Optimization, purification and characterization of recombinant L-asparaginase II in Escherichia coli

We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for L-asparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant L-asparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 312.8 U/mg. Kinetic parameters, K sub(m), V sub(max), K sub(cat) and K sub(cat)/K sub(m) of purified enzyme were found to be 0.5 mM, 500 U/mg, 14.9 10 super(3) s super(-1), and 29.9 10 super(3) mM super(-1)s super(-1), respectively. Temperature and pH optimum were observed at 45 super(o)C and pH 7.5, respectively. The enzyme exhibited about 20 and 60% retention of activity following 100 min incubation at 55 or 40 degree C, respectively. The activity of enzyme was inhibited by EDTA, Hg super(2+), Cu super(2+), Ni super(2+), and enhanced by Mg super(2+). Detergents (Tween 20, Tween 80, Triton X-100, and Triton X-114) decreased enzyme activity. DTT and DMSO at appropriate concentrations enhanced enzyme activity. In vitro anti-cancer activity was performed using different tumor cell lines. Concentration of recombinant L-asparaginase at 50 mu g/ml inhibited 45.32, 48.22, 53.68, 51.22% with HL-60, P388, P3X63Ag8, SP2/0-Ag14 cell lines. Recombinant L-asparaginase was expressed successfully in Escherichia coli with high expression level, had a high specific activity and antiproliferative effect on several tumor cell lines.