Isolation of Bacterial L‐Glutaminase as Anticancer Agent from Different Soil Environments in Egypt Using 16S rRNA Homology Technique
Authors: Kassab Mohammed MahmoudShawky 
Published: August 2024

Background:
Breast cancer and other neoplasms, such as acute lymphocytic leukaemia, are a
severe hazard to public health globally. L-glutamine-degrading enzymes are required
for the treatment of malignancies such as acute lymphocytic leukemia and breast
cancer. L-glutaminase, an amidohydrolase, has been shown to be an effective
chemotherapeutic tool for treating a variety of malignancies. The aim of the study:
Molecular characterization is used in the study and production of bacterial L- glutaminase as an anticancer agent from a variety of Egyptian soil locales. The type of the study:
Screening experimental study. Methodology:
In the current study, many bacterial isolates were tested on mineral L-glutamine
agar selective medium (MGA) to evaluate if they generated L-glutaminase. The most
effective positive bacterial isolates that produce L-glutaminase, however, were
discovered by morphological and biochemical analysis. The primary positive isolate
producing L-glutaminase was also identified utilizing molecular detection by 16S
rRNA sequencing homology technique. The properties of bacteria-produced L- glutaminase were investigated. The anticancer activity was assessed with an MTT
assay. Results:
Sole bacterial isolates that only needed L-glutamine for metabolic nitrogen grew well
on MGA. The optimal conditions for producing positive bacterial isolates were pH 7.4
and 37 ̊C. The morphological and biochemical investigations of soil samples from
diverse soil conditions in Egypt revealed that Bacillus subtilis subsp. niger (ATCC 9372)
was the primary positive bacterial isolate producing L-glutaminase. At a pH of 7.4
and a temperature of 37 °C, the activators KCL, ZnSO4, FeSO4, K2HPO4, and MgSO4
offered ideal conditions for defining L-glutaminase production. L-glutaminase from
Bacillus subtilis was detected to be a highly bioavailable and efficient anticancer
treatment. The original yield [productivity] from MGA was 6.3 U/ml, but invitro coupled
transcription and translation production raised it to 89± 1 U/ml. L-glutaminase had a
molecular mass of approximately 36 kDa. The purification of L-glutaminase resulted
total activity of 21,883 ± 15.67 (U), specific activity of 379.14 ± 7.35 (U/mg of
protein), and purification fold of 1.82 ± 3.06. The enzymatic activators Ni2+ and Mg
2+
increased L-glutaminase activity by 20%, 27%, respectively. The compound showed significant anticancer activity (IC50 = 38.72, 8.6, 6.41, 19.02, 49.83, and 9.45 μg/ml) against cancer cell lines from the liver (HepG-2), colon (HCT- 116), breast (MCF-7), lung (A-549), lymphocytic (CCL-120), and cervical (Hela). The kinetic parameters Km and Vmax were 15.2^ 10
-3 M and 119.86 μmol/ml/min,
indicating a higher affinity for the substrate. The enzyme demonstrated high efficacy at pH 6.5 and temperature 35. The enzyme
was thermostable at 58 ̊C for 1 hour.
Conclusion:
Acute lymphocytic leukemia and breast cancer are two instances of auxotrophic
malignancies for which L-glutamine is the sole metabolic source, and Bacillus subtilis- derived L-glutaminase was a suitable enzyme in their treatment.