Isolation of L-asparaginase enzyme from Bacillus subtilis bacteria isolated from native Iranian honey

Abstract

The enzyme L-asparaginase accelerates the hydrolysis of the non-essential amino acid L-asparagine into aspartic acid and ammonium. Acute lymphoblastic leukemia cells and some tumor cells are not able to synthesize the amino acid asparagine, while normal cells are able to make the asparagine they need. Therefore, leukemic cells need a large amount of asparagine and the survival of these cells depends on the presence of asparagine. Therefore, the presence of the decomposers of this amino acid in the blood circulation of patients leads to the induction of cell death.Purpose: Isolation of L-asparaginase enzyme from Bacillus subtilis bacteria isolated from native Iranian honey. Method:Bacterial species were optimized in terms of enzyme production and cultured in nutrient broth environment at 37 degrees Celsius. This culture medium contains essential materials for the production of l-asparaginase. After incubation for 72 hours, the culture medium containing the enzyme is separated from the cells by centrifugation at 6000 rpm for 20 minutes at 4 degrees Celsius. The amount of total protein is measured by the Bradford method. In the next step, using 6000 Dalton polyethylene glycol with different percentages and at different pH values, L-asparaginase is precipitated and at the end, the supernatant and the precipitated phases are analyzed using electrophoresis (SDS-PAGE). Findings: After examining all the gels and their compliance with the Bradford diagram, the obtained results indicate a very low concentration of proteins extracted from the samples, which is beyond the ability of the method used to identify these compounds. Conclusion: Bacillus subtilis species isolated from native Iranian honey are able to produce L-asparaginase enzyme. The amount of produced enzyme is small and due to dilution during the analysis processes, detection of separated enzyme is not possible in KP2, and KP6, and it is slightly visible in KH2 species. Obtained results of the Bradford assay and the assay using phenol red, the production of enzyme in KH2 is more than KP2 and it is more than KP6. According to the obtained results it is recommended to study the optimization of enzyme production in these microorganisms and also to apply HPLC methods to quantitatively analyse the concentration of the produced and precipitated enzyme.