesting that it lacks monophenolase activity. The optimum temperature and pH values vary between 20-35oC and 5.0-8.0, respectively, depending on the substrate used; for catechol, the optimum temperature and pH values were found to be 20oC and 7.0, respectively. The purified enzyme was relatively stable at 40oC but unstable at higher temperatures. Furthermore, IC50 values for various inhibitors and inhibition modes were also determined using catechol as a substrate; β-mercaptoethanol showed the strongest inhibition, followed by 2-mercapto benzothiazol, glutathione, L-cysteine and dithioerythritol, respectively

Keywords

Jerusalem artichoke Helianhtus tuberosus L., Polyphenol oxidase, Affinity chromatography, Substrate specificity, Activation energy, Heat inactivation, Inhibition

References

• Siddiq, M., Sinha, N.K., and Cash, Y.N., Characterization of a polyphenol oxidase from Stanley plums, J. Food Sci., 57, 1177-1179, 1992.
• Arslan, O., Temur, A., and Tozlu, İ., Polyphenol oxidase from Allium sp., J. Agric. Food Chem., 45, 2861-2863, 1997.
• Erat, M., Şakiroğlu, H., and Küfrevioğlu, Ö. İ., Purification and characterization of polyphenol oxidase from Ferula sp., Food Chem., 95, 503-508, 2006.
• Paul, B., and Gowda, L.R., Purification and charac- terization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab), J. Agric. Food Chem., 48, 3839- 3846, 2000.
• Arslan, O., Erzengin, M., Sinan, S., and Özensoy, Ö., Purification of mulberry (Morus alba L.) poyphenol oxidase by affinity chromatography and investigation of its kinetic and electrophoretic properties, Food Chem., 88, 479- 484, 2004.
• Zawistowski, J., Biliaderis, C.G., and Murray, E.D., Purification and characterization of Jerusalem artichoke (Helianthus tuberosus L.) polyphenol oxidase, J. Food Biochem., 12, 1-22, 1988.
• Ziyan, E., and Pekyardımcı, Ş., Characterization of polyphenol oxidase from Jerusalem Artichoke (Helianthus tuberosus.), Turkish J. Chem., 27, 217-226, 2003.
• Fujita, S., Saari, N.B., Maegawa, M., Tetsuka, T., Hayashi, N., and Tono, T., Purification and properties of polyphenol oxidase from cabbage (Brassica oleracea L.), J. Agric. Food Chem., 43, 1138-1142, 1995.
• Nagai, T., and Suzuki, N., Partial purification of polyphenol oxidase from Chinese Cabbage (Brassica rapa L.), J. Agric. Food Chem. 49, 3922-3926, 2001.
• Arslan, O., Temur, A., and Tozlu, İ., Polyphenol oxidase from Malatya Apricot (Prunus armeniaca L.). J. Agric. Food Chem., 46(4), 1239-1241, 1998.
• Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantites of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248- 254, 1976.
• Laemmli, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T, Nature, 227, 4 680-685, 1970.
• Chazarra, S., Garcia-Carmona, F., and Cabanes, J., Evidence for a tetrameric form of iceberg lettuce (Lactuca sativa L.) polyphenol oxidase: Purification and characterization, J. Agric. Food Chem., 49, 4870-4875, 2001.
• Xu, J., Zheng, T., Meguro, S., and Kawachi, S., Purification and characterization of polyphenol oxidase from Henryi chestnuts (Castanea henryi), J. Wood Sci., 50, 260-265, 2004.
• Zhou, H., and Feng, X., Polyphenol oxidase from yali pear (Pyrus bretschneideri), J. Sci. Food Agric., 57, 307-313
• Martin, F.W., and Ruberte, R., The polyphenol of Dioscorea alata (yam) tubers associated with oxidative browning, J. Agric. Food Chem., 24, 67-70, 1976.
• Sanchez-Ferrer, A., Bru, R., Cabanes, J., and Garcia- Carmona, F., Characterization of catecholase and cresolase activities of monastrell grape polyphenol oxidase, Phytochem., 27(2), 319-321, 1988.
• Şakiroğlu, H., Küfrevioğlu, İ. Ö., Kocaçalışkan, İ., Oktay, M., and Onganer, Y., Purification and characterization of Dog-rose (Rosa dumalis Rechst.) polyphenol oxidase, J. Agric. Food Chem., 44, 2982-2986, 1996.
• Whitaker, J.R., Principles of Enzymology for the Food Sciences, Second Ed., 271-556, Marcel Dekker, New York, 1994.
• Lee, P. M., Lee, K.H., and Abdul Karim, M.I., Biochemical studies of cocoa bean polyphenol oxidase, J. Sci. Food Agric., 55, 251-260, 1991.
• Vamos-Vigyazo, L., Polyphenol oxidase and peroxidase in fruits and vegetables, Crit. Rev. Food Sci. Nutr., 15, 49- 127, 1981.