Three-Phase Partitioning of α-Galactosidase from Aspergillus lentulus: Optimization of System and Characterization of Enzyme

Abstract

Üçlü-faz ayırma (TPP) tekniği α-galaktozidazın Aspergillus lentulus’dan tek adımda kısmi saflaştırılması için ilk kez başarıyla kullanıldı. Yüksek aktivite ve saflaştırma katı elde etmek için enzimin ekstraksiyon etkinliğine amonyum sülfat konsantrasyonu, ekstrakt t-butanol oranı ve pH etkisi araştırıldı. Sistemin optimum saflaştırma parametreleri %55(w/v) amonyum sülfat konsantrasyonu, 1:1 (v/v) ham ekstrakt t-butanol oranı ve pH 5.5 olarak belirlendi. Bu optimize TPP sistemi α-galaktosidaz için 5.3 saflaştırma katı ile %178 aktivite verimi oluşturdu. pH 6.5 ve 50^oC’de maksimum aktivite gözlendi. α-Galaktozidaz 25-60°C sıcaklık aralığında ve pH 2.6-5.5 aralığında oldukça iyi bir kararlılık gösterdi. K_M ve V_max değerleri sırasıyla 0.365 mM ve 0.093 U olarak belirlendi. Metal iyonları ve şekerler arasında Na₂CO₃ ve galaktoz enzim aktivitesi üzerinde kuvvetli inhibitor etkisi gösterdi. TPP ile A. lentulus’dan farklı biyokimyasal özelliklere sahip yeni bir α-galaktozidazın elde edilmesi onun çeşitli biyoteknolojik uygulamaları açısından ilgi çekici olacaktır. 

Keywords

• α-galactosidase,bioseparation,three-phase partitioning (TPP),enzyme purification

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