L- Asparaginase activity of endophytic fungi isolated from Orobanche spp. In Iran

This research focuses on evaluating endophytes as sources of bioactive compounds to identify microorganisms capable of producing the anti-cancer enzyme L-asparaginase. The plant Orobanche spp. was selected as the host, from which various compounds such as phenylthanoide glycosides, steroids, terpenoids, organic acids and their derivatives, ligands, alkaloids and flavonoids have been extracted. Endophytic fungi were isolated using standard surface sterilization techniques. Initial screening for L-asparaginase production was conducted using a qualitative assay on a modified Czapek dox agar medium. Enzyme production was identified by the formation of pink halos around the fungal colonies on the agar medium. This color change resulted from the hydrolysis of asparagine into aspartic acid and ammonia, which caused the phenol red indicator to shift from yellow (under acidic conditions) to pink (under alkaline conditions). L-asparaginase activity was also quantified using Nessler's method. The results revealed that among 34 identified endophytic morphotypes, 20 exhibited L-asparaginase production, with activities ranging from 0.701 to 3.10 µmol⁻¹ mL⁻¹ min⁻¹Furthermore, four isolates with high L-asparaginase activity were identified, exhibiting activities between 1.50 and 3.10 µmol⁻¹ mL⁻¹ min⁻¹Endophytic fungi were identified based on morphological characteristics and phylogenetic analysis of DNA sequence data, including ribosomal ITS regions and TEF1 genes. The L-asparaginase-producing species belonged to the genera Alternaria, Fusarium, Trichoderma, Penicillium, and Macrophomina. This study demonstrated that endophytic fungi isolated from Orobanche spp. possess a significant capacity for the production of L-asparaginase and can be used as an alternative source for the production of this enzyme on an industrial scale.