Purification and immobilization of L-asparaginase from Citrobacter freundii EGY-NE1

Abstract L-asparaginase is used as an antileukemic drug and as a food additive to reduce the risk of acrylamide formation. New L-asparaginases are required to reduce costs and avoid clinical side effects. Citrobacter freundii EGY-NE1represents a potential source of new L-asparaginase. We purified extracellular L-asparaginase from Citrobacter freundii EGY-NE1 (through ammonium sulfate precipitation, dialysis, Sephadex-G50 and DEAE-cellulose columns) to 5.83 fold and 25.76 % recovery. The purified L-asparaginase was a low molecular weight enzyme of 19 kDa. It was optimally active at 37 ˚C, pH 8 and 40 mM asparagine. The Km and Vmax of the enzyme were 0.0179 M and 2.66 U/ml, respectively. Ca2+, Mg2+, K+ and Ba+ 2 activated the enzyme, while Na+, Zn+ 2, EDTA, azide, tartrate and HgCl2 inhibited it. The crude and purified enzymes were immobilized by encapsulation in Ca-alginate; this improved their stability and reusability. The entrapped L-asparaginases on Ca-alginate were examined by scanning electron microscope; their protein diameters ranged from 42.17 to 47.37 nm and from 46.78 to 71.97 nm for the immobilized crude and purified enzymes, respectively. Both the immobilized enzymes kept their maximal activity for 10 minutes at 40 ˚C. After the 5th cycle of repeated use, the immobilized purified and crude enzymes kept 91% and 89 % of their activities, respectively.