Background: Acute lymphoblastic leukaemia and hepatic auxotrophic carcinoma for L-glutaminase are two of the leading causes of mortality worldwide. The bacterial L-glutaminase enzyme, which is normally produced under harsh circumstances from Escherichia coli or Bacillus cereus, was used to treat acute lymphoblastic leukaemia. Fungal L-glutaminase is preferred over bacterial L-glutaminase due to less hypersensitivity events, such as allergic responses and medicine neutralization; as well as higher efficiency against hepatic carcinoma. Aim of Study: The objective of the present work was to produce fungal L-glutaminase enzyme from different soil types in Egypt to be used as an oncolytic agent.
Patients and Methods: In the current screening experiment, mineral L-glutamine agar (MGA), a selective medium, was employed for the production of fungal L-glutaminase. Malt agar media was further used to sub-cultivate fungal L-glutaminase producing strains. L-glutaminase-producing strains were molecularly identified by using the Southern blotting method. The fungal L-glutaminase gene was firstly isolated using specific PCR primers then the gene was sub-cloned and inserted into a DNA vector to be produced via recombinant DNA technology. In order to assess anticancer activity, the MTT test was performed. Molecular mass of L-glutaminase was assessed via a mass spectrometer; then confirmed with western blot assay. Results: Aspergillus niger Strain ATCC 1015 was identified as the major fungal isolate that produced this enzyme. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay test results showed that the IC50 values for anticancer activity against the cancer cell lines CCL-120 and JHH4 were 38.9 and 40.3 g/ml, respectively. L-glutaminase had a molecular mass of 65 kDa, a specific activity of 15.3 U/mg protein, a yield of 57.6%, and a purity factor of 3.8. Extracellular L-glutaminase, 6.8U/ml, was generated. The conventional vitamin C and L-glutaminase have IC50s for their antioxidant activity of 89 g/ml and 189 g/ml, respectively. The inoculum contained 1*108 spores/ml. Conclusion: As fungus in diverse Egyptian soil environments evolved into a novel source of the enzyme L-glutaminase, the current investigation was a promising one.