Generation of fungal L-glutaminase enzyme as an antineoplastic agent from various Egyptian country soil environments

Background:  Acute lymphocytic leukaemia and auxtrophic hepatocellular 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 lymphocytic 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. Methodology: In the current screening experiment, mineral glutamine agar (MGA), a selective medium, was employed for the production of fungal L-glutaminase (only fungi that used L-glutaminase as a single metabolic source for carbon and nitrogen could grow). The incubation conditions comprised PH 6.5, 25°C, and for 3 days. Malt agar media was further used to sub-cultivate fungal L-glutaminase producing strains. Thereafter, these strains were determined by means of biochemical procedures, microscopic analysis, and morphology. L-glutaminase-producing strains were molecularly identified by using the northern blotting method to detect molecular DNA hybridization. The fungal L-glutaminase gene was firstly isolated using specific PCR primers then the gene was subcloned and inserted into a DNA vector to be produced via recombinant DNA technology. In order to assess anticancer activity, the MTT test was performed. Results: Morphological, biochemical, and DNA blotting hybridization studies identified Aspergillus niger Strain ATCC 1015 as the major fungal isolate that produced this enzyme. The in vitro performance of fungal L-glutaminase as an antileukemic and anticancer mediator for hepatic auxotrophic tumours was very good. MTT 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: The current study was a promising study because it evolved a novel source of fungal L-glutaminase from various soil environments in Egypt with high efficacy as an anticancer agent against cancers that are auxotrophic for L-glutamine, such as hepatocellular carcinoma and acute lymphocytic leukaemia. It was also free of the enormous difficulties that bacterial enzymes present, such as hypersensitivity reactions, as shown by numerous previous studies. It is advised that more studies look at how to make fungal L-glutaminase more effective as an anticancer therapy.