L-Asparaginase from Erwinia Chrysanthemi 3937: cloning, expression and characterization

Bacterial L-asparaginases (L-ASNases) catalyze the conversion of L-asparagine to L-aspartate and ammonia. In the present work, we report the cloning and expression of L-asparaginase from Erwinia chrysanthemi 3937 (ErL-ASNase) in Escherichia coli BL21(DE3)pLysS. The enzyme was purified to homogeneity in a single-step procedure involving cation exchange chromatography on an S-Sepharose FF column. The enzymatic and structural properties of the recombinant enzyme were investigated and the kinetic parameters (K(m), k(cat)) for a number of substrates were determined. In addition, we found that the enzyme can be efficiently immobilized on epoxy-activated Sepharose CL-6B. The immobilized enzyme retains most of its activity (60%) and shows high stability at 4 degrees C. The approach offers the possibility of designing an ErL-ASNase bioreactor that can be operated over a long period of time with high efficiency, which can be used in leukaemia therapy.