Agar-Kaplı Manyetik Nanopartiküller Üzerine Adsorpsiyon ile Amilaz İmmobilizasyonu

Öz

Bu çalışmada, inert atmosfer altında oda sıcaklığında agar içeren sulu bir çözelti içinde ferrik ve ferröz iyonlarının birlikte çökeltildiği, manyetik demir oksit nanopartiküllerin eşzamanlı sentezi ve kaplanması için yeni bir yöntem kullanıldı. X-ışını kırınımı (XRD) analizi, sentezlenen nanopartiküllerin kübik yapıya ve 9.38 ile 10.11 nm arasında değişen kristalit boyutuna sahip saf Fe3O4 olduğunu gösterdi. Taramalı elektron mikroskobu (SEM) görüntüleri, yüzey kaplama malzemesi konsantrasyonu arttıkça artan yüzey pürüzlülüğünü gösterdi. Üretilen manyetik partiküller adsorpsiyon yöntemiyle α-amilaz immobilizasyonu için destek olarak kullanıldı. Sonikasyon, immobilizasyon süresi (0,5; 1; 2; 4; 8; 16 saat), yüzey kaplama malzemesi konsantrasyonu (0; 0,5; %1 w/v agar), immobilizasyon pH'ının (pH 3,4,5,6, 7) protein yüklemesi, enzim aktivitesi ve spesifik aktivite üzerine etkileri araştırıldı. Sonikasyon, amilaz immobilizasyonunu arttırmadı. Enzimin spesifik aktivitesine dayalı olarak, optimum adsorpsiyon pH 4 ve 5'te 4 saatlik immobilizasyon süresinde elde edildi; bu koşullarda serbest α−amilaza kıyasla spesifik aktivitede 3 kat artış ve bağıl aktivitede %360 artış ölçüldü.

Anahtar Kelimeler

• Manyetik nanopartikül
• Fungal alfa amilaz
• Enzim immobilizasyonu
• Adsorpsiyon metodu.

Proje Numarası

20ADP099, 20AG025

Immobilization of Amylases via Adsorption on Agar-Coated Magnetic Nanoparticles

Öz

In this study, a new method for simultaneous synthesis and coating of magnetic iron oxide nanoparticles was employed, where ferric and ferrous ions were co-precipitated within an aqueous solution containing agar at room temperature under inert atmosphere. X-ray diffraction (XRD) analysis indicated that synthesized nanoparticles were pure Fe3O4 with a cubic structure and crystallite size ranging between 9.38 and 10.11 nm. The scanning electron microscope (SEM) images demonstrated the increasing surface roughness as concentration of surface coating material increased. The produced magnetic particles were used as a support for α−amylase immobilization by adsorption method. Effects of sonication, immobilization time (0.5; 1; 2; 4; 8; 16 h) concentration of surface coating material (0; 0.5; 1% w/v agar), immobilization pH (pH 3,4,5,6,7) on protein loading, enzyme activity and specific activity were investigated. Sonication did not enhance amylase immobilization. Based on the specific activity of the enzyme, the optimum adsorption was achieved at pH 4 and 5 after 4h-immobilization time, where, compared to the free α−amylase, a 3-fold increase in specific activity and 360% increase in relative activity was measured, respectively.

Anahtar Kelimeler

• Magnetic nanoparticles
• Fungal alpha amylase
• Enzyme immobilization
• Adsorption method.

Destekleyen Kurum

Eskişehir Teknik Üniversitesi, TÜBİTAK

Proje Numarası

20ADP099, 20AG025

Teşekkür

Authors would like to thank the Anadolu University Plant Drug and Scientific Research Center (AUBIBAM) for the SEM images. This work was financially supported by Eskisehir Technical University (ESTU) research fund with project number 20ADP099 and TÜBİTAK — The Scientific and Technological Research Council of Turkey with the project no. TÜBİTAK 20AG025 under program no. TÜBİTAK 20AG001. Nihal Yılmaz was supported by TUBITAK-BIDEB 2210/C National Scholarship Program for MSc students.

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