Alfa-galaktozidaz enziminin üçlü faz sistemi ile saflaştırılması
Küçük Resim Yok
Tarih
2011
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Alfa-D-Galaktozidazlar (alfa-D-galaktozid galaktohidrolaz, EC 3.2.1.22) doğada oldukça yaygın olarak bulunan hidrolaz sınıfı enzimlerdir. Bitkisel, hayvansal ve mikrobiyal kaynaklardan çok çeşitli teknikler ile izole edilerek saflaştırılan bu enzimler galaktooligosakkaritler ve polisakkaritlerde bulunan alfa-1,6-galaktoz birimlerini hidrolizlerler. Özellikle şeker endüstrisinde önemli bir potansiyele sahip olan alfa-galaktozidazlar, kompleks karbohidratların biyolojik fonksiyonlarının belirlenmesinde, organik sentezlerde, yapı analizlerinde ve medikal alanda oldukça önemli uygulama alanları olan enzimlerdir.Bu çalışmada, karpuzdan (Citrullus vulgaris) izole edilen alfa-D-galaktozidaz enzimi üçlü faz ayırma (Three Phase Partitioning; TPP) sistemi kullanılarak saflaştırıldı. TPP sisteminde organik çözgen olarak t-bütanol ve tuz olarak amonyum sülfat kullanıldı. TPP sistemini optimize etmek ve enzimi iyi bir verimle saflaştırabilmek için sistemi etkileyen temel parametreler (amonyum sülfat doygunluğu, enzim/t-bütanol oranı ve pH) incelendi. Saflaştırılan alfa-galaktozidaz enzimi karakterize edildi. Bu amaçla, enzim aktivitesine ve kararlılığına sıcaklık ve pH'ın etkisi incelenerek kinetik sabitler (Km ve Vmax) belirlendi. Enzimin saflığını belirlemek ve molekül kütlesini tayin etmek için sodyum dodesil sülfat poliakrilamid jel elektroforezi (SDS-PAGE) yapıldı.Optimize edilen koşullarda (%50 (w/v) amonyum sülfat doygunluğu, 1:1ham ekstrakt/t-bütanol oranı (v/v) ve pH 5,5) hazırlanan TPP sistemi ile alfa-galaktozidaz enzimi %76.7 aktivite verimi ile 2.67 kat saflaştırıldı. TPP sisteminden elde edilen enzimin molekül kütlesi SDS-PAGE analizi ile 45 kDa olarak belirlendi. Enzimin optimum sıcaklık ve optimum pH `sı sırasıyla 60°C ve pH 6 olarak belirlendi. Saflaştırılan enzimin, 4-50°C ve pH 4.0-6.5 aralığında oldukça kararlı olduğu belirlendi. Km ve Vmax değerleri Lineweaver-Burk diyagramından sırasıyla 0.22 M ve 0.14 U olarak hesaplandı. TPP, alfa-galaktozidazların saflaştırılmasında kullanılabilecek basit, hızlı, ekonomik ve oldukça ilgi çekici bir prosestir
?-Galactosidases (?-D-galactoside galactohydrolase, EC 3.2.1.22) are a common class of hydrolases which are widely distributed in nature. They have been isolated and purified with different techniques from various sources, such as plants, animals and microorganisms. These enzymes catalyses the hydrolysis of ?-1,6-galactose bonds which found in galactooligosaccharides and polysaccharides. They are very useful enzymes, especially for elucidation of the biological functions of complex carbohydrates, for organic synthesis, for structural analysis, for medical purpose and in sugar industry.In this study, ?-D-galactosidase isolated from watermelon (Citrullus vulgaris) was purified by three-phase partitioning (TPP). In this TPP system, t-butanol was used as organic solvent and ammonium sulfate was as a salt. Various parameters (ammonium sulfate saturation, enzyme/t-butanol ratio and pH) required for the efficient purification of the enzyme were optimized to get highest yield. The purified enzyme was also characterized. For this aim, the effect of pH and temperature on the activity and stability of the enzyme and determination of kinetic parameters (Km and Vmax) were also investigated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed to determine the purity and moleculer weight of the enzyme.Under optimised conditions (50% (w/v) ammonium sulfate saturation, 1:1 crude extract to t-butanol ratio (v/v) and pH 5,5) ?-galactosidase was purified to 2.6-fold with 76.68% activity recovery. The enzyme obtained from TPP showed considerable purification on SDS-PAGE with a moleculer weight of 45 kDa. The optimum temperature and pH were determined as 60°C and pH 6, respectively. The purified enzyme was also very stable at a temperature range of 4-50°C and a pH range of 4-6.5. The Km and Vmax values were calculatedfrom Lineweaver-Burk plot as 0.22 M and 0.14 U, respectively. TPP, is a simple, quick, economical and very attractive process for purification of ?-galactosidases.
?-Galactosidases (?-D-galactoside galactohydrolase, EC 3.2.1.22) are a common class of hydrolases which are widely distributed in nature. They have been isolated and purified with different techniques from various sources, such as plants, animals and microorganisms. These enzymes catalyses the hydrolysis of ?-1,6-galactose bonds which found in galactooligosaccharides and polysaccharides. They are very useful enzymes, especially for elucidation of the biological functions of complex carbohydrates, for organic synthesis, for structural analysis, for medical purpose and in sugar industry.In this study, ?-D-galactosidase isolated from watermelon (Citrullus vulgaris) was purified by three-phase partitioning (TPP). In this TPP system, t-butanol was used as organic solvent and ammonium sulfate was as a salt. Various parameters (ammonium sulfate saturation, enzyme/t-butanol ratio and pH) required for the efficient purification of the enzyme were optimized to get highest yield. The purified enzyme was also characterized. For this aim, the effect of pH and temperature on the activity and stability of the enzyme and determination of kinetic parameters (Km and Vmax) were also investigated. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed to determine the purity and moleculer weight of the enzyme.Under optimised conditions (50% (w/v) ammonium sulfate saturation, 1:1 crude extract to t-butanol ratio (v/v) and pH 5,5) ?-galactosidase was purified to 2.6-fold with 76.68% activity recovery. The enzyme obtained from TPP showed considerable purification on SDS-PAGE with a moleculer weight of 45 kDa. The optimum temperature and pH were determined as 60°C and pH 6, respectively. The purified enzyme was also very stable at a temperature range of 4-50°C and a pH range of 4-6.5. The Km and Vmax values were calculatedfrom Lineweaver-Burk plot as 0.22 M and 0.14 U, respectively. TPP, is a simple, quick, economical and very attractive process for purification of ?-galactosidases.
Açıklama
Anahtar Kelimeler
Biyokimya, Biochemistry, Alfa-galaktosidaz, Alpha-galactosidase, Karpuz, Watermelon
