Purification and Characterization of L-asparaginase II Production from pseudomonas aeruginosa

Abstract

From twenty clinical isolates of pseudomonas aeruginosa were collected from Al-Yarmouk Teaching Hospital in Baghdad, Iraq, were
screened for findings of L-asparaginase-producing bacterial isolate with high yield, and were cultivated in a Modified M9 medium, which contained (L-asparagine 1%) was used to produce the L–asparaginase. After 2 days of incubation at 37 ° C, pH 7.5, with 250 rpm shaking, the highest L-asparaginase productivity was obtained. The extracellular enzyme was then extracted using a cooling centrifugation process to obtain the filtrate that represents the crude enzyme. Enzyme activity was then measured at every stage of the purification process, beginning with dialysis by sucrose and followed by ion-exchange chromatography with The specific activity of 4.2unit/mg protein, with a fold of purification of 5.2, and next by Sephadex G-200 column increases enzyme purity by 10.5-fold with specific activity 8.37 unit/mg protein after using gel filtration, making gel filtration the optimum stage in the purification process due to the high specific activity of the enzyme. Purified Lasparaginase had a molecular weight of 120 kDa by SDS- PAGE. The maximum activity of the enzyme was detected at 37°C and pH 8.0 after 30 min. Besides, the enzyme had stability at 7.0-8.0 with thermal stability at 30-40 °C. L-asparaginase is activated in the presence of metal ions such as K+, and Ca 2+, and chelating agent EDTA and strongly inhibited in the presence of, Hg 2+.