Extraction, Partial Purification and Characterisation of Polyphenol Oxidase from Tea Leaf (Camellia sinensis) (in English)

Yıl 2011, Cilt: 36 Sayı: 3, 137 - 144, 01.06.2011

M. Ümit Ünal Selin N. Yabacı Aysun Sener

Öz

Polyphenol oxidase (PPO) from tea leaves was extracted and partially purified through (NH4)2SO4 precipitation, dialysis and ion exchange chromatography. Of the substrates tested, 4-methylcatechol was the best substrate for PPO with a Km value of 127.8 mM. The optimum pH for PPO activity was found to be 6.02. The enzyme showed high activity over a broad pH range of 4.03-7.00. The optimum temperature for PPO activity was 30 °C. The enzyme had more than 70% of the maximum activity between 20-80 °C. Energy of activation (Ea) and Z values were found to be 58.301 kJ/mol (r2= 0.961) and 39.68°C (r2= 0.965), respectively. Of the inhibitors tested, L-cysteine was the least potent inhibitor.

Anahtar Kelimeler

Inhibition, kinetics, purification, polyphenol oxidase, tea, thermal inactivation

Polifenol Oksidazın Çay (Camellia sinensis) Yaprağından Ekstraksiyonu, Kısmi Saflaştırılması ve Karakterizasyonu (İngilizce)

Öz

Polifenol oksidaz (PFO) çay yapraklarından ekstrakte edilip (NH4)2SO4 çökeltmesi, diyaliz ve iyon değişim kromatografisi ile kısmen saflaştırılmıştır. Test edilen substratlardan, 127.8 mM Km değeri ile 4-metilkateşol PFO için en iyi substrat olarak belirlenmiştir. PFO aktivitesi için optimum pH 6.02 olarak saptanmıştır. Enzim, 4.03-7.00 gibi geniş bir pH aralığında aktivite göstermiştir. PFO aktivitesi için optimum sıcaklık 30°C olarak bulunmuştur. 20-80 °C’ler arasında enzim maksimum aktivitesinin %70’den fazlasını korumuştur. Aktivasyon enerjisi (Ea) ve Z değerleri, sırasıyla, 58.301 kJ/mol (r2= 0.961) ve 39.68 °C (rr2= 0.965) olarak bulunmuştur. Test edilen inhibitörlerden L-sisteinin en düşük inhibisyon derecesine sahip olduğu belirlenmiştir.

Anahtar Kelimeler

Çay, inhibisyon, kinetik, polifenol oksidaz, saflaştırma, termal inaktivasyon

Kaynakça

• Robinson DS, Eskin NAM (eds), Zawistowski J, Biliaderis CG, Eskin NAM. 1991. Oxidative Enzymes in Foods. Elsevier, New York, pp. 217- 273.
• Halder J, Tamuli P, Bhaduri AN. 1998. Isolation and characterization of polyphenol oxidase from Indian tea leaf (Camellia sinensis). J Nutr Biochem,9:75-80.
• Subramanian N, Venkatesh P, Ganguli S, Sinkar VP. 1999. Role of polyphenol oxidase and peroxidase in the generation of black tea theaflavins. J Agric Food Chem, 47:2571-2578.
• Finger A. 1994. In-Vitro studies on the effect of polyphenol oxidase and peroxidase on the formation of polyphenolic black tea constituents. J Sci Food Agric, 66:293-305.
• Singh HP, Ravindranath SD. 1994. Occurrence and distribution of PPO activity in floral organs of some standard and local cultivars of tea. J Sci Food Agric,64:117- 120.
• Demir A. 2002. Çay, TEAE (Tarımsal Ekonomi Arafltırma Enstitüsü) 1:1-4 (In Turkish).
• 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.
• Serradell MA, Rozenfeld PA, Martinez GA, Civello PM, Chaves AR, Anon MC. 2000. Polyphenoloxidase activity from strawberry fruit (Fragaria x ananassa, Duch., cv Selva): characterization and partial purification. J Sci Food Agric, 80:1421-1427.
• 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. 10. Bradford MM. 1976. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem, 72:248-254.
• Ünal MÜ, fiener A. 2006. Determination of biochemical properties of polyphenol oxidase from Emir grape (Vitis vinifera L. cv. Emir). J Sci Food Agric,86:2374-2379.
• Ünal MÜ. 2007. Properties of polyphenol oxidase from Anamur banana (Musa cavendishii). Food Chem,100:909-913.
• Arogba SK, Ajiboye OL, Ugboko LA, Esienette SY, Afolabi PO. 1998. Properties of polyphenol oxidase in mango (Mangifera indica) kernel. J Sci Food Agric,77:459-462.
• Wissemann KW, Lee CY. 1981. Characterization of polyphenoloxidase from Ravat 51 and Niagara grapes. J Food Sci, 46:506-508.
• Rapeanu G, Loey A.V, Smout C, Hendrickx M. 2005. Effect of pH on thermal and/or pressure inactivation of Victoria grape (Vitis vinifera sativa) polyphenol oxidase: A kinetic study. J Food Sci, 70:301-307.
• Lee CY, Smith NL, Pennesi AP. 1983. Polyphenol oxidase from Dechaunac grapes. J Sci Food Agric,34:987-991.
• Ünal MÜ, fiener A, fien K. 2007. Characterization of polyphenol oxidase from Sultaniye grapes (Vitis vinifera cv Sultana). Int J Food Sci Technol, 42:1123-1127.
• Palmer, JK. 1995. Understanding Enzymes. Prentice Hall/Ellis Horwood, Herdfordshire.
• Orenes-Pinero E, Garcia-Caromana F, Sanchez-Ferrer A. 2006. Latent polyphenol oxidase from quince fruit pulp (Cydonia oblonga): purification, activation and some properties. J Sci Food Agric, 86:2172-2178.
• Duangmal K, Apenten RKO. 1999. A comparative study of polyphenoloxidases from taro (Colocasia esculenta) and potato (Solanum tuberosumvar. Romano). Food Chem, 64:351-359. 21. Vamos-Vigyazo, L. 1981. Polyphenol oxidase and peroxidase in fruits and vegetables. Crit Rev Food Sci,15:49-127.
• Yoruk R, Marshall MR. 2003. Physicochemical properties and function of plant polyphenol oxidase: a review. J Food Biochem, 27:361-422.
• Yang C-P, 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.
• 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.
• Gomez-Lopez VM. 2002. Some biochemical properties of polyphenol oxidase from two varieties of avocado. Food Chem, 77:163-169.
• Dogan M., Arslan O., Dogan S. 2002. Substrate specificity, heat inactivation and inhibition of polyphenol oxidase from different aubergine cultivars. Int J Food Sci Technol, 37:415-423.