Characterization and Cytotoxic Activity Evaluation of Superoxide Dismutase (SOD) Enzyme Purified From Escherichia coli.

Superoxide dismutases (SODs) are antioxidant metalloenzymes produced by all living cells, including E. coli. SODs enzymes have several roles in treatment of different diseases, including tumors. Its catalysing the dismutation of superoxide free radicals (O2•–) to H2O2 and H2O to protect cells against their harmful effects. This might prevent damage to tissues. Objective: Production and purification of antitumor enzyme (superoxide dismutase) from local isolate Escherichia coli. Methods: SOD was partially purified from E. coli using ammonium sulphate precipitation, DEAE-cellulose ion exchange and sephadex G-150 Gel filtration chromatography. Subsequently, the molecular weight of the purified SOD was determined using SDS-PAGE. Moreover, the activity and stability of SOD were examined under different pH and temperature values. Finally, cytotoxic potential assessed against colon cancer and normal cell lines. Results: Crude extract of SOD was partially purified using ammonium sulphate precipitation, DEAE-cellulose ion and sephadex G-150 Gel filtration chromatography, whereas specific activity of SOD was increased from 314.2 into 1357 U/mg protein with 3.5-fold and yield (52.5%). The activity and stability of SOD were examined under different pH and temperature values. The maximum SOD activity was 95 U/ml at pH 6, while the stability of enzyme (90 – 100%) at pH range (6-8). The maximum activity at 37oC. At 32-37oC, SOD enzyme was stable with remaining activity of 100%. Various agents (NaCl, CaCl2, H2O2, MnSO4 and urea) were used to estimate their effects on enzyme stability. The enzyme was maintaining its activity (100%) in presence of NaCl, CaCl2 and H2O2, while completely lose its activity in presence of urea (0.1M), and this activity was increased into 140% in presence of MnSO4 (0.1 M). Cytotoxicity of purified SOD was exanimated on primary dermal fibroblast normal (Hdfn) and a human colorectal adenocarcinoma cell-line (Caco-2) cell lines. The viability of (Caco-2) was decreased into 45% after treatment with SOD enzyme at 400 µg/ml after 24 hrs. On the other hand, the viability of HdFn was decreased into 75% after treatment with SOD enzyme at concentration of 400 µg/ml after 24 hrs. Conclusion: Lower IC₅₀ values indicate a stronger inhibition of cell viability at higher doses of SOD. Which suggesting its potential therapeutic for future applications.