Material and microorganisms:
Diacetyl monoxime, L-citrulline and Thiocarbazide were purchased from Alnasr chemical company, Egypt. Glucose, Lactose, Mannitol, Maltose, Soluble starch, NaCl, L-arginine, KH2PO4, MgCl2, MnCl2, CoCl2, NiCl2, FeSO4, FeCl3, 90% Phosphoric acid and 85% Sulfuric acid were purchased from Algumhoria chemical company, Egypt. The bacterial isolates were obtained from different soil environments in Egypt. The chemicals and markers used for the molecular identification of ADI were obtained from Bio-Rad laboratories, USA. All chemical reagents utilized in the present study were of analytical grades.
Collection of samples:
50 soil samples in a depth 0–10 cm were collected from different governorates in Egypt.
Date and the place of the study:
The present study was conducted in the faculty of Pharmacy, Cairo University, Egypt between August 2022 and March 2024.
Screening of Arginine degrading bacteria using the plate technique:
The test samples were collected from various regions in Egypt at a depth 0–10 cm; besides the addition of 100 ml sterile water to the soil samples was achieved. On the other hand, 1 ml of each diluent sediment sample was transferred to the selective screening medium, Mineral Arginine Agar(MAA), and incubated for 24 at 37 ℃. The MAA plate consisted of 2% L-arginine, 0.01% glucose, 0.002% NaCl, 0.05% MgCl2, 0.075% KH2PO4, 0.02% MnCl2, 0.0001% FeSO4, 0.2 M CaCO3, 2.5% Phenol red and 1.2% Agar. Colonies which were able to utilize L-arginine as sole metabolic Nitrogen rootage were only grown on MAA plates and were considered ADI producing bacterial isolates which were further subcultured for numerous times until pure colonies were gotten.20
The produced colonies on MAA plates were further subjected to subculture on Nutrient agar plates.
Determination of activity of Arginine deiminase using nesslerization test:
The yielded pure colonies were subjected to further activity analysis of Arginine deiminase utilizing Nessler reagent to standardize the Ammonia concentration as the product of Arginine degrading enzymes. L-arginine was exploited as the sole metabolic Nitrogen source in the culture media. The soil samples were cultured in 20 ml of the defined medium; then incubated at 37℃for 24 hours. Afterwards, 2 ml of each growth medium was collected and centrifuged to assess the concentration of Ammonia in the supernatants; then 45 µl of Nessler reagent was added to each1 ml of obtained supernatants. The concentration of the produced Ammonia was quantified via measuring the optical density at 480 nm wavelength utilizing UV Spectrophotometer according to the standard curve of Ammonium sulfate. The calculated values were correlated to the activity of L-arginine deiminase within 24 hours of incubation.21
Diacetyl monoxime Thiocarbazide assay for assessment of ADI activity:
Based on the photometric assessment of the Citrulline concentration—which was produced after the ADI enzyme hydrolyzed L-arginine—the ADI enzyme activity was examined. The applicable reagents were made as following; Acid-ferric solution; 300 ml of concentrated Sulfuric acid( 90%) and 150 ml of concentrated Phosphoric acid( 90%) were added to 550 ml of distilled water. After allowing the mixture to cool to room temperature, 3 mg/L of FeCl3 was added to it. Diacetylmonoxime solution; 100 ml of distilled water was mixed with 500 mg of Diacetylmonoxime. Chromatogenic reagent; 50 ml of reagent 2 was mixed with 5 mg of Thiosemicarbazide, and then 100 ml of reagent 1 was added. The solution was applied within an hour of the setup. The culture medium intended for the selected colony was cultivated in 20 milliliters. 2 ml of each growth media was collected after 24 hours of incubation, the amount of freed Citrulline in the supernatants was measured by centrifugation.1ml of the supernatant and 2 ml of the Chromogenic solution were combined, and the mixture was heated for 5 minutes at 100ºC. After cooling to room temperature, the absorbance at 530 nm was calculated. Based on the L-citrulline standard curve, which was determined using the same technique, the amount of Citrulline was computed and associated with the activity of the ADI enzyme, which converted L-arginine to L-citrulline.22
Identification of the major bacterial isolate producing ADI:
This was performed using Gram staining technique and different biochemical reactions; as well as the determination of morphological characters and molecular detection using 16S rRNA sequencing technique were employed. The genomic DNA was extracted using PureLink™ genomic DNA Purification kit( purchased from ThermoFisher scientific, USA) on Silica Columns. The DNA extraction procedure was performed according to the DNA Purification Kit manufacture instructions.
Procedure of 16S rRNA sequencing technique:
The QIAGEN Multiplex PCR Kit, Catalog number 206143 was acquired from QIAGEN,USA.Molecular characterization of ADI producing bacteria was carried out via 16S rRNA sequence analysis using the forward and reverse primers with the sequences of( 5−-CAGCCGCGGTAATAC−3−) and( 5−-ACGGGGGGTGTGTAC−3−), respectively. Using polymerase chain reaction( PCR), 16S rRNA gene was cloned. Initial denaturation was at 98℃ for 5 minutes. This was followed by 25 cycles of denaturation at 94℃ for 30 sec. Annealing stage was performed at 54℃ for 30 sec. Finally, the extension phase was adjusted at 72℃ for 10 minutes. The size of the resulted amplicons were confirmed using Agarose gel electrophoresis. The yielded sequences were compared with sequences deposited in Gen-bank database of national centre for biotechnology information( NCBI) using nucleotide basic local alignment search tool( BLASTn).23
Determination of environmental and physiological factors affecting the productivity of ADI:
This was applied using different temperatures, pH, Metal ions, metabolic carbon and nitrogen sources, inoculum volumes, aeration conditions and substrate concentrations. Nesslerization technique was included to determine the amount of ADI produced spectrophotometrically at 480 nm wavelength.
Purification of ADI:
The cold crude enzyme was diluted with Ammonium Sulfate until 70% saturation was achieved. After being stored at 4°C for the whole night, the mixture was centrifuged for 25 minutes at 4000 ×g. The residue was dissolved in a minimum volume of 50 mM Tris-HCl buffer( pH 7.4) and dialyzed against the same buffer for a whole night at 4°C to get rid of the salts. A 45 x 1.5 cm Sephadex G-100 column pre-equilibrated with 0.05M Tris-HCl buffer( pH 8.6) was loaded with the dialyzed fraction. 0.05M Tris-HCl buffer (pH 7.4) with 0.1M KCl was used to elute the protein. The protein and enzyme activity of the fractions was measured after they were collected. After lyophilizing the fraction with the maximum amount of enzyme activity, the powder was kept at 4°C. Following that, SDS-PAGE, or Sodium Dodecyl Sulphate Polyacrylamide gel electrophoresis, was used. The cells were resuspended in 200 µl of lysis buffer 62.5 mM Tris/HCl, pH 6.8, 15% Glycerol, 5% 2-Mercaptoethanol, 2% Sodium Dodecyl Sulphate( SDS), and 0.001% Bromophenol blue in order to produce complete cell lysates. After 5 minutes of boiling at 100°C, the complete cell lysates were put onto a 15% SDS Polyacrylamide gel and run at 100 V. Coomassie blue staining was applied to the isolated proteins. The gel was then photographed using a Gel Documentation System after being moved to a de-staining solution to eliminate any remaining binding dye and leaving the stained proteins as visible blue bands.24
Estimation of protein content:
This was applied according to Bradford assay. Bovine Albumin was utilized as a standard drug.25
Determination of anticancer activity of ADI using MTT assay:
Human renal( Caki-1) and leukemic( K-562) cancer cell lines purchased from ATTC,UK were used to conduct the cytotoxicity assay. On the other hand Vero CCL-81™ cell lines( Purchased from ATCC, USA) were utilized also in MTT assay as standard natural cell lines. The cells were kept in Roswell Park Memorial Institute( RPMI) medium supplemented with 10% heat-inactivated foetal bovine serum, 100 µg/ml streptomycin, and 100U/ml penicillin at 37°C in a humidified environment with 5% CO2 V/V. Furthermore, the cells underwent twice-weekly subculturing. The MTT( 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was performed. The cells and test compounds were prepared in 96-well plates containing a final volume of 100 µl/well; then were incubated for 2 hours. 10 µl MTT Solution was added per well to achieve a final concentration of 0.50 mg/ml then incubated for 3 hours at 37°C. 100 µl Solubilization solution was added to each well to dissolve formazan crystals. The components were mixed to ensure complete solubilization. The absorbance was recordered at 570 nm.26 In vitro determination of biological half life of ADI was performed using nesslerization assay.
Determination of antioxidant activity of ADI:
This was applied via rapid DPPH radical scavenging assay using dot-blot. With a few minor adjustments, the Soler-Rivas et al., 2000 approach was used to qualitatively evaluate ADI extracts for anti-oxidant activity using DPPH radical. In summary, a small amount(5 µl) of every ADI extract dilution and standard antioxidant was meticulously placed onto a thin-layer chromatography( TMC) plate( Silica gel 60 F254, Merck) and left to air dry. After that, the sheets were sprayed with DPPH( 0.2% W/V in ADI to demonstrate the extract's antioxidant capacity. The scavenging potential and antioxidant actions of the extract were assessed by the intensity of the yellow colour and the rate at which the extract spots' colour changed from purple to yellow.27
Determination of apoptosis induced via ADI:
Caspase 3 assay was utilized for this purpose. The procedure was performed as described before according to Kim RH et al, 2009.28
Production of ADI via bacterial recombinant DNA technology:
Forward primer: 5−-TGACCCAATGCCAAACCTAT−3−
Reverse primer: 5−-CGTCTCCACCTTCGATTGAT−3−
The QIAGEN Multiplex PCR Kit, Catalog number 206143 was purchased from QIAGEN,USA. pET-26b(+), which was acquired from Novagene in the USA, served as the expression vector. GenSmart™ Design software was used to create the expression vector. The tagged protein, 6x histidine attached to the C terminus of an ADI molecule, was promotor T7 Lac. As the expression host, Escherichia coli BL21[DE3] polys S was used. They were all employed in the bacterial recombinant DNA method to produce ADI. The inducer of the transcription process was IPTG. Escherichia coli DH5, which was procured from Stratagene Corporation in the USA, served as the primary host for plasmid manufacture and replication. Using restriction endonuclease type II enzymes( DNA cutting enzymes) Xbal and ACCI, the predominant bacterial isolate found in various soil conditions in Egypt was subjected to genomic DNA isolation. The German company Sigma-Aldrich supplied these cutting enzymes. Additionally, using the same restriction endonuclease type II enzymes that were used to extract genomic DNA from the primary bacterial isolates, genomic DNA was amplified using the polymerase chain reaction( PCR) technique and then sub-cloned to the prokaryotic expression vector PET26b(+). After then, Escherichia coli BL21[DE3] poly S was transformed from PET26b(+). Gene expression was started when IPTG was added because it increased transcription at the promotor site T7 Lac. For standard bacterial culture, LB and LA broth were employed, and the incubation temperature was set at 37°C for 24 hours. In accordance with the manufacturer's instructions, Ampicillin and/or Kanamycin were added to the culture medium. Ion exchange column chromatography, dialysis, and 70% Ammonium Sulphate were used to purify the recombinant protein.29
Kinetic properties of the purified ADI:
A pH range of 5.0 to 13.0 was used to establish the ideal pH for ADI activity. Enzyme preparations at different pH values were incubated for one hour to conduct pH stability tests. Following this, the relative activity was measured using conventional assay procedures. By testing ADI activity at various temperatures between 30 and 100°C, the optimum temperature for enzyme activity was found. Furthermore, the enzyme's thermal stability was assessed by assaying ADI activity after the lyophilized enzyme was incubated for one hour at the same temperatures. With concentrations ranging from 0.0 to 0.1M, L-arginine was used as a substrate to calculate the kinetic parameters( Km and Vmax) for ADI.Using the equation obtained from the linear-regression analysis of the curve, the Michaelis-Menten parameters were ascertained from Lineaweaver-Burk plots.30
STATISTICAL ANALYSIS
The average and standard deviation of each experiment were reported. Each experiment was carried out in triplicate. Minitab software was utilized for conducting statistical analyses, which involved one-way analysis of variance( ANOVA) with a p-value of less than 0.05.