Cloning, Over-Expression, and Purification of ?–Carbonic Anhydrasefrom an Extremophilic Bacterium: Deinococcus radiodurans

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 $CO_2$ 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 $Zn^{2+}$, which resulted in 5-fold increases of $CO_2$ hydration activity. Also sulfonamide showed inhibitory effect on the pure enzyme. The apparent Km and Vmax for $CO_2$ as substrate were 8.4 mM and 637 WAU/mg for DrCA respectively. The $CO_2$ hydration assay demonstrated that the specific activity of purified recombinant enzymes (DrCA) was significantly high.