Ekstremofilik bir bakteri olan Deinococcus radiodurans’dan β–Karbonik Anhidraz’ın klonlanması, Aşırı İfadesi ve Saflaştırılması

Abstract

Bu çalışmada, radyasyona ekstrem dirençli bir bakteri olan Deinococcus radiodurans’dan direnç fizyolojisinde önemli olduğunu düşündüğümüz karbonik anhidrazın (DrCA) enziminin klonlanması, saflaştırılması ve başlangıç karakterizasyonu gerçekleştirilmiştir. Ayrıca, artan dozlarda gama radyasyonun pH ile ilişkili DrCA enzim aktivitesi üzerindeki etkisi belirlenmiştir. Radyasyon uygulamasından sonra DrCA aktivitesi, 800 Gy’a kadar sürekli artarak 6 kat, artmıştır, bu noktadan sonra aktivitede azalma olduğu gözlenmiştir. DrCA enzimi için maksimum CO2 hidrasyon aktivitesi, pH 7.0 ve 40°C’de gözlenmiştir. Homo-dimer kompleks yapısında olan DrCA enzimi hafif termostabildir. DrCA’nın CO2 hidrasyon aktivitesini, çeşitli metal iyonları, özellikle Zn2+ önemli ölçüde, 5 kat artırmıştır. Ayrıca sülfonamid saf enzim üzerinde inhibe edici etki göstermiştir. DrCA’nın substrat olarak CO2 için belirlenen Km ve Vmax değerleri sırasıyla 8.4 mM ve 637 WAU/mg idi. CO2 hidrasyon deneyi saflaştırılmış rekombinant enzimin (DrCA) spesifik aktivitesinin önemli ölçüde yüksek olduğunu göstermiştir.

Keywords

• Deinococcus radiodurans
• karbonik anhidraz
• iyonlaştırıcı radyasyon

Cloning, Over-Expression, and Purification of β–Carbonic Anhydrase from an Extremophilic Bacterium: Deinococcus radiodurans

Abstract

In this study, the cloning, purification and initial characterization of carbonic anhydrase (DrCA) enzyme which we consider to be important in the resistance physiology from extremely radioresistant bacteria Deinococcus radiodurans is performed. In addition, the effect of increased gamma irradiation doses on pH-related DrCA enzyme activity was determined. DrCA activity after radiation treatment showed that the activity continuously increased by 6 fold, up to the first 800 Gy, which a decrease in activity was observed thereafter. The maximum CO2 hydration activity for DrCA enzyme was observed at pH 7.0 and 40°C. DrCA enzyme, homo-dimer complex, is slightly thermostable. The activity of DrCA was significantly enhanced by several metal ions, especially Zn2+, which resulted in 5-fold increases of CO2 hydration activity. Also sulfonamide showed inhibitory effect on the pure enzyme. The apparent Km and Vmax for CO2 as substrate were 8.4 mM and 637 WAU/mg for DrCA respectively. The CO2 hydration assay demonstrated that the specific activity of purified recombinant enzymes (DrCA) was significantly high.

Keywords

• Deinococcus radiodurans
• carbonic anhydrase
• ionizing radiation

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