Journals / Iğdır Üniversitesi Fen Bilimleri Enstitüsü Dergisi / 2018 / Cilt: 8 - Sayı: 4
The Partial Purification and Characterization of Polyphenol Oxidase in Native Grape Kirmizi Kismisi Cultivar (Vitis vinifera L.) Grown in Iğdır Province
- Pages
- 153–161
- DOI
- —
Abstract
The native grape Kirmizi Kismisi cultivar was gathered from the Necefali village of Igdir provincein Turkey, and then caried into the laboratory and stored in deep-frozen at -20°C. Polyphenol oxidase (PPO) waspartial purified 5.45 times using the cold acetone precipitation from Kirmizi Kismisi Grape and was characterized.50 gr of Kirmizi Kismisi Grape were placed in a porcelain mortar and pestled in order to decompose cell membranes.The frozen Kirmizi Kismisi Grapes were homogenised by using a porcelain mortar in 100 mL of 50 mM sodiumacetate buffer (pH 5.0) containing 1% (w/v) polyethylene glycol (PEG). Then homogenate was percolated through4-fold muslin and the resulting filtrate was santrifuged for 30 minutes at 10,000 rpm at 4°C. The supernatantobtained after centrifugation was mixed with cold acetone as much as the volume of the supernatant in the icebath and the mixture was incubated overnight at 4°C for precipitation of proteins. After centrifugation at 10,000rpm for 30 min at 4°C, the precipitate was redissolved in 20 mL 50 mM sodium acetate buffer (pH 5.0). Proteinconcentration was determined according to the Lowry method. Protein concentrations of crude extract and acetoneprecipitation were determined as 7.04 and 3.83 mg/mL, respectively. Optimum pH and temperature values werefound to be 6.0 and 20°C, respectively, using catechol as a substrate. In addition, some biochemical propertiessuch as Km and Vmax values were investigated. PPO activity was inhibited from ascorbic acid, citric acid, sodiummetabisulfite and benzoic acid and the IC50 for each inhibitor was calculated.The data obtained from this studyshowed that this enzyme could be useful for food industrial purposes.
Özet
Yerel üzüm çeşidi Kırmızı Kişmişi, Türkiye’nin Iğdır ilinin Necefali köyünden toplandı ve daha sonra