Purification and biochemical characterization of assimilative nitrate reductase from Haloferax alexandrinus

The present thesis focuses on the purification and determination of biochemical properties of assimilative nitrate reductase (Nas) enzyme.Km values for H. alexandrinus Nas enzyme for nitrate and methyl viologen were 0,045 and 0,0065 mM. The optimal pH for the enzyme was 9.5 and the temprature was 50 oC. Isoelectric point of the enzyme was determined as 5.3. Nas activity increased directly proportional to the NaCl concentration and maximum activity was determined at 3 M NaCl concentration.The study was carried on with different concentrations of NaCl and H. alexandrinus Nas enzyme activity was found to be increased over 100 %with KCl when compared to NaCI. MgCI2 and AICI3 salts inhibited the enzyme activity.Nas activity of H. alexandrinus decreased by cyanide, azide, EDTA and sulphite but it was not effected by DTT.The enzyme was purified 70 fold at the end of purification steps. It was found to be a monomer with 72 ± 1.8 kDa molecular weight by SDS-PAGE analysis.