Purification, characterization and dertermination of kinetic features of carbonic anhydrase from turbot (Psetta Maxima) muscle tissue

Öz

In this study, firstly, carbonic anhydrase purification from turbot (Psetta maxima) muscle tissue, together with analysis of the kinetic behaviour and some enzyme properties are described. The purification steps comprised hemolssate preparation, Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography and dialyze. The yield was 69.05% and the enzyme was found to be specific activity of 755.2 EU/mg proteins. The overall purification was about 50.65-fold. Temperature of +40C was maintained during the purification process. The molecular mass of the subunit was determined to be 29.7 kDa by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimal pH at 8.0 and stable pH at 8.0 and optimal temperature at 30ºC. Km and Vmax for p-nitrophenylacetate as a substrate were also determined.

Anahtar Kelimeler

• Fish
• carbonic anhyrase
• muscle
• turbot

Kalkan balığı (Psetta Maxima) kas dokusundan karbonik anhidrazın saflaştırılması, karakterizasyonu ve kinetik özelliklerinin belirlenmesi

Öz

Bu çalışmada öncelikle kalkan (Psetta maxima) kas dokusundan karbonik anhidrazın saflaştırılması, enzimin kinetik davranışının analizi ve bazı özellikleri belirlenmiştir.Saflaştırma basamakları; hemolizat hazırlanması ve Sepharose-4B-L tirozin-sülfanilamid afinite jel kromatografisi işlemidir. CA enziminin saflaştırılmasında, enziminin spesifik aktivitesi 755.2 EU/mg protein, verim 69.05 ve saflaştırma katsayısı 50.65 olarak belirlenmiştir ve yapılan saflaştırma işlemlerinin hepsi +4ºC'de gerçekleşmiştir. SDS poliakrilamid jel elektroforezi ile saflaştırılan CA enziminin molekül ağırlığı 29.7 kDa olarak belirlenmiştir. Kalkan balığı (Psetta maxima) kas dokusundan saflaştırılan CA enziminin optimum iyonik şiddeti 0.020 M Tris-SO4 tamponu, optimum pH’sı 8.0, stabil pH’sı 8.0 ve optimum sıcaklığı 30ºC olarak gözlenmiştir. Enzimin kinetik özellikleri içerisinde yer alan KM ve Vmax değerleri belirlenmiştir.

Anahtar Kelimeler

• Balık
• karbonik anhidraz
• kas
• kalkan balığı

Kaynakça

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