Isolation and identification of bacterial isolates producing Arginine
deiminase from assorted soil environments in Egypt using 16S rRNA
sequencing technique
Abstract
Background: Auxotrophic cancers for Arginine are a
leading cause of death worldwide. The manufacture of novel arginine
degrading enzymes such as Arginine deiminase enzyme is mandatory
due to this crisis. Aim of the study: Since certain tumor cells
are auxotrophic for Arginine, the depletion of the extracellular
Arginine by means of Arginine deiminase enzyme was
exploited in the present study to target such tumors.
Methodology: Selective recovery of some bacterial isolates from
different environmental sites in Egypt and assessment their capabilities
for Arginine deiminase production. Studying environmental and
physiological factors affecting Arginine deiminase production by
some selected isolates. Characterization of of activity Arginine
deiminase produced by certain selected isolates as well as its
production through bacterial recombinant DNA technology.
Results: The major bacterial isolates grown on mineral
Arginine agar( MAA) plates producing ADI were further identified
as Bacillus subtilis DE 111 using 16S rRNA sequencing technique.
The Arginine deiminase production and activity were optimal at
40℃ and alkaline pH. Mn+2,
Ni+2 and Co+2 metal
ions as cofactors were optimum activators for production and activity
of ADI. The results showed that the potent cytotoxic consequences
of ADI were exerted on the renal and leukemic cancer cell lines.
ADI produced via bacterial recombinant DNA technology
showed efficacious IC50 10.31± 0.2
and 16.08± 0.3 µg/ml against renal(
Caki-1) and leukemic( K-562) cancer cell lines,
respectively. The purified monomeric ADI was 36.18 KDa
molecular mass as determined using SDS-PAGE, the specific
activity reached 36.07 U/mg. Km, Vmax and
Kcat were 0.05871 M, 40.36 µmol/ml/min and
5.014min-1respectively. Optimum pH and
temperature for productivity and activity ranged from 6-10 and
37-70℃,respectively. Total protein estimation using
Bar-ford assay was determined to be 5.68 mg during the
initial culture. ADI purification was achieved using 70%
Ammonium Sulfate followed by Ni+2-immobilized
affinity column chromatography with a final purification fold of
15.03. In vitro determination of biological half life of
ADI using nesslerization assay was observed to be nearly
300 min. Conclusion: ADI produced from
Bacillus subtilis DE111 demonstrated efficacious anticancer
activities against leukemic( K-562) and renal( Caki-1)
auxotrophic cancers for Arginine due to the depletion of
L-arginine from the external surrounding environments.