Screening and Characterization of L-asparaginase Producing Bacterial Strains from the Soil in Bangladesh

Although the enzyme L-asparaginase (L-ASNase) from Escherichia coli, Erwinia and Serratia has been applied to treat certain lymphomas and leukemias, several medical complications such as severe immunological responses leading to hypersensitivity, anaphylaxis, etc. have limited its application. The researchers have documented that such impediments are due to the different biochemical and kinetic properties of L-ASNase, which are directly dependent on genetic variations in microbial strains. Thus, there is a compelling need to explore novel L-ASNase producing microorganisms that would exhibit different serological properties while retaining similar and/or better therapeutic effects against cancer cells. Heretofore, L-ASNase producing bacterial strains from Bangladesh have never been isolated and characterized. Therefore, the main objective of this research was to isolate and characterize these strains from unexplored and ecologically different habitats that could lead to developing a potential therapeutic drug with fewer immunological responses and side effects over the existing drugs in order to treat cancer patients in the near future. Two L-ASNase producing bacterial strains were successfully isolated from the soil of Hatirjheel lake in Dhaka for the first time. Molecular characterization revealed that both strains belonged to Pseudomonas aeruginosa and their DNA sequences were submitted to NCBI GenBank. The accession number OK446669 was obtained for the strain of P. aeruginosa EWUKR-1 and OL307081 for P. aeruginosa EWUKR-2. The specific activity of L-ASNase from EWUKR-2 (212.1 ± 14.8 U/mg protein) was significantly higher than that of EWUKR-1 (16.3 ± 0.8 U/mg protein) when they were grown in modified M9 media containing 0.5 g/l glucose at 370C for 24 hours. The experimental results revealed that both of these bacterial strains were extracellular L-ASNase producers. The enzyme from P. aeruginosa EWUKR-2 was partially purified using saturated ammonium sulfate followed by dialysis and concentrated using Vivaspin-20 centrifugal concentrator having MWCO of 30 kDa. The optimum temperature and pH of the partially purified enzyme were 370C and 7.5, respectively. The purification-fold after ammonium sulfate precipitation and yield of the concentrated enzyme were 2.8 and 101%, respectively. SDS-PAGE analysis revealed that the molecular weight of L-ASNase from P. aeruginosa EWUKR-2 was around 43 kDa.