Publication: Pektinaz enziminin farklı polimerik destekler üzerine klik reaksiyonu ile immobilizasyonu ve karakterizasyonu
Abstract
Meyve suyu endüstrisinde vizkoziteyi düşürerek bulanıklığın azaltılması gibi işlemlerde kullanılan pektinazlar, poligalakturonik asidin a-1,4-glikosidik bağının hidrolizini katalize eder. Pektinaz gibi endüstriyel uygulamaları olan enzimler yüksek üretim maliyeti, çalışma koşullarında kararsızlıklar, ürünlerin reaksiyon ortamından çekilememesi, yeniden kullanılabilirlik ve depolama sırasında aktivitede düşüşler gibi sınırlamaları vardır. Bu sınırlamaları en aza indirmek için alternatif olarak enzimlerin katı bir destek üzerinde immobilizasyonu kullanılmaktadır. Bu çalışmada, asetilen fonksiyonilitesine sahip pektinaz enzimi azid fonksiyonilitesine sahip PVC, PVC-PEG, PVC-Kitosan ve PVC-PEG-Kitosan desteklere klik reaksiyonu ile immobilize edildi. Polimerik destek üzerindeki fonksiyonel gruplar ATR-FTIR ile karakterize edildi. Serbest ve immobilize enzim üzerine aktivite çalışmaları farklı şartlarda yapıldı. Ayrıca immobilize enzimin taze sıkılmış elma suyunda bulanıklığa etkisi türbidimetrik olarak incelendi. Serbest enzim pH 6’da maksimum aktivite gösterirken farklı desteklere immobilize edilmiş enzimin pH 7’de maksimum aktivite gösterdiği bulundu. Ayrıca pH değişikliklerine karşı immobilize enzimin dirençli olduğu ve serbest enzime göre daha yüksek aktivite gösterdiği tayin edildi. Tüm desteklerde immobilize enzimin sıcaklık değişikliklerine karşı daha iyi direnç gösterdiği bulundu. 60 gün sonunda serbest enzim ilk aktivitesinin %40’ını korurken bu değer immobilize enzimlerde yaklaşık %70 olarak tayin edildi. 12 tekrar kullanımdan sonra immobilize enzimlerin 62-76 aralığında değişen yüzdelerde aktivite gösterdikleri bulundu.
Pectinases, which are used in processes such as reducing turbidity by lowering viscosity in the juice industry, catalyze the hydrolysis of the α-1,4-glycosidic bond of polygalacturonic acid. Enzymes with industrial applications such as pectinase have limitations such as high production cost, instability in operating conditions, inability to withdraw products from the reaction medium, reusability and decreases in activity during storage. To minimize these limitations, immobilization of enzymes on a solid support is used as an alternative. In this study, pectinase enzyme with acetylene functionality was immobilized to PVC, PVC-PEG, PVC-Chitosan and PVC-PEG-Chitosan supports with azide functionality by click reaction. The functional groups on the polymeric support were characterized by ATR-FTIR. Activity studies on free and immobilized enzymes were carried out under different conditions.In addition, the effect of immobilized enzyme on turbidity in freshly squeezed apple juice was investigated turbidimetrically. It was determined that while the free enzyme showed maximum activity at pH 6.0, the enzyme immobilized on different supports showed maximum activity at pH 7.0. In addition, it was determined that the immobilized enzyme was resistant to pH changes and showed higher activity than the free enzyme. It was found that the immobilized enzyme showed better resistance to temperature changes in all supports. At the end of 60 days, while the free enzyme retained 40% of its initial activity, this value was determined as approximately 70% in immobilized enzymes. After 12 reuses, it was found that the immobilized enzymes showed activity at varying percentages in the range of 62-76.
Pectinases, which are used in processes such as reducing turbidity by lowering viscosity in the juice industry, catalyze the hydrolysis of the α-1,4-glycosidic bond of polygalacturonic acid. Enzymes with industrial applications such as pectinase have limitations such as high production cost, instability in operating conditions, inability to withdraw products from the reaction medium, reusability and decreases in activity during storage. To minimize these limitations, immobilization of enzymes on a solid support is used as an alternative. In this study, pectinase enzyme with acetylene functionality was immobilized to PVC, PVC-PEG, PVC-Chitosan and PVC-PEG-Chitosan supports with azide functionality by click reaction. The functional groups on the polymeric support were characterized by ATR-FTIR. Activity studies on free and immobilized enzymes were carried out under different conditions.In addition, the effect of immobilized enzyme on turbidity in freshly squeezed apple juice was investigated turbidimetrically. It was determined that while the free enzyme showed maximum activity at pH 6.0, the enzyme immobilized on different supports showed maximum activity at pH 7.0. In addition, it was determined that the immobilized enzyme was resistant to pH changes and showed higher activity than the free enzyme. It was found that the immobilized enzyme showed better resistance to temperature changes in all supports. At the end of 60 days, while the free enzyme retained 40% of its initial activity, this value was determined as approximately 70% in immobilized enzymes. After 12 reuses, it was found that the immobilized enzymes showed activity at varying percentages in the range of 62-76.