Production, isolation and purification with high efficiency of L- Asparaginase from bacteria E-Coli O55-B5

L-Asparaginase is now known to be a potent antineoplastic agent in animals and has given complete remission in some human leukemias. Extensive clinical trials of this enzyme, however, were not possible in the past because of inadequate production of this substance. We have developed practical procedures for producing L-asparaginase in yields of sufficient quantity and purity. The nutritional requirements for optimal production of biologically activeL-asparaginase by a strain of Escherichia coli have been ascertained. The purification was performed at 4ºC. The bacterium was cultured in nutrient broth at 370 C for 12-18 h to reach the end of the logarithmic phase. The cell suspensions were ruptured using a probe sonicator. E-Coli have two types of asparaginase I and II. The only type II enzyme have anti-leukemia activity. To remove the L-aspraginase I, the crude extract was heated at 550 C for 30 min at 40 C. The enzyme was purified using ammonium sulfate salt 35-65% final saturation, gel-filtration, DEAE-Cellulose anion exchanger culomn, and CM-Cellulose cation exchanger culomn, respectivelyWith the procedure described for isolation of biologically active L-asparaginase from E. coli, stable L-asparaginase preparations with a specific activity of 360 IU per mg of protein (315 -fold purification with 40%total recovery) were obtained

Keywords:

L- Asparaginase , purification. E-Coli , kinetic properties , Production