الفهرس 
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Abstract
Asparaginases are known to be the cornerstone for treatment of acute lymphoblastic leukemia (ALL) and are used for treatment in all pediatric regimens as well as in the majority of adult treatment protocols. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. The optimization of different process parameters for L-asparaginase production by Streptomyces fradiae NEAE-82 using Plackett–Burman experimental design and response surface methodology was carried out. Sixteen assigned variables (temperature, incubation time, inoculum size, agitation speed, pH, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO4.7H2O, NaCl, FeSO4. 7H2O, ZnSO4 and CaCl2) and three dummy variables were screened using Plackett–Burman experimental design. The most positive significant independent variables affecting enzyme production (pH values, L-asparagine and NaCl) were further optimized by the Box-Behnken experimental design. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L asparaginase activity were found to be 8.5, 45 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml−1min−1, respectively. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS–PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The amino acid contents of the purified L-asparaginase produced by Streptomyces fradiae NEAE-82 was determined using amino acid analyzer. Histidine was the most represented amino acid in the quantified L-asparaginase (17.09 µg/mL). The safety pattern of purified L-asparaginase was scanned on human fibroblast cells. The anti-proliferative activity of L-asparaginase on cancer cells was quantitatively estimated on HepG2, Hep2 and CaCo2 cells. the activity of the extract against CaCo2 was superior to that with both HepG2 or Hep2 cells and recorded inhibition percentage reached up to 80.9 with cancer cell selectivity index reached 4.