Enhanced production and purification of L-asparaginase from Bacillus paralicheniformis AUMC B-516 with potent cytotoxicity against MCF-7 cell lines.

Abstract

L-asparaginase is an important enzyme used in the pharmaceutical and food industries. Nowadays, bacterial species represent the main source of microbial synthesis for L-asparaginase. But studies aimed at improving production yields and new methods that use various microbes to expand the scope of application for the generated enzyme are also necessary for industrial manufacturing. This study focused on the isolation of Bacillus paralicheniformis AUMC B-516 and the optimization of L-asparaginase production under submerged fermentation. The enzyme was purified and characterized, followed by an evaluation of its cytotoxic effects against the MCF-7 human breast cancer cell line. The results revealed potent anticancer activity, highlighting the potential application of the purified enzyme in cancer therapeutics. Bacillus paralicheniformis AUMC B-516 was utilized for the biosynthesis of L-asparaginase (116.4 U/mL) after 48 h in the presence of 0.2% glucose and 1.0% L-asparagine at 35 °C and pH 8.0. Two-step chromatography (DEAE-cellulose and Sephacryl S200 HR) achieved a 12-fold purification, resulting in an enzyme specific activity of 4087.6 U/mg. For pure L-asparaginase that contained L-asparagine, the Km and Vmax values were 6.22 × 10^-2 mM and 120.75 µmol/min, respectively. Quantitative assessment of DNA fragmentation in MCF‑7 cells treated with B. paralicheniformis B-516' pure L-asparaginase was performed (22.2 ± 1.36%) and the drug doxorubicin (23.9 ± 0.93%) were significantly greater than those in the negative control cells (8.9 ± 0.83%). MCF-7 cells treated with 1000, 500, 250, 125, 62.5, or 31.25 µg/mL B. paralicheniformis AUMC B-516' pure L-asparaginase showed considerable cytotoxicity, with an IC₅₀ of 49.3 µg/mL. Biochemical analyses revealed significant increases in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, while blood glucose, other electrolyte levels, and indicators of renal function remained unchanged. These findings highlight B. paralicheniformis AUMC B-516 as a promising source of L-asparaginase for future biotechnological and pharmaceutical applications.