L-asparaginase (E.C. 3.5.1.1), an anti-cancer drug has been used in the treatment of acute lymphoblastic leukemia. A novel source of L-asparaginase from Pseudomonas fluorescens was addressed in the present studies. The ANS gene in Pseudomonas fluorescens MTCC 8127 which produces L-asparaginase was cloned and expressed in E. coli BL21 (DE3). The expressed recombinant protein (PfAns) which was purified, showed L-asparaginase activity. The enzyme was further characterized. The pH and temperature optima were found to be 7.5 and 37 degrees C. The recombinant enzyme was stable to temperature and pH. The enzyme was homotetramer with the molecular weight of the monomer being 35 kDa and a whole protein molecular weight of 140 kDa. The purified L-asparaginase had a specific activity of 26 U/mg with a K-m and V-max of 0.050 M and 4.032 mol(-1)min. The enzyme exhibited a high affinity towards L-asparagine and showed a very minimal activity with glutamine as a substrate. The enzyme activity was inhibited by PMSF, suggesting the presence of serine at the active site. The presence of Mg2+ enhanced PfAns activity by 49%, and SDS strongly inhibited the enzyme activity. The in vitro half-life of the recombinant enzyme was similar to 40 h. The enzyme demonstrated deglycosylation activity which could exhibit an additional barrier for proliferating cancer cells. (C) 2017 Published by Elsevier Inc.