Production, chemical characterization and in vitro application of anti-cancerogenic l-asparaginase from different gram-negative bacteria

Bacterial L-asparaginase is an enzyme of high therapeutic value, due to its use in certain kinds of cancer therapies, mainly in acute lymphoblastic leukemia (ALL). This study was carried out to further clarify how the synthesis of this enzyme is affected from various culture conditions, given the fact that L-asparaginase is an enzyme regulated by oxygen and carbon catabolite repression. For this objective, Enterobacter aerogenes, Pseudomonas aeruginosa ve Escherichia coli and their recombinant strains carrying a recombinant oxygen uptake system, the Viteroscilla hemoglobin (VHb), were used. In this study, these host bacteria and their recombinants carrying the VHb were studied with respect to carbon catabolite repression inflicted by various carbon sources (glucose, fructose, sucrose, lactose and glycerol). Furthermore, the effect of different nitrogen sources (L-asparagine, L-glutamine, urea and sodium nitrite) on the enzyme synthesis was investigated. The strain with the highest enzyme activity was used for L-asparaginase isolation and purification (ammonium sulfate precipitation and ion-exchange chromatography). The purified enzyme was analyzed for various chemical characteristics (the temperature range for optimum and maximum activities, optimal pH, kinetic parameters and stability). Moreover, an in vitro setting simulating that of in vivo system was utilized to determine the stability of the enzyme, an important parameter for possible therapeutic application of the enzyme. It has been determined that the production of L-asparaginase in different bacteria is governed by complex regulatory mechanisms and conditions required for the optimal synthesis of the enzyme in one bacterium may not be applicable to other (e.g., except glutamine in nitrogen source, the presence of VHb in E. coli has a stimulatory effect on the enzyme synthesis, while in E. aerogenes its presence is inhibitory). This positive effect of VHb on L-asparaginase synthesis is an important finding since the enzyme with cancer therapeutic activity has mainly been produced and commercialized using E. coli. The chemical characterization of purified enzyme revealed an optimal activity at 40 oC and pH 9.0 while the maximum activity was recorded at 60 oC a finding in accordance with its known thermostabile nature. In this context, enzyme was determined to have a high stability during its storage at different temperatures and similar kinetic parameters to that of other Lasparaginases. Key words: L-asparaginase, Vitreoscilla hemoglobin, catabolic repression, therapeutic enzymes, enzyme purification.