Purification and Biochemical characterization of a novel Recombinant L-glutamin-(asparagin-)ase from E.coli BL21 (DE3)

The recombinant enzyme containing a 6xHistidin-tag can be easily purified by Ni-NTA chromatography. Besides, its molecular weight was determined to be 40 KDa by SDS-PAGE .The biochemical properties of a novel recombinant L-glutamin-(asparagin-)ase was studied in the presence of L-glutamine(L-Gln) and L- asparagine (L-Asn) as substrates. The highest activity of the enzyme has been shown at pH 8 and 7. It has also been noticed the maximum activity L-glutamin-(asparagin-)ase was o.994 and 1.006 IU/ml towards L-asparagine and L-glutamine, respectively. On the other hand, the maximum L-glutamin-(asparagin-)ase activity was at 37 and 40 °C. Moreover, the enzyme kept more than 90% of its activity at temperature degrees ranged between 25-37 °C for 30 min. Metal ions (2 and 5 mM), such as MgSO4.7H2O, CuSO4.5H2O, FeSO4.7H2O, CaCl2 2H2O and KCl have a slight effect on the activity of L-glutamin-(asparagin-)ase at a high concentration of ions. Accordingly, both enzymatic activities were not modified in the presence of EDTA at 2 and 5 mM while the remaining enzyme activity % was reduced into 74% and 68% in the presence of each 5mM of 2-mercaptoethanol (2-ME) and Dithiothreitol (DTT) , respectively. The enzymatic L-glutamin-(asparagin-)ase activities increased in the presence of 5% of NaCl in the reaction buffer. Finally, the results of enzyme Kinetics have showed that the Km and Vmax values of L-glutamin-(asparagin-)ase were 0.067 mM and 0.042 mM /min towards L-asparagine and 0.054 mM and 0.041 mM/min towards L-glutamine, respectively.