Kanser tedavisinde kullanılan l-asparagınase used for cancer treatment to magnetic nanoparticles

Abstract

Bu tezde, anti-lösemik enzim E. coli kaynaklı L-asparajinaz (EC 3.5.1.1) manyetik poli(HEMA-GMA) nanopartiküllerine immobilize edilmiştir. Karakterizasyon çalışmaları ile manyetik nanopartiküllerin ortalama boyutunun 117.5 nm olduğu ve asparajinazın sentezlenen manyetik nanopartiküllere kovalent olarak bağlandığı bulunmuştur. immobilize edilen asparajinaz enzimi miktarı 66.43 mg asparajinaz/g nanopartikül olarak hesaplanmıştır. immobilize asparajinazın aktivitesine pH, sıcaklık ve substrat derişiminin etkisi incelenmiştir. Serbest asparajinazın optimum pH‟ı 7.5 bulunurken, immobilize asparajinazın pH‟ı 6.5 olarak bulunmuştur. immobilize asparajinazın optimum sıcaklığı serbest asparajinaza göre 10°C artmış ve 55°C olarak gözlenmiştir. Serbest asparajinazın kcat değeri (47356 dk-1), immobilize asparajinazınkinden (497 dk-1) daha yüksek bulunmuştur. Ayrıca serbest ve immobilize asparajinazın ısıl ve depo kararlılıkları incelenmiş ve immobilize asparajinazın işlemsel kararlılığı belirlenmiştir. Bu tezde immobilize asparajinazın yapay insan serumundaki etkinliğini belirlemek üzere serbest ve immobilize enzimin aktivitesi ölçülmüş ve immobilize asparajinazın serbest asparajinaza göre %74.74 aktivite gösterdiği gözlenmiştir. Elde edilen sonuçlar hazırlanan asparajinaz immobilizasyon işleminin başarılı olduğunu göstermektedir.

In this thesis, anti-leukemic enzyme L-asparaginase (E.C. 3.5.1.1.) from E. coli was immobilized on the magnetic poly(HEMA-GMA) nanoparticles. Characterization studies revealed that, the average size of the magnetic nanoparticles was 117.5 nm and asparaginase was covalently immobilized to the synthesized magnetic nanoparticles. Immobilized enzyme was calculated to be 66.43 mg asparaginase/g nanoparticles. The effects of pH, temperature and substrate concentration on the activity of the immobilized asparaginase were investigated. Optimum pH of free asparaginase was found to be 7.5, while optimum pH of the immobilized asparaginase was found to be 6.5. Optimum temperature of the immobilized asparaginase increased 10 °C, and was observed to be 55°C. kcat value of free asparaginase (47356 min-1) was found to be higher than that of immobilized asparaginase (497 min-1). Additionally, thermal and storage stabilities of free and immobilized asparaginase were investigated, and operational stability of immobilized asparaginase was determined. In this presented thesis, in order to detect the efficiency of the immobilized asparaginase in artificial human serum, activities of free and immobilized asparaginase were measured, and it was observed that, immobilized asparaginase carried 74.74 % of its initial activity. These results showed that, asparaginase immobilization process in this work was successful.