Expression and characterization of recombinant l-asparaginase from Pseudomonas fluorescens

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 °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 Km and Vmax of 0.050 M and 4.032 mol−1min. 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 ∼40 h. The enzyme demonstrated deglycosylation activity which could exhibit an additional barrier for proliferating cancer cells. •A novel recombinant l-asparaginase from Pseudomonas fluorescens was cloned and expressed in E. coli.•The expressed recombinant protein was purified in a single-step by affinity chromatography.•The purified enzyme showed l-asparaginase activity.•The enzyme exhibited a high affinity towards l-asparagine and showed a very minimal glutaminase activity.•The recombinant l-asparaginase caused deglycosylation of glycoprotein.