The Investigations of Some Characteristics and the Prufocolion of Poliphenol Oxidase Enzyme From Iğdır Apple
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2009
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Bu çalışmada Iğdır Elması'ndan polifenol oksidaz enzimi (E.C. 1.14.18.1;PPO) afinite kromatografisi yöntemi ile saflaştırılmış ve polifenol oksidaz enziminin çeşitli özellikleri incelenmiştir. Bu çalışma için Iğdır yöresinden elma temin edilmiştir. Iğdır elmasından polifenol oksidaz enzimi saflaştırmak için pH'sı 7.3 olan fosfat tamponu kullanılarak, gerekli santrifüj ve diğer işlemler yapılarak kolona uygulanacak homojenat hazırlanmıştır. Bu homojenat aktifleşmiş Sepharose 4-B-L-tirozin-p-aminobenzoik asit afinite kolonuna uygulanmıştır. Kolondan alınan fraksiyonlardan aktivite gösterenler, kantitatif protein tayinleri için Coomassie Blue yöntemiyle ve 595 nm'de yapılmıştır. Bunun yanı sıra enzimin optimum pH'sı, optimum sıcaklığı, iyonik şiddet etkisi ve bazı kimyasalların inhibisyon etkisi incelenmiştir. Enzimin optimum pH'sı 6.0 , optimum sıcaklığı 50 0C olarak bulundu. Ayrıca iyonik şiddetin etkisi üzerine yapılan çalışmada ise en yüksek aktivite 0.16 M (NH4)2SO4 konsantrasyonunda görülmüştür.Anahtar Kelimeler: Afinite kromatografisi, Iğdır Elması, İnhibisyon, Polifenol oksidaz, Saflaştırma.
In this study, polyphenol oxidase (E.C. 1.14.18.1; PPO) enzyme obtained from Igdır Apple?s was purified with affinity chromatography and various properties of poliphenol oxidase enzym were stated. The apple was provided from Igdır province. To purify polyphenol oxidase enzyme obtained from apple, phosphate buffer at 7.3 pH was used, necessary santrifuging and other processes were carried out and the homogenate will be applied to the column was prepared. The homogenate was applied to activated Sepharose 4B-Tyrosine-p-amnobenzoic asid affinity column. Activity showing fractions obtained from column, for quantitative protein analyse was performed at 595 nm with Coomassie Blue method. In addition, optimum pH of enzyme, optimum temperature, ionic strength effect and inhibition kinetics of some chemicals on enzyme were investigated. Optimum pH of enzyme and optimum temperature was found as 6 and 50oC respectively. Furthermore, the work carried out on the ionic strength the highest activity was seen in concentration of 0.16 M(NH4)2SO4.Key words: Affinity Chromatography, Igdır Apple, Inhibition, Polyphenol oxidase, Purification.
In this study, polyphenol oxidase (E.C. 1.14.18.1; PPO) enzyme obtained from Igdır Apple?s was purified with affinity chromatography and various properties of poliphenol oxidase enzym were stated. The apple was provided from Igdır province. To purify polyphenol oxidase enzyme obtained from apple, phosphate buffer at 7.3 pH was used, necessary santrifuging and other processes were carried out and the homogenate will be applied to the column was prepared. The homogenate was applied to activated Sepharose 4B-Tyrosine-p-amnobenzoic asid affinity column. Activity showing fractions obtained from column, for quantitative protein analyse was performed at 595 nm with Coomassie Blue method. In addition, optimum pH of enzyme, optimum temperature, ionic strength effect and inhibition kinetics of some chemicals on enzyme were investigated. Optimum pH of enzyme and optimum temperature was found as 6 and 50oC respectively. Furthermore, the work carried out on the ionic strength the highest activity was seen in concentration of 0.16 M(NH4)2SO4.Key words: Affinity Chromatography, Igdır Apple, Inhibition, Polyphenol oxidase, Purification.
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Kimya, Enzim Saflaştırması, Enzim-Inhibisyon Yöntemi, Polifenol Oksidaz, Chemistry, Enzyme Purification, Enzyme-Inhibition Method, Polyphenol Oxidase
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