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

https://izlik.org/JA95DU23TS

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)

https://izlik.org/JA95DU23TS

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.