Determination of Biochemical Properties of Polyphenol Oxidase from Domat Olives (In English)

Öz

 Enzymatic browning of damaged tissues of fruits and vegetables occurs from the oxidation of phenolic compounds and contributes significantly to quality loss. The primary enzyme responsible for the browning reaction is polyphenol oxidase (PPO). PPO from Domat olives was extracted and purified through (NH4)2SO4 precipitation, dialysis and ion exchange chromatography. Optimum pH and temperature for olive PPO were found to be 4.5 and 30 °C, respectively, using 4-methyl catechol as substrate. Km and Vmax values for olive PPO using 4-methyl catechol as substrate were found to be 14.52 mM (r2= 0.985) and 1.78 Abs/min, respectively. Of the inhibitors tested, the most potent inhibitors were L-cysteine and sodium disulfite. The Z and Ea values obtained in this study were 18.6 °C (r2 = 0.953) and 121.0 kj/mol (r2= 0.950), respectively. 

Anahtar Kelimeler

• Olive, polyphenol oxidase, enzymatic browning, kinetics, thermal inactivation

Domat Zeytini Polifenol Oksidazının Biyokimyasal Özelliklerinin Belirlenmesi (İngilizce)

Öz

Zarar görmüş meye ve sebzelerdeki enzimatik esmerleşme fenolik bileşiklerin oksidasyonu sonucu meydana gelir ve önemli kalite kayıplarına neden olur. Esmerleşme reaksiyonuna neden olan en önemli enzim polifenol oksidaz (PFO)’dır. Domat zeytininden polifenol oksidaz enzimi ekstrakte edilmiş ve amonyum sülfat çökeltmesi, dializ ve iyon değişim kromatografisi ile saflaştırılmıştır. Zeytin PFO’sunun optimum pH ve sıcaklığı sırasıyla 4-metil kateşol substratı kullanılarak 4.5 ve 30 °C bulunmuştur. Zeytin PFO’su Km ve Vmax değerleri sırasıyla 4-metil kateşol substratı kullanılarak 14.52 mM (r2= 0.985) ve 1.78 Abs/dak bulunmuştur. Test edilmiş inhibitörlerin en etkili olanları sistein ve sodyum disülfittir. Bu çalışmada elde edilmiş Z ve Ea değerleri sırasıyla 18.6 °C (r2 = 0.953) ve 121.0 kj/mol (r2= 0.950) bulunmuştur.

Anahtar Kelimeler

• Zeytin, polifenol oksidaz, enzimatik esmerleşme, kinetik, termal inaktivasyon

Kaynakça

1. Martinez MV, Whiataker JR. 1995. The biochemistry and control of enzymatic browning. Trends Food Sci Technol, 6: 195-200.
2. Ramirez EC, Whitaker JR, Virador VM. 2004. Handbook of Food Enzymology. Marcel Dekker, New York, pp. 509-522.
3. Lee CY, Whitaker JRAS (eds), Whitaker JR, Lee C.Y. 1995. Recent advances in chemistry of enzymatic browning. Symposium Series 600, American Chemical Society, Washington, pp. 2-7. 4. Chararra S, Carcia-Carmona F, Cabanes J. 2001. Hysteresis and positive cooperativity of iceberg lettuce PPO. Biochem Biophys Res Commun, 289: 769-775.
4. Lamikanra O (ed), Lamikanra O. 2002. Fresh-Cut Fruits and Vegetables: Science, Technology and Market. CRC Press, London, pp. 125-185.
5. Segovia-Bravo KA, Jareän-Galaä NM, Garcıäa- Garcıäa P. 2007. Antonio Garrido-Fernaä ndez A. Characterization of polyphenol oxidase from the Manzanilla cultivar (Olea europaea pomiformis) and prevention of browning reactions in bruised olive fruits. J Agric Food Chem, 55: 6515-6520.
6. Coseteng MY, Lee CY. 1987. Changes in apple polyphenol oxidase and polyphenol concentrations in relation to degree of browning. J Food Sci, 52: 985-989.
7. Ünal MÜ, Şener A. 2010. Bozdoğan A. A comparative study of polyphenol oxidase from two varieties of quince (Cydonia oblonga). J Food Biochem, 34: 356-367.
8. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 7: 248-254. 10. Ünal MÜ. 2007. Properties of polyphenol oxidase from Anamur banana (Musa cavendishii). Food Chem, 100: 909-913.

9. Vamos-Vigyazo L. 1981. Polyphenol oxidase and peroxidase in fruits and vegetables. Crit Rev Food Sci, 15: 49-127.
10. Zawistowski J, Bilideris CG, Eskin NAM. 1991. Oxidative Enzymes in Foods. Elsevier, New York, pp. 217-273.
11. Nakao H, Shinoda S, Yamamoto S. 1991. Purification and some properties of carboxynorspermidine synthase participating in a novel biosynthetic pathway for norspermidine in Vibrio alginolyticus. J Gen Microbiol, 137:1737-1742.
12. Nakao H, Shinoda S, Yamamoto S. 1990. Purification and properties of carboxynorspermidine decarboxylase, a novel enzyme involved in norspermidine biosynthesis, from Vibrio dginolyticus. J Gen Microbiol, 136:1699-1704.
13. Shiiba K, Negishi Y, Okada K, Nagao S. 1991. Purification and Characterization of Lipoxygenase Isozymes from Wheat Germ. Cereal Chem, 68:115-122.
14. Lourenço EJ, Neves VA, Da Silva MA. 1992. Polyphenol Oxidase from Sweet Potato: Purification and Properties. J Agric Food Chem, 40:2369-2373.
15. Gawlik-Dziki U, Szymanowska U, Baraniak B. 2007. Characterization of polyphenol oxidase from broccoli (Brassica oleracea var. botrytis italica) florets. Food Chem, 105: 1047-1053.
16. Chase MR, Raina K, Bruno J, Sugumaran M. 2000. Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata. Insect Biochem Molec Biol, 30: 953-967.
17. Gülçin I, Küfrevioğlu OI, Oktay M. 2005. Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity. J Enzyme Inhib Med Chem, 20: 297-302.
18. Gawlik-Dziki U, Zlotek U, Swieca M. 2008. Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.). Food Chem, 107: 129-135.
19. Shi C, Dai Y, Xia B, Xu X, Xie Y, Liu Q. 2001. The purification and spectral properties of polyphenol oxidase I from Nicotina tabacum. Plant Molec Biol Rep, 19: 381-383.
20. Erat M, Şakiroğlu H, Küfrevioğlu OI. 2006. Purification and characterization of polyphenol oxidase from Ferula sp. Food Chem, 95: 503-508. 23. Yang CP, Fujita S, Kohno K, Kusubayashi A, Ashrafuzzaman MD, Hayashi N. 2001. Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L) peel. J Sci Food Agric, 49: 1446-1449.
21. Ding CK, Chachin K, Ueda Y, Imahori Y. 1998. Purification and properties of polyphenol oxidase from loquat fruit. J Agric Food Chem, 46: 4144-4149.
22. Ünal MÜ, Şener A. 2006. Determination of biochemical properties of polyphenol oxidase from Emir grape (Vitis vinifera L. cv. Emir). J Sci Food Agric, 86: 2374-2379.
23. Serradell MA, Rozenfeld PA, Martinez GA, Civello PM, Chaves AR, Anon M.C. 2000. Polyphenoloxidase activity from strawberry fruit (Fragaria x ananassa, Duch., cv Selva): characterization and partial purification. J Sci Food Agric, 80: 1421-1427.
24. Arslan O, Temur A, Tozlu I. 1998. Polyphenol oxidase from Malatya apricot (Prunus armeniaca L.). J Agric Food Chem, 46: 1239-1241.
25. Yemenicioğlu A, Cemeroğlu B. 2003. Consistency of polyphenol oxidase (PPO) thermostability in ripening apricots (Prunus armenica L.): Evidence for the presence of thermostable PPO forming and destabilizing mechanisms in apricots. J Agric Food Chem, 51: 2371-2379.
26. Ortega-Garci’a F, Peragon J. 2009. Phenylalanine ammonia-lyase, polyphenol oxidase, and phenol concentration in fruits of Olea europea L. cv. Picual, Verdial, Arbequina, and Frantoio during ripening. J Agric Food Chem, 57: 10331-10340.
27. Marshall MR, Jeongmok K, Wei C-I. 2000. Enzymatic Browning in Fruits, Vegetables and Seafoods, FAO, www.fao.org/ag/ags/agsi/ ENZYMEFINAL/Enzymatic %20Browning.html.