Isolation and partial purification of the clotamine synthetase from the black bean plant (Nigella sativa L.)

ABSTRACT Callus induction occurred on stem segments of Nigella sativa L. seedlings grown on a modified Murashige and Skoog medium containing 10-6 M of 2, 4- dichlorophenoxy acetic acid (2, 4 D). Glutamine synthetase (EC 6. 3 . 1. 2) was isolated and partially purified from Nigella sativa L. callus the partially purified enzyme from the aqueous extract by ammonium sulfate precipitation, dialysis and gel filtration on sephadex G 200 revealed that the activity increased by 37 fold. The molecular weight by gel filtration chromatography was found to be around 50000 Da. The optimum conditions of the partially purified glutamine synthetase from callus were obtained by using Tris HCl (80 mM) at pH 2.7 as a buffer and 10 mM of glutamate as substrate with incubation temperature 30 C. The Michaelis Menten constants for glutamate, adenosinetriphophate and ammonium chloride were 10 10-3M, 2 10-3 M and 0.280 10-3 M respectively.