Abstract:
L-Methionase has emerged as a potent enzyme with promising applications in cancer therapy due
to its ability to selectively deplete methionine an essential amino acid for methionine-dependent
tumor cells. This study aimed to isolate and characterize fungal strains capable of producing Lmethionase,
optimize its production, purify the enzyme, and evaluate its in vitro anticancer
potential. Soil samples were collected from diverse ecological regions across Gujarat, India
including marine, riverine, and agricultural sites to explore fungal biodiversity. A total of 50 fungal
isolates were obtained, and qualitative screening using modified Czapek-Dox agar identified
Aspergillus fumigatus MF13 as the most potent L-methionase producer. Quantitative assessment
through enzyme assay and specific activity estimation further confirmed MF13’s enzymatic
potential, with a maximum activity of 4.31 U/mL/min and a specific activity of 1.48 U/mg.
Molecular identification using ITS sequencing validated MF13’s identity as Aspergillus fumigatus
(GenBank accession: OQ690549). Optimization of enzyme production was achieved using a
combination of One-Factor-at-a-Time (OFAT), Plackett-Burman Design (PBD), and Central
Composite Design (CCD), culminating in a 2.57 U/mL/min yield under optimal conditions: 30°C,
pH 8.0, 2.4 g/L yeast extract, and 1.2 g/L dipotassium phosphate. Purification via cold acetone
precipitation and Sephadex G-75 chromatography resulted in a 10.5-fold increase in purity, with a
specific activity of 40.0 U/mg and molecular weight of ~45 kDa, as confirmed by SDS-PAGE.
Biochemical characterization showed optimal activity at pH 7.5 and 30°C, and notable stability
under alkaline and moderate thermal conditions. Enzyme kinetics revealed a Km of 0.674 mM and
Vmax of 0.871 U/mL, indicating strong substrate affinity. In vitro cytotoxicity assays (MTT)
demonstrated dose-dependent anticancer activity of purified L-methionase. HT-29 (colon cancer)
cells were highly sensitive (IC₅₀ ≈ 175 μg/mL), while MDA-MB-231 (breast cancer) cells showed
resistance (IC₅₀ ≈ 390 μg/mL), suggesting variable methionine dependency. This research
highlights Aspergillus fumigatus MF13 as a promising source of L-methionase and reinforces the
enzyme's potential as a selective anticancer agent. The successful optimization and purification
pave the way for further development in therapeutic applications, with future work focusing on
overcoming resistance mechanisms and evaluating in vivo efficacy.
