A-Galaktozidaz yüklü mikropartiküller: sentez, karakterizasyon ve In vitro salım çalışmaları
Küçük Resim Yok
Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Enzimler uzun zamandır çeşitli endüstriyel sektörlerde kullanılmaktadır. Biyorafineride yenilenebilir malzemeleri kullanmak üzere son yıllarda artan ihtiyaç ve ilgi, enzimlerin endüstriyel uygulamasına daha fazla dikkat çekmiştir. ?-Galaktozidaz (?-D-galaktozid galaktohidrolaz, EC 3.2.1.22), hem basit hem de kompleks oligosakkaritlerin terminal ?-galaktozil kısımlarının hidrolizini katalize eden glikozid hidrolaz enzim grubudur. ?-Galaktozidazlar doğada yaygın halde bulunur. Enzimlerin immobilizasyonu, temel olarak enzim maliyetlerinin işlem ekonomisi üzerindeki payını en aza indirmek amacıyla, enzimin birçok kez tekrar kullanılmasını mümkün kılan yaygın bir uygulamadır. İmmobilize edilmiş bir enzimin kullanımı ayrıca genellikle alt akım işlemini kolaylaştırır, çünkü ortamdan kolayca uzaklaştırılabilir. Enzimler ve destek taşıyıcıları arasındaki etkileşim düşünüldüğünde, enzim immobilizasyon yöntemleri fiziksel yöntemler ve kimyasal yöntemler olarak sınıflandırılabilir. Adsorpsiyon ve tutuklama, enzimler ve destek taşıyıcıları arasında kovalent etkileşimlerin olmadığı fiziksel yöntemlere dahil edilirken, kovalent bağlanma ve çapraz bağlanma kimyasal yöntemlere aittir. Bu projede, mısır unundan izole edilen ve gradient amonyum sülfat çöktürmesi ile kısmi olarak saflaştırılan ?-galaktozidaz enzimi tutuklama yöntemi ile kalsiyum aljinat-kitosan mikropartiküllerde immobilize edildi. Bu amaçla, mikropartiküllerin sentezi, karakterizasyonu ve son aşamada da in vitro ortamda salım çalışmaları gerçekleştirildi. İmmobilize enzim preparatının hazırlanması için belirlenen optimum koşullar; 0.1 mg protein, % 3 (w/v) aljinat, 0.15 M CaCl2, % 0.5 (w/v) kitosan, 1 saat çalkalama, %0.05 (v/v) glutaraldehit ve 1 saat inkübasyon olarak belirlenmiştir. Optimize edilen koşullarda hazırlanan immobilize ve serbest ?-galaktozidaz enzimlerinin karakterizasyon çalışmaları yapılmıştır. Serbest ve immobilize enzim için optimum sıcaklık 40°C olarak belirlenmiştir. Termal kararlılık denemesinde serbest enzim 60°C, immobilize enzim ise 70°C sıcaklıkta hala aktivitesinin %50' sini korumuştur. Optimum pH değeri serbest ve immobilize enzimler için sırasıyla pH 4.5 ve 5.5 olarak bulunmuştur. Serbest enzim pH 3.0 – 6.5 aralığında aktivitesinin %80'inden fazlasını korurken, immobilize enzim pH 4.5 – 6.0 arasında aktivitesinin %70' den fazlasını korumuştur. Tekrar kullanılabilirlik denemesinde immobilize enzim 2 kere kullanılabilmiştir. İmmobilize enzim için in vitro ortamda salım çalışması yapılmıştır
Enzymes have been used in various industrial sectors for a long time. The growing need and interest in the use of renewable materials for biorefinery in recent years has brought more attention to the industrial application of enzymes. ?-Galactosidase (?-D-galactoside galactohydrolase, EC 3.2.1.22) is a group of glycoside hydrolase enzymes that catalyze the hydrolysis of terminal ?-galactosyl moieties of both simple and complex oligosaccharides. ?-Galactosidases are common in nature. Immobilization of enzymes is a common practice that makes it possible to reuse the enzyme several times, mainly to minimize the share of enzyme costs on the process economy. The use of an immobilized enzyme also generally facilitates downstream processing because it can be easily removed from the medium. Considering the interaction between enzymes and support carriers, enzyme immobilization methods can be classified as physical and chemical methods. Adsorption and encapsulation are included in physical methods without covalent interactions between enzymes and support carriers, while covalent binding and cross-linking belong to chemical methods. In this project, ?-galactosidase enzyme isolated from corn flour and partially purified by gradient ammonium sulfate precipitation was immobilized in calcium alginate chitosan microparticles with encapsulation method. For this purpose, synthesis, characterization and in vitro release of microparticles were carried out. Optimum conditions for the preparation of immobilized enzyme were determined as; 0.1 mg protein amount, 3% (w/v) alginate, 0.15 M CaCl2, 0.5% (w/v) chitosan, 1 hour shaking, 0.05% (v/v) glutaraldehyde and 1 hour incubation. Characterization studies of free and immobilized ?-galactosidase prepared under optimized conditions were performed. The optimum temperature for the free and immobilized enzyme was determined as 40°C. In the thermal stability test, the free and immobilized enzyme maintained %50 of their initial activity at 60°C and 70°C, respectively.. The optimum pH values for free and immobilized enzymes were found to be pH 4.5 and 5.5, respectively. The free enzyme maintained more than 80% of its activity in the pH range of 3.0 - 6.5, while the immobilized enzyme maintained more than 70% of its activity between pH 4.5 - 6.0. In the reuseability assay, immobilized enzyme could be used twice. In vitro release studies were performed for the immobilized enzyme.
Enzymes have been used in various industrial sectors for a long time. The growing need and interest in the use of renewable materials for biorefinery in recent years has brought more attention to the industrial application of enzymes. ?-Galactosidase (?-D-galactoside galactohydrolase, EC 3.2.1.22) is a group of glycoside hydrolase enzymes that catalyze the hydrolysis of terminal ?-galactosyl moieties of both simple and complex oligosaccharides. ?-Galactosidases are common in nature. Immobilization of enzymes is a common practice that makes it possible to reuse the enzyme several times, mainly to minimize the share of enzyme costs on the process economy. The use of an immobilized enzyme also generally facilitates downstream processing because it can be easily removed from the medium. Considering the interaction between enzymes and support carriers, enzyme immobilization methods can be classified as physical and chemical methods. Adsorption and encapsulation are included in physical methods without covalent interactions between enzymes and support carriers, while covalent binding and cross-linking belong to chemical methods. In this project, ?-galactosidase enzyme isolated from corn flour and partially purified by gradient ammonium sulfate precipitation was immobilized in calcium alginate chitosan microparticles with encapsulation method. For this purpose, synthesis, characterization and in vitro release of microparticles were carried out. Optimum conditions for the preparation of immobilized enzyme were determined as; 0.1 mg protein amount, 3% (w/v) alginate, 0.15 M CaCl2, 0.5% (w/v) chitosan, 1 hour shaking, 0.05% (v/v) glutaraldehyde and 1 hour incubation. Characterization studies of free and immobilized ?-galactosidase prepared under optimized conditions were performed. The optimum temperature for the free and immobilized enzyme was determined as 40°C. In the thermal stability test, the free and immobilized enzyme maintained %50 of their initial activity at 60°C and 70°C, respectively.. The optimum pH values for free and immobilized enzymes were found to be pH 4.5 and 5.5, respectively. The free enzyme maintained more than 80% of its activity in the pH range of 3.0 - 6.5, while the immobilized enzyme maintained more than 70% of its activity between pH 4.5 - 6.0. In the reuseability assay, immobilized enzyme could be used twice. In vitro release studies were performed for the immobilized enzyme.
Açıklama
Anahtar Kelimeler
Biyokimya, Biochemistry, Enzimler, Enzymes, Galaktosidazlar, Galactosidases, Kalsiyum aljinat, Calcium alginate, Kitosan, Chitosan, Mısır, Maize, Mısır unu, Corn flour, Zea mays, ; İmmobilizasyon, Immobilization