Purification and Biochemical Characterization of L-Methioninase Produced from Klebsiella pneumoniae and Evaluation of In Vitro Cytotoxicity Against HepG2 and A549 Cancer Cell Lines with a Potential Pediatric Use

Abstract

In the present study, L-methioninase was produced from the Klebsiella pneumoniae isolated from clinical samples and was evaluated for its enzymatic and anticancer potential. A total number of 150 clinical samples were collected from different infected areas, from which 27 bacterial isolates were selected. One showed the highest L-methioninase production and was selected for further research.
Screening the modified M9 medium confirmed enzyme production through a characteristic color change, indicating methionine degradation. The quantitative DTNB assay revealed a progressively increased enzyme activity which was correlated with the protein concentration measured by the Bradford estimation method. These results confirmed the efficient enzyme biosynthesis. Optimization studies showed that maximum enzyme production was achieved at pH of 7.0, temperature of 37°C, and incubation period of 24 hours, with glucose and peptone as the most favorable carbon and nitrogen sources, respectively.
Purification of L-methioninase using ammonium sulfate precipitation followed by ion-exchange and gel filtration chromatography could significantly increase specific activity, reaching 55.5 U/mg with a purification fold of 9.2 and yield of 58.3%. The purified enzyme showed optimal activity at physiological conditions (37°C and pH 7.0), proving its suitability for biomedical uses.
The anticancer potential of enzyme was evaluated using MTT assay, showing a clear dose-dependent reduction in viability of A549 and HepG2 cancer cell lines. However, normal HdFn cells showed significantly higher survival rates, indicating selective cytotoxicity. High-Content Screening (HCS) analysis further revealed low cell viability, nuclear damage, increased membrane permeability, and mitochondrial dysfunction.
The results showed that the L-methioninase derived from K. pneumoniae has a strong enzymatic activity, stability under physiological conditions, and significant selective anticancer effects. These findings proved it as a promising therapeutic agent targeting methionine-dependent tumors to support further in vivo and clinical investigations.