Tehran University of Medical Sciences Acta Medica Iranica 0044-6025 63 2 2025 07 09 73 82 Abbas Abed Noor Al-Owaidi Department of Biology, College of Sciences, University of Babylon, Babylon, Hillah, 51001, Iraq. Mohammed Abdullah Jebor Department of Biology, College of Sciences, University of Babylon, Babylon, Hillah, 51001, Iraq 2025 01 05 2025 04 10 The employment of L-Methioninase (L-Met), an enzyme degrading L-methionine, as a possible anticancer therapy has been provided that it selectively destroys cancer cells, which require methionine to grow. Many tumor tissues have a limited capacity for methionine production and depend on exogenous supplies of this amino acid. Therefore, they could be selectively attacked by methioninase-based treatment. The present study was carried out to investigate the effect of L-Met on cancer cells, especially colorectal cancer (HCT-116) and renal cell carcinoma (A498), and its potential as a therapeutic agent. Various techniques, such as ammonium sulfate precipitation, dialysis, ion-exchange chromatography, and gel filtration chromatography, were employed in this study for enzyme purification of L-Met. Cytotoxicity testing was performed against HCT-116 and A498 cells in the range of 25-200 µg/mL concentration by the MTT assay for viable cell quantification, Total Nuclear Intensity (TNI), and Cell Membrane Permeability (CMP). The statistical analysis was done using one-way ANOVA and Dunnett's multiple comparisons test to compare multiple groups. The optimal temperature for enzyme activity was 37° C, with pH 7 offering the best enzyme stability. Further investigation into incubation time revealed that a 48-hour period maximized enzyme yield. The enzyme was purified using a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration, achieving a 4.6-fold increase in purity. Characterization of the purified enzyme revealed a molecular weight of approximately 55 kDa, with optimal activity at pH 7 and 37°C. The enzyme demonstrated strong potential for therapeutic applications, showing dose-dependent cytotoxicity against colorectal cancer (HCT-116) and renal cancer (A498) cell lines, with significant inhibitory effects at concentrations as low as 25 µg/mL for colorectal cancer cells. The study highlights the potential of P. aeruginosa-derived L-Met showed strong activity in colorectal cancer, while activity in renal cell carcinoma was lower or inconclusive. https://acta.tums.ac.ir/index.php/acta/article/view/11461