Asparaginases are the cornerstone therapy of many successful combination regimens for the treatment of acute lymphoblastic leukemia (ALL), the most common malignancy in children and adolescents. The aim of this work was to produce recombinant Erwinia carotovora L-asparaginase II in Escherichia coli fed-batch cultures. Using a robust fed-batch technique with pre-determined exponential feeding rates, our bioreactor culture system yielded 30.7 grams of dry cell weight and 0.9 grams of soluble rErAII protein per liter of culture broth. The homogeneous rErAII activity was determined by isothermal titration calorimetry (ITC). The enzyme K m values for the main substrates L-Asn and L-Gln were 33x10 -6 M and 10x10 -3 M, respectively. Our work shows that it is possible to produce an active homogeneous rErAII enzyme in the soluble cell fraction through IPTG-induced E. coli fed-batch cultivation.