Üreazın modifiye edilmiş florisile kovalent immobilizasyonu ve serbest ve immobilize üreazın karakterizasyonu

Year 2019, Volume: 44 Issue: 3, 811 - 818, 30.09.2019

Özlem Alptekin

https://doi.org/10.17826/cumj.453980

Cited By: 1

Abstract

Amaç:Bu çalışmada Canavalia
ensiformis kaynaklı üreazın modifiye edilmiş Florisil’e (magnezyum
silikat)  kovalent immobilize edilmesi ve
immobilize üreazın karakterizasyonunun yapılarak biyoreaktör uygulamalarında
kullanılabilirliğinin araştırılması amaçlanmıştır.

Gereç ve Yöntem:Florisil, 3-aminopropiltrietoksisilan ile aktifleştirilmiş daha sonra
glutaraldehit ile modifiye edilmiştir. Modifiye edilmiş desteğe üreaz kovalent immobilize edilmiştir. İmmobilize
enzimin ve desteğin taramalı elektron mikroskobunda görüntüleri incelenmiştir. Serbest
ve immobilize üreazın optimum pH’sı, sıcaklığı, kinetik parametreleri (Km, Vmax, kcat/Km) belirlenmiş, 4°C’de ve
oda sıcaklığında depolama kararlılıkları incelenmiştir. İmmobilize üreazın
kesikli tip reaktörde tekrar kullanılabilirliği araştırılmıştır.

Bulgular:Serbest ve immobilize
üreaz için optimum pH sırasıyla 7,0 ve 6,5; sıcaklık ise 50 ve 60°C olarak
belirlenmiştir. İmmobilizasyondan sonra enzimin Km değeri 2,2 kat artmıştır.
İmmobilize üreazın katalitik etkinliği, serbest üreazın katalitik etkinliğinin %.0,1’i
kadar bulunmuştur. Oda sıcaklığında ve 4°C’de serbest üreaz 5 gün sonunda ve aktivitesini
tamamen kaybederken immobilize üreaz 19 gün sonunda aktivitesini
kaybetmemiştir. İmmobilize üreaz kesikli tip biyoreaktörde 10 kullanım sonunda
başlangıç aktivitesinin %50’sini korumuştur. 

Sonuç:Modifiye Florisile kovalent immobilize edilen üreazın serbest üreaza
göre düşük katalitik aktivite göstermesine rağmen, immobilize üreazın serbest üreaza
göre depolama kararlılığının yüksek olması ve biyoreaktör uygulamalarında
tekrar kullanılabilmesi nedeniyle immobilize üreazın kullanım potansiyeli
yüksektir.

Keywords

Üreaz, immobilizasyon, bioreaktör

Covalent immobilization of urease onto modified florisil and characterization of free and immobilized urease

Abstract

Purpose: In this study, it was aimed to immobilize urease from Canavalia ensiformis onto modified Florisil (magnesium silicate) by covalently and to characterize immobilized urease and also to investigate reusability of immobilized urease in a batch type bioreactor.

Materials and Methods: Florisil was activated with 3-aminopropyltriethoxysilane and then modified with glutaraldehyde. Urease was covalently immobilized onto modified support. Images of support and immobilized urease were examined on a scanning electron microscope. The optimum pH and temperature, kinetic parameters (Km, Vmax, kcat/Km) and storage stabilities at 4°C and room temperature of free and immobilized ureases were determined. Reusability of immobilized urease was investigated in a batch type bioreactor.

Results:  Free and immobilized ureases showed their maximum activities at pHs 7.0 and 6.5, and at temperatures 50 and 60°C, respectively. The Km value of immobilized urease increased 2.2 fold compared to free urease. The catalytic efficiency of immobilized urease was about 0.1% of the free urease.  Free urease completely lost its activity at the end of 5 days stored at 4°C and room temperature; however, immobilized urease did not lose its activity at the end of 19 days at the same conditions. Immobilized urease maintained 50% of its initial activity at the end of 10 reuses in batch type bioreactor.  

Conclusion: Although urease activity significantly decreased upon covalent immobilization onto modified Florisil. However, high storage stability and reusability of immobilized urease makes it potentially useful immobilized form. 

Keywords

Urease, immobilization, bioreactor, Florisil

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