Extraction, Optimization, and Purification of Anti-Cancer L-Glutaminase Enzyme from Bacillus Subtilis B7

This study investigated the production, purification, and vitro cytotoxic potential of L-glutaminase derived from Bacillus subtilis. Twenty-five bacterial isolates were recovered from diverse soil samples. Identification of B. subtilis was confirmed using standard biochemical assays, differential media, and the VITEK 2 automated system. Screening for L-glutaminase production identified B. subtilis isolate B7 as exhibiting the highest activity, with a crude enzyme-specific activity of 15.8 U/mg. Optimization of enzyme production parameters revealed glucose and yeast extract as optimal carbon and nitrogen sources, respectively, under controlled conditions of 40 °C and pH 6. L-glutaminase was purified using a four-step protocol: ammonium sulfate precipitation, ion exchange chromatography, gel filtration chromatography, and salt precipitation. Gel filtration chromatography resulted in the highest specific activity (754.6 U/mg), representing a 15.2-fold purification and a 21.1% yield relative to the crude extract. The in vitro cytotoxic effects of crude and purified L-glutaminase were evaluated using the MTT assay against the A549 (human lung adenocarcinoma) and WRL 68 (normal human liver) cell lines. Paclitaxel, a known chemotherapeutic agent, was a positive control for cytotoxicity against A549 cells. Treatment of A549 cells with purified L-glutaminase induced a dose-dependent reduction in cell viability, with a concentration of 400 µg/ml achieving maximal inhibition. These findings suggest that purified L-glutaminase from B. subtilis B7 demonstrates significant in vitro cytotoxic activity against A549 lung cancer cells and merits further investigation as a potential therapeutic candidate.