Ceviz (Juglans regia L.) tohumu lipazının saflaştırılması biyokimyasal özelliklerinin belirlenmesi
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Dosyalar
Tarih
2008
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Lipazlar (triaçilgliserol açilhidrolazlar, EC 3.1.1.3), su-yağ arayüzeyinde trigliseridlerin hidrolizini katalizlerler. Lipazların biyolojik proseslerdeki kullanım alanları, önemi ve yeri gün geçtikçe artmaktadır. Bu çalışmada 'şebin' türü olarak bilinen ceviz tohumundan lipaz enziminin izole edilmesi, saflaştınlması ve karakterizasyonu amaçlandı. Ceviz proteinlerinin yağsızlaştırılmasından sonra amonyum sülfat çöktürmesi (60-90), diyaliz ve jel filtrasyon metodlan kullanılarak ceviz tohumu lipazı yaklaşık olarak 29 kat saflaştırıldı. Her saflaştırma basamağında Lowry metoduna göre protein tayini ve her fraksiyon için titrimerik metod kullanılarak lipaz aktivitesi tayini yapıldı. Ceviz tohumu lipazının optimum reaksiyon süresi 13 dakika olarak belirlendi. Ayrıca enzimin en uygun substratı triolein olarak belirlendi. Saflaştınlan enzim pH: 9.0 ve 70°C'de maksimum aktivite gösterdi. Ceviz tohumundan elde edilen lipazın depo kararlılığını belirlemek amacıyla, bir yıl süreyle enzim denemeleri gerçekleştirildi. -20 C'de 12. ayın sonunda enzim aktivitesinin yaklaşık % 85'inin korunduğu gözlendi. Aktivatör olarak CaCİ2 eklendiğinde lipaz aktivitesini en fazla artıran 0.5 mM'lık CaCİ2 konsantrasyonu (% 287) oldu. Substrat olarak triolein kullanılan ceviz tohumu lipazının Km ve Vmax değerleri sırasıyla 48 mM ve 23.06 (x 10"3) U/dk.mg.enzim olarak hesaplandı. Ceviz tohumu lipazının aktivasyon enerjisi Ea= 3.26 kj/mol olarak hesaplandı. Metal iyonu etkisi incelendiğinde, EDTA bağıl aktiviteyi arttırırken, Cu+ , Hg+ , Mn+ , Ca+2 ve Ni+2 iyonlarının inhibisyona neden olduğu, Zn+2 ve Mg+2nin ise aktiviteyi değiştirmediği belirlendi. Deterjanlarla yapılan çalışmada ise SDS, Triton X-100 ve Tween-80'in 5, 15, 25 ve 35 mM'da lipaz enzimi üzerindeki etkileri belirlendi. Bağıl aktivite üzerine inhibitör etkisi araştırıldığında ise sadece «-bromosüksinimid, üre, potasyumsiyanür ve potasyumferrisiyanür'ün inhibisyona neden olduğu gözlendi. Son olarak ise farklı organik çözücülerin enzim üzerinde 24 saat ve 48 saat sonraki etkileri belirlendi.
Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) catalyze the hydrolyze of triglyserids in water-fat interface. Application areas on bioprocessing of the lipases, their values and importance increase day by day. in this study, it was aimed that lipase enzyme from walnut seed identified as "şebin" species was isolated, purified and characterized. Lipase enzyme from walnut seed was purified about 29 fold to homogeneous state by ammonium sulphate precipitation (60-90) and dialyse after by defatting from walnut proteins. At each purification step, protein concentration was determined by the Lowry method and the lipase activity was defined by using titrimetric method. it was determined that the optimum reaction time of walnut lipase is 13 minutes. it was also determined that the triolein was the most suitable substrate for the lipase from walnut seed. Purified lipase from walnut seed exhibited the maximum activity at pH: 9.0 and 70°C. in order to determine the storage stability of lipase from walnut seed, the activity assays were performed for a period of one year. it was observed that about 85 % of its activity was retained at the end of 12* months at -20°C. When the CaCİ2 was added as activator, it was observed that the lipase activity was considerably increased by 0.5 mM CaCl2 concentration (287 %). it was calculated as 48 mM and 23.06 (xl0~) U/min.mg.enzyme that the Km and Vmax values of lipase from walnut seed for triolein as substrate. Activation energy of lipase from walnut seed was calculated as 3.26 kg/mol. When the effect of metal ions were examined, the hydrolytic activity was enhanced by EDTA, but was reduced by Cu+2, Hg+2, Mn+2, Ca+2, and Ni+2, while Zn+2, and Mg+2 had no effect on activity of lipase from walnut seed. in the assay with detergents, the effects on lipase enzyme of SDS, Triton X-100, and Tween-80 were determined for 5, 15, 25, and 35 mM detergent concantrations. When the effect of inhibitors on relative activity was investigated, it was observed that only n-bromosuksinimyd, urea, potassium cyanide, and potassium ferri-cyanide caused an inhibition. Finally, it was determined that the effects of different organic solvents on the enzyme from walnut seed at the end of 24 and 48 hours.
Lipases (triacylglycerol acylhydrolases, E.C. 3.1.1.3) catalyze the hydrolyze of triglyserids in water-fat interface. Application areas on bioprocessing of the lipases, their values and importance increase day by day. in this study, it was aimed that lipase enzyme from walnut seed identified as "şebin" species was isolated, purified and characterized. Lipase enzyme from walnut seed was purified about 29 fold to homogeneous state by ammonium sulphate precipitation (60-90) and dialyse after by defatting from walnut proteins. At each purification step, protein concentration was determined by the Lowry method and the lipase activity was defined by using titrimetric method. it was determined that the optimum reaction time of walnut lipase is 13 minutes. it was also determined that the triolein was the most suitable substrate for the lipase from walnut seed. Purified lipase from walnut seed exhibited the maximum activity at pH: 9.0 and 70°C. in order to determine the storage stability of lipase from walnut seed, the activity assays were performed for a period of one year. it was observed that about 85 % of its activity was retained at the end of 12* months at -20°C. When the CaCİ2 was added as activator, it was observed that the lipase activity was considerably increased by 0.5 mM CaCl2 concentration (287 %). it was calculated as 48 mM and 23.06 (xl0~) U/min.mg.enzyme that the Km and Vmax values of lipase from walnut seed for triolein as substrate. Activation energy of lipase from walnut seed was calculated as 3.26 kg/mol. When the effect of metal ions were examined, the hydrolytic activity was enhanced by EDTA, but was reduced by Cu+2, Hg+2, Mn+2, Ca+2, and Ni+2, while Zn+2, and Mg+2 had no effect on activity of lipase from walnut seed. in the assay with detergents, the effects on lipase enzyme of SDS, Triton X-100, and Tween-80 were determined for 5, 15, 25, and 35 mM detergent concantrations. When the effect of inhibitors on relative activity was investigated, it was observed that only n-bromosuksinimyd, urea, potassium cyanide, and potassium ferri-cyanide caused an inhibition. Finally, it was determined that the effects of different organic solvents on the enzyme from walnut seed at the end of 24 and 48 hours.
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Anahtar Kelimeler
Kimya, Chemistry
