Fungal L-Asparaginase from environmental isolates with antimicrobial and antibiofilm activities

Fourteen fungal species were isolated from sewage water and soil, of which ten strains showed positive extracellular L-asparaginase production by plate assay. Among them, Aspergillus nidulans AUMC17371, A. flavus AUMC17373, A. flavus AUMC17374, and A. terreus AUMC17372 exhibited the highest enzyme activities (1.35, 1.25, 0.97, and 0.79 U/mL, respectively). Optimization of culture conditions revealed that maximum production was achieved using fructose as carbon source, asparagine as nitrogen source, pH 6, and 30 °C. SDS-PAGE analysis confirmed enzyme production, with protein bands ranging from 35 to 45 kDa. Antimicrobial screening revealed strong activity against Gram-positive and Gram-negative bacteria; however, only limited antifungal effectiveness against Candida albicans was exhibited. MIC results showed that L-asparaginase from A. nidulans AUMC17371 showed the strongest antibacterial effect against Staphylococcus aureus, followed by A. flavus AUMC17373 against S. aureus and Pseudomonas aeruginosa. Antibiofilm assays indicated inhibition ranging from 22 to 69%, with A. flavus AUMC17373 showing the most potent effect. Cytotoxicity testing revealed that A. flavus AUMC17374 enzyme exhibited anticancer activity against MCF-7 and HepG2 cell lines with IC₅₀ values of 184 and 450 µg/mL, respectively. Computational analysis supported these findings, with molecular docking demonstrating stable binding of L-asparagine to A. nidulans AUMC17371 L-asparaginase (- 4.67 kcal/mol), and molecular dynamics indicating a structurally stable enzyme-substrate complex. Collectively, these results suggest that fungal L-asparaginase may serve as a promising candidate with antimicrobial, antibiofilm, and cytotoxic activities.