Isolation and identification of bacterial isolates producing Arginine deiminase from assorted soil environments in Egypt using16S rRNA sequencing technique

Isolation and identification of bacterial isolates producing Arginine deiminase from assorted soil environments in Egypt using16S rRNA sequencing technique | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Isolation and identification of bacterial isolates producing Arginine deiminase from assorted soil environments in Egypt using 16S rRNA sequencing technique Mohammed Mahmoud Shawky Kassab This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4126905/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version 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 IC 50 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 -1 respectively . 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. Applied & Industrial Microbiology Cancer Arginine Purification 16S rRNA degrading enzymes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 INTRODUCTION Auxotrophic cancers for L-arginine such as renal and hepatic carcinoma are overwhelming cause of mortality globally. 1 Arginine Auxotrophy occurs certain neoplasms. 2 Its usual cause is the silencing of arginosuccinate synthetase I 3 or Arginine lyase genes. 4 Certain tumors need the extracellular origins of certain aminoacids which are considered nonessential in normal cells due to metabolic deficiencies. 5 Enzymatic collapse of auxotrophic cancer aminoacids is considered efficacious strategy in diminishing of such neoplasms. 6 L-arginase is considered an example of aminoacid depriving enzymes which was recently exploited in the management of melanoma and hepatic cellular carcinoma globally. 7 A comparable approach probably be victimized for different types of Arginine auxotrophic cancers. 8 Arginine degrading enzymes comprise 3 main types; L-arginase , Arginine decarboxylase and Arginine deiminase ( ADI ). 9 To explore novel origins of Arginine depriving enzymes, a sole possible way was isolation, screening as well as the characterization of microbial soil environments. 10 ADI , among the Arginine depleting enzymes, was detected as antitumor enzyme to manage auxotrophic cancers for L-arginine like melanomas and hepatocellular carcinoma. 11 The catabolism of L-arginine to L-citrulline and Ammonia was noticed to occur via ADI pathway. 12 A number of bacterial genotypes such as Mycoplasma , 13 Pseudomonas , 14 Lactobacillus , 15 Halo-bacteria , 16 Lactococcus 17 and Streptococci 18 was observed to utilize ADI pathway as energy source. 19 The aim of the present study was to isolate bacterial species-es from different soil environments in Egypt were able to secrete ADI as anticancer agent using 16S rRNA sequencing technique; besides its bacterial recombinant DNA technology manufacture; moreover the characterization of its auxotrophic anticancer activity. MATERIAL AND METHODS 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 C itrulline 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 S ulfuric 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( BLAST n ). 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 µ l 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 . RESULTS Bacterial isolates were grown on MAA plates with characterized Pink zones due to Arginine deiminase activity. Maximum Arginine deiminase production was achieved using an incubation period of 24 hours , optimum pH 8 and temperature 40℃ and agitation rate 200 rpm . On the other hand, Mannitol and Peptone were used as the sole metabolic carbon and nitrogen sources, respectively which contributed to the highest production of ADI . The purified ADI demonstrated final specific activity of approximately 36.07 U/mg and molecular mass was detected to be 36.18 kDa using SDS-PAGE gel filtration technique . The maximum activity and stability of the purified enzyme occurred at pH 9 and 8 , respectively as well as temperatures 40℃ and 37℃ .Complete thermal stability was confirmed at 70℃ for one hour . The Km and Vmax of ADI were 0.05871 M and 40.36 µmol/ml/min , respectively; while, Kcat reached about 5.014min − 1 . The anticancer activity was determined through MTT assay. ADI demonstrated significant cytotoxic actions versus renal ( Caki-1 ) and leukemic ( K-562 ) cancer cell lines( IC 50 10.31 µg/ml and 16.08 µg/ml , respectively). Negligible ADI cytotoxicity effect on Vero CCL-81™ cell lines were observed during MTT assay. IC 50 of standard Doxorubicin anticancer drug against renal ( Caki-1 ) and leukemic ( K-562 ) cancer cell lines( IC 50 9.87 µg/ml and 7 .33 µg/ml , respectively). Standard L-asparaginase demonstrated only IC 50 ~ 5 µg /ml against leukemic ( K-562 ) cancer cell line but failed to exert significant cytotoxicity against Vero and renal cancer cell lines. DPPH scavenging free radical assay using Ascorbic acid as a standard drug revealed that ADI possess no antioxidant activities. 5.68 mg total protein was produced during the initial culture of ADI on MAA medium as estimated via Bradford assay utilizing Bovine Albumin as a standard agent. In vitro resoluteness of biological half life of ADI exploiting nesslerization assay was ascertained to be closely 300 min . Only 29 bacterial isolates producing different arginine degrading enzymes were detected to be grown on MAA , from which 17 bacterial isolates showed the highest production of ADI . Morphological, biochemical reactions detected that Bacillus subtilis was the major bacterial species producing ADI . 16S rRNA gene sequencing technique using BLASTn demonstrated that Bacillus subtilis DE111 was the predominant Bacillus secreting ADI in the present study. The specific activity of recombinant ADI was 140.09 U/mg . The biochemical processes profile of Bacillus subtilis DE111 is shown in Table 8 . Table 7 presents a BLASTn analysis of 16S rRNA sequencing. Aeration impacts on ADI production are seen in Table 6 . The carbon and nitrogen growth variables that impact ADI production are displayed in Table 1 . The effects of metal cations on ADI activity and production are shown in Table 4 . Table 5 . It illustrates how varying substrate concentrations affect the productivity of ADI . The effects of pH and temperature on ADI production are shown in Table 2 . The effects of pH and temperature on ADI activity are shown in Table 3 . Bacillus subtilis DE111 isolates that produce ADI under a standard microscope are shown in Fig. 14 . Figure 1 shows the ADI monomer 3D structure using the SWISS-MODEL software. There are 418 amino acids in ADI. The evaluation of ADI-induced apoptosis is shown in Fig. 7 using colorimetric Caspase 3 activity detection. The Michaelis Menten plot of ADI is shown in Fig. 11 . The Bradford test is shown in Fig. 12 and is used to measure the protein concentration in ADI productivity studies. The absorbance of ADI at 480 nm wavelength caused by ammonia leakage is seen in Fig. 5 . More ADI activities were seen in response to Ammonia releases. The optical density of ADI activity at 530 nm is shown in Fig. 6 . As the ADI concentration grew, absorbance increased as well. The test ADI 's phylogenetic guide tree, created by Clustal Omega software, is shown in Fig. 13 . The MTT test of ADI on a leukemic cancer cell line is shown in Fig. 10 . Figure 8 shows how to determine whether ADI -induced apoptosis occurred by looking for Caspase 3 activity at 450 nm . The MTT test of ADI on a kidney cancer cell line is shown in Fig. 9 . Using CB-DOCk2 software, Fig. 3 illustrates the strong attraction of the Mn + 2 metal cation to ADI . The optical density of ADI activity at 530 nm is shown in Fig. 6 . As the ADI concentration grew, absorbance increased as well. Figure 2 illustrates how ADI is docked with CB-DOCK 2 software. Figure 4 illustrates the purification of ADI produced by Bacillus subtilis DE111 using SDS-PAGE . Nearly 85% of the sample was pure. DISCUSSION ADI catalyzing the hydrolysis of L-arginine to L-citrulline and Ammonia was found to be an enzyme of therapeutic importance in the management of cancer, especially auxotrophic cancers for L-arginine such as Melanoma and renal cell carcinoma. The present study described the recombinant production, properties and anticancer potential of the enzyme from Bacillus subtilis DE111 . About 58% of bacterial isolates grown on MAA showed high productivity of arginine degrading enzymes, from which 34% bacterial isolates produced ADI . Morphological characterization and biochemical reactions; besides 16S rRNA sequencing technique revealed that Bacillus subtilis DE111 bacterial isolates were the potent ADI producers. A 1230 base pair gene encoding 410 aminoacids was PCR amplified and cloned into BL21(DE3) poly sS strain of E.col i expression host using pET26b(+) plasmid which was the expression vector. The creation of the recombinant ADI was induced under the influence of 0.5 mM IPTG . On the other hand, ADI was purified via 70% ammonium sulfate salting out from the supernatant of the centrifuge tube. Thereafter, ADI was extracted as a refined enzyme( 85% purity ) using cation exchange chromatography and gel filtration through SDS-PAGE . Purified enzyme was analyzed for the kinetics in Silico studies and experimentally. Through MTT assay, ADI showed anticancer potential against auxotrophic cancers for L-arginine such as renal cancer; as well as It demonstrated significant antileukaemic activities. The recombinant ADI enzyme showed a molecular mass of 36.18 kDa , specific activity of 140.09 U/mg which was fourth fold more than the initial culture . The Km and Vmax of ADI were 0.05871 M and 40.36 µmol/ml/min , respectively; while, Kcat reached about 5.014min − 1 reflecting high affinity of the enzyme to L-arginine substrate. Optimum temperature and pH were 37–40 ℃ and 6–10, respectively. ADI demonstrated the highest activity at 37 ℃ and pH 9 . On the other hand, ADI showed thermostability at 70 ℃ for 1 hour .During the initial culture on MAA plates, the productivity and the activity were detected to be increased in presence of Co + 2 , Ni + 2 and Mn + 2 metal ions; while they were diminished significantly in presence of Fe + 3 metal ions. No detectable enzyme activity was detected when either L- glutamine or L-asparagine was used as the substrate for ADI . Peptone and Mannitol were the optimal activators for ADI producing bacterial isolates. Ebrahimi N et al study, 2016 demonstrated that ADI was produced via Enterbacter sp. Sgn 1 as a potential anticancer agent;3 1 whereas in the present study ADI was produced from Bacillus subtilis DE111 . In Silico studies confirmed that ADI existed as a monomer having Arg 9 , ALa 85 , Thr 107 , His 108 , Gln 139 , Leu 161 and Lys 253 being the putative active site residues. The free energy change calculated via molecular docking studies of the enzyme and the substrate was observed to be ∆G−5.1 KJ/mole indicating the optimum affinity of ADI with L-arginine substrate . The optimal shaking speed for the production of ADI was noticed at 200 rpm after 24 hours incubation time. The more the concentration of L-arginine as a substrate the more ADI was produced during the initial culture on MAA plates. The significant rise in Caspase 3 activity was demonstrated using Caspase 3 activity assay suggesting that ADI induced programmed cell death of cancer cells( apoptosis) selectively. ADI chiefly destroyed the renal cancer cell line through its inhibition of L-arginine from the external media surrounding these auxotrophic cancer cells for L-arginine . On the other hand the leukemic cancer cell line which was not auxotrophic for L-arginine( due to abundance of Arginosuccinate synthetase I was broke down through apoptosis induced via recombinant ADI. ADI exhibited 68% and 51% growth inhibition of leukemic and renal cancer cell lines, respectively as indicated using MTT assay. In the present study, MTT assay revealed that ADI ascertained comparable efficacy to standard L-asparaginase and Doxorubicin against leukemic cancer cell line and renal cancer cell line. Neither ADI antioxidant nor scavenging free radical activities were detected via DPPH assay. CONCLUSION The present work was a promising study due to the evolution of novel bacterial recombinant ADI from different soil environments in Egypt as a potential anti-neoplastic mediator versus renal auxotrophic cancer cells for L-arginine as well as leukaemia cancers. Declarations Conflict of interest: There is no conflict of interest. Funding: The present study was funded by me. References Thakker DP, Narayanan R. Arginine deiminase produced by lactic acid bacteria as a potent anti-cancer drug. Med Oncol. 2023 May 12;40(6):175. doi: 10.1007/s12032-023-02043-4. PMID: 37171497. Xiong L, Teng JL, Botelho MG, Lo RC, Lau SK, Woo PC. Arginine Metabolism in Bacterial Pathogenesis and Cancer Therapy. Int J Mol Sci. 2016 Mar 11;17(3):363. doi: 10.3390/ijms17030363. PMID: 26978353; PMCID: PMC4813224. Feun LG, Kuo MT, Savaraj N. Arginine deprivation in cancer therapy. Curr Opin Clin Nutr Metab Care. 2015 Jan;18(1):78-82. doi: 10.1097/MCO.0000000000000122. PMID: 25474015. Kuo MT, Savaraj N, Feun LG. Targeted cellular metabolism for cancer chemotherapy with recombinant arginine-degrading enzymes. Oncotarget. 2010 Aug;1(4):246-51. doi: 10.18632/oncotarget.135. PMID: 21152246; PMCID: PMC2998341. Tsai WB, Aiba I, Lee SY, Feun L, Savaraj N, Kuo MT. Resistance to arginine deiminase treatment in melanoma cells is associated with induced argininosuccinate synthetase expression involving c-Myc/HIF-1alpha/Sp4. Mol Cancer Ther. 2009 Dec;8(12):3223-33. doi: 10.1158/1535-7163.MCT-09-0794. PMID: 19934275; PMCID: PMC2795119. Long Y, Tsai WB, Chang JT, Estecio M, Wangpaichitr M, Savaraj N, Feun LG, Chen HH, Kuo MT. Cisplatin-induced synthetic lethality to arginine-starvation therapy by transcriptional suppression of ASS1 is regulated by DEC1, HIF-1α, and c-Myc transcription network and is independent of ASS1 promoter DNA methylation. Oncotarget. 2016 Dec 13;7(50):82658-82670. doi: 10.18632/oncotarget.12308. PMID: 27765932; PMCID: PMC5347722. Feun L, You M, Wu CJ, Kuo MT, Wangpaichitr M, Spector S, Savaraj N. Arginine deprivation as a targeted therapy for cancer. Curr Pharm Des. 2008;14(11):1049-57. doi: 10.2174/138161208784246199. PMID: 18473854; PMCID: PMC3096551. Thongkum A, Wu C, Li YY, Wangpaichitr M, Navasumrit P, Parnlob V, Sricharunrat T, Bhudhisawasdi V, Ruchirawat M, Savaraj N. The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma. Int J Mol Sci. 2017 Jun 1;18(6):1175. doi: 10.3390/ijms18061175. PMID: 28587170; PMCID: PMC5485998. Savaraj N, Wu C, Li YY, Wangpaichitr M, You M, Bomalaski J, He W, Kuo MT, Feun LG. Targeting argininosuccinate synthetase negative melanomas using combination of arginine degrading enzyme and cisplatin. Oncotarget. 2015 Mar 20;6(8):6295-309. doi: 10.18632/oncotarget.3370. PMID: 25749046; PMCID: PMC4467438. Kim RH, Coates JM, Bowles TL, McNerney GP, Sutcliffe J, Jung JU, Gandour-Edwards R, Chuang FY, Bold RJ, Kung HJ. Arginine deiminase as a novel therapy for prostate cancer induces autophagy and caspase-independent apoptosis. Cancer Res. 2009 Jan 15;69(2):700-8. doi: 10.1158/0008-5472.CAN-08-3157. PMID: 19147587; PMCID: PMC2629384. Bowles TL, Kim R, Galante J, Parsons CM, Virudachalam S, Kung HJ, Bold RJ. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase. Int J Cancer. 2008 Oct 15;123(8):1950-5. doi: 10.1002/ijc.23723. PMID: 18661517; PMCID: PMC4294549. Ensor CM, Holtsberg FW, Bomalaski JS, Clark MA. Pegylated arginine deiminase (ADI-SS PEG20,000 mw) inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo. Cancer Res. 2002 Oct 1;62(19):5443-50. PMID: 12359751. Ni Y, Schwaneberg U, Sun ZH. Arginine deiminase, a potential anti-tumor drug. Cancer Lett. 2008 Mar 8;261(1):1-11. doi: 10.1016/j.canlet.2007.11.038. Epub 2008 Jan 7. PMID: 18179862. Shen LJ, Shen WC. Drug evaluation: ADI-PEG-20--a PEGylated arginine deiminase for arginine-auxotrophic cancers. Curr Opin Mol Ther. 2006 Jun;8(3):240-8. PMID: 16774044. Ascierto PA, Scala S, Castello G, Daponte A, Simeone E, Ottaiano A, Beneduce G, De Rosa V, Izzo F, Melucci MT, Ensor CM, Prestayko AW, Holtsberg FW, Bomalaski JS, Clark MA, Savaraj N, Feun LG, Logan TF. Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies. J Clin Oncol. 2005 Oct 20;23(30):7660-8. doi: 10.1200/JCO.2005.02.0933. Erratum in: J Clin Oncol. 2006 Aug 20;24(24):4047. PMID: 16234528. Izzo F, Marra P, Beneduce G, Castello G, Vallone P, De Rosa V, Cremona F, Ensor CM, Holtsberg FW, Bomalaski JS, Clark MA, Ng C, Curley SA. Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I/II studies. J Clin Oncol. 2004 May 15;22(10):1815-22. doi: 10.1200/JCO.2004.11.120. PMID: 15143074. Glazer ES, Piccirillo M, Albino V, Di Giacomo R, Palaia R, Mastro AA, Beneduce G, Castello G, De Rosa V, Petrillo A, Ascierto PA, Curley SA, Izzo F. Phase II study of pegylated arginine deiminase for nonresectable and metastatic hepatocellular carcinoma. J Clin Oncol. 2010 May 1;28(13):2220-6. doi: 10.1200/JCO.2009.26.7765. Epub 2010 Mar 29. PMID: 20351325. Curley SA, Bomalaski JS, Ensor CM, Holtsberg FW, Clark MA. Regression of hepatocellular cancer in a patient treated with arginine deiminase. Hepatogastroenterology. 2003 Sep-Oct;50(53):1214-6. PMID: 14571701. Chu YD, Lai MW, Yeh CT. Unlocking the Potential of Arginine Deprivation Therapy: Recent Breakthroughs and Promising Future for Cancer Treatment. Int J Mol Sci. 2023 Jun 26;24(13):10668. doi: 10.3390/ijms241310668. PMID: 37445845; PMCID: PMC10341449. Wang W, Li M, Miao M, Zhang T. Characterization of a recombinant arginine deiminase from Halothermothrix orenii and its application in citrulline production. Biotechnol Appl Biochem. 2023 Apr;70(2):526-536. doi: 10.1002/bab.2375. Epub 2022 Jun 27. PMID: 35761421. Rashad YM, Hafez M, Rashad M. Diazotrophic Azotobacter salinestris YRNF3: a probable calcite-solubilizing bio-agent for improving the calcareous soil properties. Sci Rep. 2023 Nov 23;13(1):20621. doi: 10.1038/s41598-023-47924-w. PMID: 37996572; PMCID: PMC10667278. Wei X, Chow HY, Chong HC, Leung SL, Ho MK, Lee MY, Leung YC. Arginine Is a Novel Drug Target for Arginine Decarboxylase in Human Colorectal Cancer Cells. Int J Mol Sci. 2023 Sep 6;24(18):13741. doi: 10.3390/ijms241813741. PMID: 37762044; PMCID: PMC10531272. Zhao MM, Du SS, Li QH, Chen T, Qiu H, Wu Q, Chen SS, Zhou Y, Zhang Y, Hu Y, Su YL, Shen L, Zhang F, Weng D, Li HP. High throughput 16SrRNA gene sequencing reveals the correlation between Propionibacterium acnes and sarcoidosis. Respir Res. 2017 Feb 1;18(1):28. doi: 10.1186/s12931-017-0515-z. PMID: 28143482; PMCID: PMC5286795. Alrumman SA, Mostafa YS, Al-Izran KA, Alfaifi MY, Taha TH, Elbehairi SE. Production and Anticancer Activity of an L-Asparaginase from Bacillus licheniformis Isolated from the Red Sea, Saudi Arabia. Sci Rep. 2019 Mar 6;9(1):3756. doi: 10.1038/s41598-019-40512-x. PMID: 30842557; PMCID: PMC6403232. Kielkopf CL, Bauer W, Urbatsch IL. Bradford Assay for Determining Protein Concentration. Cold Spring Harb Protoc. 2020 Apr 1;2020(4):102269. doi: 10.1101/pdb.prot102269. PMID: 32238597. Buranaamnuay K. The MTT assay application to measure the viability of spermatozoa: A variety of the assay protocols. Open Vet J. 2021 Apr-Jun;11(2):251-269. doi: 10.5455/OVJ.2021.v11.i2.9. Epub 2021 May 8. PMID: 34307082; PMCID: PMC8288735. Nile SH, Khobragade CN, Park SW. Optimized and comparative antioxidant assays and its applications in herbal and synthetic drug analysis as an antioxidants. Mini Rev Med Chem. 2012 Sep 1;12(10):1007-14. doi: 10.2174/138955712802762310. PMID: 22625419. Kim RH, Coates JM, Bowles TL, McNerney GP, Sutcliffe J, Jung JU, Gandour-Edwards R, Chuang FY, Bold RJ, Kung HJ. Arginine deiminase as a novel therapy for prostate cancer induces autophagy and caspase-independent apoptosis. Cancer Res. 2009 Jan 15;69(2):700-8. doi: 10.1158/0008-5472.CAN-08-3157. PMID: 19147587; PMCID: PMC2629384. Jeong SJ, Heo K, Park JY, Lee KW, Park JY, Joo SH, Kim JH. Characterization of AprE176, a fibrinolytic enzyme from Bacillus subtilis HK176. J Microbiol Biotechnol. 2015 Jan;25(1):89-97. doi: 10.4014/jmb.1409.09087. PMID: 25315053. Shrivastava A, Khan AA, Shrivastav A, Jain SK, Singhal PK. Kinetic studies of L-asparaginase from Penicillium digitatum. Prep Biochem Biotechnol. 2012;42(6):574-81. doi: 10.1080/10826068.2012.672943. PMID: 23030468. Ebrahimi N, Gharibi S, Ghoshoon MB, Karimi Z, Gholami A, Nezafat N, Mohkam M, Ghasemi Y. Selective Isolation and Identification of Arginine Degrading Bacteria; the Optimized Arginine Deaminase Production by Enterobacter sp. sgn1 as a New Source of This Potentially Anti-Tumor Enzyme. J App Pharm Sci, 2016; 6 (09): 093-101. Tables Table 1 It shows Carbon and Nitrogen growth factors affecting ADI productivity: Carbon source( 2%W/V) Nitrogen source( 1% W/V) ADI productivity( U/ml) Glucose Yeast 181 Mannitol Peptone 193 Lactose L-arginine 162 Maltose Na Nitrate 127 Soluble Starch Ammonium Sulfate 155 Table 2 It represents the effects of temperature and pH on ADI productivity: Temperature( ℃ ) pH ADI productivity( U/ml ) 20 5 0 25 6 7 35 6.5 93 40 7 191 45 8 181 60 8.5 170 70 9 162 80 10 156 82 11 19 Table 3 It demonstrates impacts of pH and temperature on ADI activity: Temperature( ℃ ) pH ADI activity( U/ml ) 25 4 0 30 5 4 33 6 42 35 8 179 37 9 191 40 10 187 50 11 140 70 12 131 80 13 0 Table 4 It demonstrates influences of metal cations on ADI productivity and activity: Metal Cation( 5mM ) ADI productivity( U/ml ) Metal Cation( 10 mM ) ADI activity( U/ml ) Na + 1 157 Fe + 3 0 K + 1 160 K + 1 163 Co + 2 181 Na + 1 151 Ni + 2 185 Ni + 2 190 Mn + 2 192 Co + 2 193 Mg + 2 164 Mn + 2 185 Fe + 2 149 Mg + 2 173 Fe + 3 0 Fe + 2 145 Table 5 It demonstrates the influence of different substrate concentrations on ADI productivity: Substrate concentration( mM ) ADI productivity( U/ml) 2 83 4 101 8 125 15 139 30 148 40 157 80 192 Table 6 It shows aeration effects on ADI productivity: Shaking speed( rpm ) ADI productivity( U/ ml) 50 103 100 117 200 192 400 159 600 131 Table 7 It indicates 16S rRNA sequencing analysis using BLASTn . Description Max Score Total Score Query Cover E value Per. identity Bacillus subtilis strain DE111 1530 1530 100% 0 100 Bacillus sp. (in: Bacteria) strain HA15-23 1520 1520 100% 0 99.76 Bacillus subtilis strain KMB31 1520 1520 99% 0 99.88 Bacillus subtilis strain LXA7 1520 1520 99% 0 100 Bacillus subtilis strain bacs2 1519 1519 99% 0 99.88 Bacillus sp. (in: Bacteria) strain ITI09 1519 1519 100% 0 99.76 Bacillus sp. (in: Bacteria) strain AU3b4 1519 1519 99% 0 99.88 Bacillus subtilis strain MRHB1-205 1519 1519 99% 0 100 Bacillus sp. (in: Bacteria) strain BGS3 1517 1517 99% 0 99.88 Table 8 It represents Bacillus subtilis DE111 biochemical reactions profile: Biochemical reaction Findings Citrate +ve Catalase +ve Flagella +ve Gelatin hydrolysis +ve Gas -ve Gram staining +ve Motility +ve Indole -ve Oxidase +ve Nitrate reduction +ve Methyl red( MR) +ve Vogues proskaeur( VP) +ve Spore +ve Shape Rods Urease -ve Pigment -ve Fructose fermentation +ve Arabinose fermentation +ve Glucose fermentation +ve Inositol fermentation +ve Maltose fermentation +ve Mannitol fermentation +ve Starch fermentation +ve Rhamanose fermentation +ve Arabitol fermentation -ve Dulcitol fermentation -ve Additional Declarations The authors declare no competing interests. Supplementary Files MTTASSAYofADIonrenalcancercellline.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4126905","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":281159467,"identity":"9b321ef8-63be-4c5b-9241-acaca3778023","order_by":0,"name":"Mohammed Mahmoud Shawky Kassab","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYBACCQYGNgbGBgbGfvnHBx8ABXj4iNYysyEt2QCkhY1oLRsactQkQCIEtUj2H3726OaObbIbGM6wVX7NsZNhY2B++OgGHi3SEmnmxrlnbhtvZ+w9dlt2WzLQYWzGxjl4tMhJMJhJ57bdTtzZzJd2W3IbM1ALD5s0Xi38x7+BtWw4xmNWLLmtnrAWaYYciC0bzvCYMX7cdpiwFskZOeXGQC3GM2ewJUszbjvOw8ZMwC8S549vewzUItsvwXzw489t1fb87M0PH+PTggKYecAkscpBgPEHKapHwSgYBaNgxAAA7lhKFgrxlaAAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-2554-0663","institution":"Faculty of Pharmacy, Cairo University","correspondingAuthor":true,"prefix":"","firstName":"Mohammed","middleName":"Mahmoud Shawky","lastName":"Kassab","suffix":""}],"badges":[],"createdAt":"2024-03-19 03:15:54","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4126905/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4126905/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53074454,"identity":"7dae45d0-d30e-49b5-b846-327ffa52b572","added_by":"auto","created_at":"2024-03-20 09:17:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":199095,"visible":true,"origin":"","legend":"\u003cp\u003eIt demonstrates Monomer \u003cem\u003e3D structure \u003c/em\u003eof \u003cem\u003eADI \u003c/em\u003eusing\u003cem\u003e SWISS-MODEL software\u003c/em\u003e. \u003cem\u003eADI\u003c/em\u003e consists of \u003cem\u003e418\u003c/em\u003e \u003cem\u003eaminoacids\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/2b071d034860bf1133f03b54.png"},{"id":53074441,"identity":"2253b080-d8ea-4488-addc-6588d50dd5c2","added_by":"auto","created_at":"2024-03-20 09:17:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":913374,"visible":true,"origin":"","legend":"\u003cp\u003eIt shows the docking of \u003cem\u003eADI\u003c/em\u003e using \u003cem\u003eCB-DOCK software\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/e0fbef67f81e75c05ec73ab1.png"},{"id":53075087,"identity":"d4410691-a4b3-4aab-aa5d-6354bc5fe66e","added_by":"auto","created_at":"2024-03-20 09:25:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":560624,"visible":true,"origin":"","legend":"\u003cp\u003eIt demonstrates the high affinity of\u003cem\u003e Mn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+2\u003c/em\u003e\u003c/sup\u003e metal cation to \u003cem\u003eADI using CB-dock2 software.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/b4eaa33f170b5f587eb80a0b.png"},{"id":53074449,"identity":"82e8154a-f59d-4043-a2d0-b0fea0883199","added_by":"auto","created_at":"2024-03-20 09:17:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":445392,"visible":true,"origin":"","legend":"\u003cp\u003eIt demonstrates the \u003cem\u003eSDS-PAGE\u003c/em\u003e purification of \u003cem\u003eADI\u003c/em\u003e secreted from \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e. The percentage of purity was close to \u003cem\u003e85%\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/60288575a203bef9cb9cce9d.png"},{"id":53074451,"identity":"3b3fb2c2-2589-4ad0-97f7-acdfa162fa46","added_by":"auto","created_at":"2024-03-20 09:17:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":308817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 13\u003c/strong\u003e. It shows gene cloning using \u003cem\u003ePCR\u003c/em\u003e during \u003cem\u003e16S rRNA\u003c/em\u003e sequencing technique.\u003c/p\u003e","description":"","filename":"41.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/813cf9b90dc8a722846b250d.png"},{"id":53074445,"identity":"98729d73-6c3f-4a71-9657-ecf0c68ffe00","added_by":"auto","created_at":"2024-03-20 09:17:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":36697,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 5. \u003c/strong\u003eIt shows the absorbance of \u003cem\u003eADI\u003c/em\u003e at \u003cem\u003e480 nm\u003c/em\u003e wavelength due to the release of \u003cem\u003eAmmonia. \u003c/em\u003eThe more\u003cem\u003e Ammonia \u003c/em\u003ewere released, The more\u003cem\u003e ADI \u003c/em\u003eactivities were detected\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/5bb3a947c51efdd73b2b0868.png"},{"id":53075086,"identity":"c26ec245-c84a-43a3-9929-a2e0a2f565d6","added_by":"auto","created_at":"2024-03-20 09:25:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":30844,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 6. \u003c/strong\u003eIt demonstrates the optical density of \u003cem\u003eADI\u003c/em\u003eactivity at \u003cem\u003e530 nm\u003c/em\u003e. \u003cem\u003eAbsorbance\u003c/em\u003e was increased with increasing the \u003cem\u003eADI\u003c/em\u003econcentration.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/1b5400e17d4d082e3c33cdcb.png"},{"id":53074452,"identity":"844f5d0a-9f94-40a8-b29c-fb1a8e4f5a97","added_by":"auto","created_at":"2024-03-20 09:17:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":32329,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 7.\u003c/strong\u003e It represents \u003cem\u003eADI\u003c/em\u003e induced apoptosis assessment via the detection of \u003cem\u003eCaspase 3\u003c/em\u003e activity colorimetrically.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/da05268faba3d73cb5431946.png"},{"id":53074447,"identity":"6213bb1c-3205-441d-be48-1ba04af9c435","added_by":"auto","created_at":"2024-03-20 09:17:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":20626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 8. \u003c/strong\u003eIt demonstrates \u003cem\u003eADI\u003c/em\u003e induced apoptosis determination via the detection of \u003cem\u003eCaspase 3\u003c/em\u003e activity at \u003cem\u003e450 nm.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/2b759bbcc33bf58e61bc4c98.png"},{"id":53074456,"identity":"7a5e237d-e924-4d13-badd-3dcab60c2b3d","added_by":"auto","created_at":"2024-03-20 09:17:50","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":21112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 9\u003c/strong\u003e\u003cem\u003e. It shows MTT assay of ADI on renal cancer cell line.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/19e89798e7a56a4c19cd6d8a.png"},{"id":53074446,"identity":"42f9911d-0062-4968-8396-32cc950a7127","added_by":"auto","created_at":"2024-03-20 09:17:49","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":22787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 10\u003c/strong\u003e\u003cem\u003e. It shows MTT assay of ADI on leukemic cancer cell line.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/b212f3e0302c07db3572a284.png"},{"id":53074457,"identity":"110fc44e-5902-4ff1-9ce9-13c182d94006","added_by":"auto","created_at":"2024-03-20 09:17:51","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":28093,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 11. \u003c/strong\u003eIt reflects \u003cem\u003eMichaelis Menten plot\u003c/em\u003e of \u003cem\u003eADI\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/41aadce538bf36fc4e564d42.png"},{"id":53074443,"identity":"7e4842e0-cd22-4f0a-953e-c15bb7f55c92","added_by":"auto","created_at":"2024-03-20 09:17:49","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":22010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 12. \u003c/strong\u003eIt demonstrates \u003cem\u003eBradford assay\u003c/em\u003e for the determining protein content during the assessment of\u003cem\u003e ADI\u003c/em\u003e productivity study.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/449e4f1f3fb7a1d29f2197a4.png"},{"id":53074450,"identity":"cb9e488f-eb92-4aff-b933-dc6b0a0d856a","added_by":"auto","created_at":"2024-03-20 09:17:50","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":496847,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 13.\u003c/strong\u003e It demonstrates \u003cem\u003ephylogenetic guide tree\u003c/em\u003eof the test \u003cem\u003eADI using Clustal omega software.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/d76130eddce4cae1a28351e1.png"},{"id":53074442,"identity":"12329369-f2cc-4006-aa63-8ed7382095bb","added_by":"auto","created_at":"2024-03-20 09:17:49","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":600955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 14.\u003c/strong\u003e It refers to \u003cem\u003eBacillus subtilis DE111\u003c/em\u003eisolates producing \u003cem\u003eADI\u003c/em\u003e under normal microscope.\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/a94d83d0dca31b148d1c2afe.png"},{"id":53075533,"identity":"323533e8-2bb3-4b83-94dd-1562a129f449","added_by":"auto","created_at":"2024-03-20 09:33:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3899102,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/7660ac76-f031-4479-a9a9-3e36e528c0b4.pdf"},{"id":53074455,"identity":"13a4b03f-37b7-46cc-acd9-4f27d24069ac","added_by":"auto","created_at":"2024-03-20 09:17:50","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15312,"visible":true,"origin":"","legend":"","description":"","filename":"MTTASSAYofADIonrenalcancercellline.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4126905/v1/dc6da2b591f8c942c3843f24.xlsx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eIsolation and identification of bacterial isolates producing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArginine deiminase\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e from assorted soil environments in Egypt using\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e16S rRNA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003esequencing\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etechnique\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAuxotrophic cancers for \u003cem\u003eL-arginine\u003c/em\u003e such as renal and hepatic carcinoma are overwhelming cause of mortality globally.\u003csup\u003e1\u003c/sup\u003e \u003cem\u003eArginine\u003c/em\u003e Auxotrophy occurs certain neoplasms.\u003csup\u003e2\u003c/sup\u003e Its usual cause is the silencing of arginosuccinate synthetase I\u003csup\u003e3\u003c/sup\u003e or \u003cem\u003eArginine\u003c/em\u003e lyase genes.\u003csup\u003e4\u003c/sup\u003e Certain tumors need the extracellular origins of certain aminoacids which are considered nonessential in normal cells due to metabolic deficiencies.\u003csup\u003e5\u003c/sup\u003e Enzymatic collapse of auxotrophic cancer aminoacids is considered efficacious strategy in diminishing of such neoplasms.\u003csup\u003e6\u003c/sup\u003e \u003cem\u003eL-arginase\u003c/em\u003e is considered an example of aminoacid depriving enzymes which was recently exploited in the management of melanoma and hepatic cellular carcinoma globally.\u003csup\u003e7\u003c/sup\u003e A comparable approach probably be victimized for different types of \u003cem\u003eArginine\u003c/em\u003e auxotrophic cancers.\u003csup\u003e8\u003c/sup\u003e \u003cem\u003eArginine\u003c/em\u003e degrading enzymes comprise 3 main types; \u003cem\u003eL-arginase\u003c/em\u003e, \u003cem\u003eArginine decarboxylase\u003c/em\u003e and \u003cem\u003eArginine deiminase\u003c/em\u003e( \u003cem\u003eADI\u003c/em\u003e).\u003csup\u003e9\u003c/sup\u003e To explore novel origins of \u003cem\u003eArginine\u003c/em\u003e depriving enzymes, a sole possible way was isolation, screening as well as the characterization of microbial soil environments.\u003csup\u003e10\u003c/sup\u003e \u003cem\u003eADI\u003c/em\u003e, among the \u003cem\u003eArginine\u003c/em\u003e depleting enzymes, was detected as antitumor enzyme to manage auxotrophic cancers for L-arginine like melanomas and hepatocellular carcinoma.\u003csup\u003e11\u003c/sup\u003e The catabolism of L-arginine to \u003cem\u003eL-citrulline\u003c/em\u003e and \u003cem\u003eAmmonia\u003c/em\u003e was noticed to occur via \u003cem\u003eADI\u003c/em\u003e pathway.\u003csup\u003e12\u003c/sup\u003e A number of bacterial genotypes such as \u003cem\u003eMycoplasma\u003c/em\u003e,\u003csup\u003e13\u003c/sup\u003e \u003cem\u003ePseudomonas\u003c/em\u003e,\u003csup\u003e14\u003c/sup\u003e \u003cem\u003eLactobacillus\u003c/em\u003e,\u003csup\u003e15\u003c/sup\u003e \u003cem\u003eHalo-bacteria\u003c/em\u003e,\u003csup\u003e16\u003c/sup\u003e \u003cem\u003eLactococcus\u003c/em\u003e\u003csup\u003e17\u003c/sup\u003e and \u003cem\u003eStreptococci\u003c/em\u003e\u003csup\u003e18\u003c/sup\u003e was observed to utilize \u003cem\u003eADI\u003c/em\u003e pathway as energy source.\u003csup\u003e19\u003c/sup\u003e The aim of the present study was to isolate bacterial species-es from different soil environments in Egypt were able to secrete \u003cem\u003eADI\u003c/em\u003e as anticancer agent using \u003cem\u003e16S rRNA\u003c/em\u003e sequencing technique; besides its bacterial recombinant \u003cem\u003eDNA\u003c/em\u003e technology manufacture; moreover the characterization of its auxotrophic anticancer activity.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":" \u003cp\u003e \u003cb\u003eMaterial and microorganisms\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003cem\u003eDiacetyl monoxime\u003c/em\u003e, \u003cem\u003eL-citrulline\u003c/em\u003e and \u003cem\u003eThiocarbazide\u003c/em\u003e were purchased from Alnasr chemical company, Egypt. \u003cem\u003eGlucose\u003c/em\u003e, \u003cem\u003eLactose\u003c/em\u003e, \u003cem\u003eMannitol\u003c/em\u003e, \u003cem\u003eMaltose\u003c/em\u003e, \u003cem\u003eSoluble starch\u003c/em\u003e, \u003cem\u003eNaCl\u003c/em\u003e, \u003cem\u003eL-arginine\u003c/em\u003e, \u003cem\u003eKH2PO4\u003c/em\u003e, \u003cem\u003eMgCl2\u003c/em\u003e, \u003cem\u003eMnCl2\u003c/em\u003e, \u003cem\u003eCoCl2\u003c/em\u003e, \u003cem\u003eNiCl2\u003c/em\u003e, \u003cem\u003eFeSO4\u003c/em\u003e, \u003cem\u003eFeCl3\u003c/em\u003e, \u003cem\u003e90% Phosphoric acid\u003c/em\u003e and \u003cem\u003e85% Sulfuric acid\u003c/em\u003e 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 \u003cem\u003eADI\u003c/em\u003e were obtained from Bio-Rad laboratories, USA. All chemical reagents utilized in the present study were of analytical grades.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCollection of samples\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e50 soil samples in a depth \u003cem\u003e0\u0026ndash;10 cm\u003c/em\u003e were collected from different governorates in Egypt.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDate and the place of the study\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe present study was conducted in the faculty of Pharmacy, Cairo University, Egypt between \u003cem\u003eAugust 2022\u003c/em\u003e and \u003cem\u003eMarch 2024.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eScreening of\u003c/b\u003e \u003cb\u003eArginine\u003c/b\u003e \u003cb\u003edegrading bacteria using\u003c/b\u003e \u003cb\u003ethe plate technique\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe test samples were collected from various regions in Egypt at a depth \u003cem\u003e0\u0026ndash;10 cm\u003c/em\u003e; besides the addition of \u003cem\u003e100 ml sterile water\u003c/em\u003e to the soil samples was achieved. On the other hand, \u003cem\u003e1 ml\u003c/em\u003e of each diluent sediment sample was transferred to the selective screening medium, Mineral Arginine Agar(MAA), and incubated for \u003cem\u003e24\u003c/em\u003e at \u003cem\u003e37 ℃\u003c/em\u003e. The \u003cem\u003eMAA\u003c/em\u003e plate consisted of \u003cem\u003e2% L-arginine\u003c/em\u003e, \u003cem\u003e0.01% glucose\u003c/em\u003e, \u003cem\u003e0.002% NaCl\u003c/em\u003e, \u003cem\u003e0.05% MgCl2\u003c/em\u003e, \u003cem\u003e0.075% KH2PO4\u003c/em\u003e, \u003cem\u003e0.02% MnCl2\u003c/em\u003e, \u003cem\u003e0.0001% FeSO4\u003c/em\u003e, \u003cem\u003e0.2 M CaCO3\u003c/em\u003e, \u003cem\u003e2.5% Phenol red\u003c/em\u003e and \u003cem\u003e1.2% Agar\u003c/em\u003e. Colonies which were able to utilize \u003cem\u003eL-arginine\u003c/em\u003e as sole metabolic \u003cem\u003eNitrogen\u003c/em\u003e rootage were only grown on \u003cem\u003eMAA\u003c/em\u003e plates and were considered \u003cem\u003eADI\u003c/em\u003e producing bacterial isolates which were further subcultured for numerous times until pure colonies were gotten.\u003csup\u003e20\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe produced colonies on \u003cem\u003eMAA plates\u003c/em\u003e were further subjected to subculture on Nutrient agar plates.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of activity of\u003c/b\u003e \u003cb\u003eArginine deiminase\u003c/b\u003e \u003cb\u003eusing nesslerization test\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe yielded pure colonies were subjected to further activity analysis of \u003cem\u003eArginine deiminase\u003c/em\u003e utilizing \u003cem\u003eNessler reagent\u003c/em\u003e to standardize the \u003cem\u003eAmmonia\u003c/em\u003e concentration as the product of \u003cem\u003eArginine\u003c/em\u003e degrading enzymes. \u003cem\u003eL-arginine\u003c/em\u003e was exploited as the sole metabolic \u003cem\u003eNitrogen\u003c/em\u003e source in the culture media. The soil samples were cultured in \u003cem\u003e20 ml\u003c/em\u003e of the defined medium; then incubated at \u003cem\u003e37℃\u003c/em\u003efor \u003cem\u003e24 hours\u003c/em\u003e. Afterwards, \u003cem\u003e2 ml\u003c/em\u003e of each growth medium was collected and centrifuged to assess the concentration of \u003cem\u003eAmmonia\u003c/em\u003e in the supernatants; then \u003cem\u003e45 \u0026micro;l\u003c/em\u003e of \u003cem\u003eNessler reagent\u003c/em\u003e was added to each1 ml of obtained supernatants. The concentration of the produced \u003cem\u003eAmmonia\u003c/em\u003e was quantified via measuring the optical density at \u003cem\u003e480 nm\u003c/em\u003e wavelength utilizing \u003cem\u003eUV Spectrophotometer\u003c/em\u003e according to the standard curve of \u003cem\u003eAmmonium sulfate\u003c/em\u003e. The calculated values were correlated to the activity of \u003cem\u003eL-arginine deiminase\u003c/em\u003e within \u003cem\u003e24 hours\u003c/em\u003e of incubation.\u003csup\u003e21\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDiacetyl monoxime Thiocarbazide assay for assessment of\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e \u003cb\u003eactivity\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eBased on the photometric assessment of the C\u003cem\u003eitrulline\u003c/em\u003e concentration\u0026mdash;which was produced after the \u003cem\u003eADI\u003c/em\u003e enzyme hydrolyzed \u003cem\u003eL-arginine\u003c/em\u003e\u0026mdash;the \u003cem\u003eADI\u003c/em\u003e enzyme activity was examined. The applicable reagents were made as following; \u003cem\u003eAcid-ferric solution\u003c/em\u003e; \u003cem\u003e300 ml\u003c/em\u003e of concentrated S\u003cem\u003eulfuric acid\u003c/em\u003e( 90%) and \u003cem\u003e150 ml\u003c/em\u003e of concentrated \u003cem\u003ePhosphoric acid\u003c/em\u003e( \u003cem\u003e90%\u003c/em\u003e) were added to \u003cem\u003e550 ml\u003c/em\u003e of \u003cem\u003edistilled water\u003c/em\u003e. After allowing the mixture to cool to room temperature, \u003cem\u003e3 mg/L\u003c/em\u003e of \u003cem\u003eFeCl3\u003c/em\u003e was added to it. \u003cem\u003eDiacetylmonoxime solution; 100 ml of distilled water\u003c/em\u003e was mixed with \u003cem\u003e500 mg\u003c/em\u003e of \u003cem\u003eDiacetylmonoxime\u003c/em\u003e. \u003cem\u003eChromatogenic reagent\u003c/em\u003e; \u003cem\u003e50 ml\u003c/em\u003e of \u003cem\u003ereagent 2\u003c/em\u003e was mixed with \u003cem\u003e5 mg\u003c/em\u003e of \u003cem\u003eThiosemicarbazide\u003c/em\u003e, and then \u003cem\u003e100 ml\u003c/em\u003e of \u003cem\u003ereagent 1\u003c/em\u003e was added. The solution was applied within an hour of the setup. The culture medium intended for the selected colony was cultivated in \u003cem\u003e20 milliliters\u003c/em\u003e. \u003cem\u003e2 ml\u003c/em\u003e of each growth media was collected after \u003cem\u003e24 hours\u003c/em\u003e of incubation, the amount of freed \u003cem\u003eCitrulline\u003c/em\u003e in the supernatants was measured by centrifugation.\u003cem\u003e1ml\u003c/em\u003e of the supernatant and 2 \u003cem\u003eml\u003c/em\u003e of the Chromogenic solution were combined, and the mixture was heated for \u003cem\u003e5 minutes\u003c/em\u003e at \u003cem\u003e100\u0026ordm;C\u003c/em\u003e. After cooling to room temperature, the absorbance at \u003cem\u003e530 nm\u003c/em\u003e was calculated. Based on the \u003cem\u003eL-citrulline\u003c/em\u003e standard curve, which was determined using the same technique, the amount of \u003cem\u003eCitrulline\u003c/em\u003e was computed and associated with the activity of the \u003cem\u003eADI\u003c/em\u003e enzyme, which converted \u003cem\u003eL-arginine\u003c/em\u003e to \u003cem\u003eL-citrulline.\u003c/em\u003e\u003csup\u003e\u003cem\u003e22\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of the major bacterial isolate producing\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThis was performed using \u003cem\u003eGram staining\u003c/em\u003e technique and different biochemical reactions; as well as the determination of morphological characters and molecular detection using \u003cem\u003e16S rRNA\u003c/em\u003e sequencing technique were employed. The genomic DNA was extracted using \u003cem\u003ePureLink\u0026trade;\u003c/em\u003e \u003cem\u003egenomic DNA Purification kit\u003c/em\u003e( purchased from \u003cem\u003eThermoFisher scientific, USA\u003c/em\u003e) on \u003cem\u003eSilica Columns\u003c/em\u003e. The \u003cem\u003eDNA\u003c/em\u003e extraction procedure was performed according to the \u003cem\u003eDNA Purification Kit\u003c/em\u003e manufacture instructions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProcedure of\u003c/b\u003e \u003cb\u003e16S rRNA\u003c/b\u003e \u003cb\u003esequencing technique\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe QIAGEN Multiplex \u003cem\u003ePCR Kit\u003c/em\u003e, Catalog number \u003cem\u003e206143\u003c/em\u003e was acquired from \u003cem\u003eQIAGEN,USA.\u003c/em\u003eMolecular characterization of \u003cem\u003eADI\u003c/em\u003e producing bacteria was carried out via \u003cem\u003e16S rRNA\u003c/em\u003e sequence analysis using the forward and reverse primers with the sequences of( \u003cem\u003e5\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-CAGCCGCGGTAATAC\u0026minus;3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e) and( \u003cem\u003e5\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-ACGGGGGGTGTGTAC\u0026minus;3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e), respectively. Using polymerase chain reaction( \u003cem\u003ePCR\u003c/em\u003e), \u003cem\u003e16S rRNA gene\u003c/em\u003e was cloned. Initial \u003cem\u003edenaturation\u003c/em\u003e was at \u003cem\u003e98℃\u003c/em\u003e for \u003cem\u003e5 minutes\u003c/em\u003e. This was followed by \u003cem\u003e25 cycles\u003c/em\u003e of \u003cem\u003edenaturation\u003c/em\u003e at \u003cem\u003e94℃\u003c/em\u003e for \u003cem\u003e30 sec\u003c/em\u003e. \u003cem\u003eAnnealing stage\u003c/em\u003e was performed at \u003cem\u003e54℃\u003c/em\u003e for \u003cem\u003e30 sec\u003c/em\u003e. Finally, \u003cem\u003ethe extension phase\u003c/em\u003e was adjusted at \u003cem\u003e72℃\u003c/em\u003e for \u003cem\u003e10 minutes\u003c/em\u003e. The size of the resulted amplicons were confirmed using \u003cem\u003eAgarose gel electrophoresis\u003c/em\u003e. The yielded sequences were compared with sequences deposited in Gen-bank database of national centre for biotechnology information( \u003cem\u003eNCBI\u003c/em\u003e) using nucleotide basic local alignment search tool( \u003cem\u003eBLAST\u003c/em\u003e\u003csub\u003e\u003cem\u003en\u003c/em\u003e\u003c/sub\u003e).\u003csup\u003e23\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of environmental and physiological factors affecting the productivity of\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThis was applied using different temperatures, \u003cem\u003epH\u003c/em\u003e, Metal ions, metabolic carbon and nitrogen sources, inoculum volumes, aeration conditions and substrate concentrations. Nesslerization technique was included to determine the amount of \u003cem\u003eADI\u003c/em\u003e produced spectrophotometrically at \u003cem\u003e480 nm\u003c/em\u003e wavelength.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePurification of\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe cold crude enzyme was diluted with \u003cem\u003eAmmonium Sulfate\u003c/em\u003e until \u003cem\u003e70% saturation\u003c/em\u003e was achieved. After being stored at \u003cem\u003e4\u0026deg;C\u003c/em\u003e for the whole night, the mixture was centrifuged for \u003cem\u003e25 minutes\u003c/em\u003e at \u003cem\u003e4000 \u0026times;g\u003c/em\u003e. The residue was dissolved in a minimum volume of \u003cem\u003e50 mM Tris-HCl buffer\u003c/em\u003e( \u003cem\u003epH 7.4\u003c/em\u003e) and dialyzed against the same buffer for a whole night at \u003cem\u003e4\u0026deg;C\u003c/em\u003e to get rid of the salts. A \u003cem\u003e45 x 1.5 cm Sephadex G-100 column\u003c/em\u003e pre-equilibrated with \u003cem\u003e0.05M Tris-HCl buffer\u003c/em\u003e( \u003cem\u003epH 8.6\u003c/em\u003e) was loaded with the dialyzed fraction. \u003cem\u003e0.05M Tris-HCl buffer (pH 7.4)\u003c/em\u003e with \u003cem\u003e0.1M KCl\u003c/em\u003e 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 \u003cem\u003e4\u0026deg;C\u003c/em\u003e. Following that, \u003cem\u003eSDS-PAGE\u003c/em\u003e, or \u003cem\u003eSodium Dodecyl Sulphate Polyacrylamide gel electrophoresis\u003c/em\u003e, was used. The cells were resuspended in \u003cem\u003e200 \u0026micro;l of lysis buffer 62.5 mM Tris/HCl, pH 6.8\u003c/em\u003e, \u003cem\u003e15% Glycerol\u003c/em\u003e, 5% \u003cem\u003e2-Mercaptoethanol, 2% Sodium Dodecyl Sulphate( SDS)\u003c/em\u003e, and \u003cem\u003e0.001% Bromophenol blue\u003c/em\u003e in order to produce complete cell lysates. After \u003cem\u003e5 minutes\u003c/em\u003e of boiling at \u003cem\u003e100\u0026deg;C\u003c/em\u003e, the complete cell lysates were put onto a \u003cem\u003e15% SDS Polyacrylamide gel\u003c/em\u003e and run at \u003cem\u003e100 V\u003c/em\u003e. \u003cem\u003eCoomassie blue\u003c/em\u003e 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.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstimation of protein content\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThis was applied according to \u003cem\u003eBradford\u003c/em\u003e assay. \u003cem\u003eBovine Albumin\u003c/em\u003e was utilized as a standard drug.\u003csup\u003e25\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of anticancer activity of\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e \u003cb\u003eusing\u003c/b\u003e \u003cb\u003eMTT\u003c/b\u003e \u003cb\u003eassay\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eHuman \u003cem\u003erenal\u003c/em\u003e( \u003cem\u003eCaki-1\u003c/em\u003e) and \u003cem\u003eleukemic\u003c/em\u003e( \u003cem\u003eK-562\u003c/em\u003e) cancer cell lines purchased from \u003cem\u003eATTC,UK\u003c/em\u003e were used to conduct the cytotoxicity assay. On the other hand \u003cem\u003eVero CCL-81\u0026trade;\u003c/em\u003e cell lines( Purchased from \u003cem\u003eATCC\u003c/em\u003e, \u003cem\u003eUSA\u003c/em\u003e) were utilized also in \u003cem\u003eMTT\u003c/em\u003e assay as standard natural cell lines. The cells were kept in Roswell Park Memorial Institute( \u003cem\u003eRPMI\u003c/em\u003e) medium supplemented with \u003cem\u003e10%\u003c/em\u003e heat-inactivated \u003cem\u003efoetal bovine serum\u003c/em\u003e, \u003cem\u003e100 \u0026micro;g/ml\u003c/em\u003e streptomycin, and 100U/ml penicillin at \u003cem\u003e37\u0026deg;C\u003c/em\u003e in a humidified environment with \u003cem\u003e5% CO2 V/V\u003c/em\u003e. Furthermore, the cells underwent twice-weekly subculturing. The \u003cem\u003eMTT\u003c/em\u003e( 3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was performed. The cells and test compounds were prepared in \u003cem\u003e96-well plates\u003c/em\u003e containing a final volume of \u003cem\u003e100 \u0026micro;l/well\u003c/em\u003e; then were incubated for \u003cem\u003e2 hours\u003c/em\u003e. \u003cem\u003e10 \u0026micro;l MTT\u003c/em\u003e Solution was added per well to achieve a final concentration of \u003cem\u003e0.50 mg/ml\u003c/em\u003e then incubated for \u003cem\u003e3\u003c/em\u003e hours at \u003cem\u003e37\u0026deg;C\u003c/em\u003e. \u003cem\u003e100 \u0026micro;\u003c/em\u003el Solubilization solution was added to each well to dissolve formazan crystals. The components were mixed to ensure complete solubilization. The absorbance was recordered at \u003cem\u003e570 nm\u003c/em\u003e.\u003csup\u003e26\u003c/sup\u003e In vitro determination of biological half life of \u003cem\u003eADI\u003c/em\u003e was performed using nesslerization assay.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of antioxidant activity of\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThis was applied via rapid \u003cem\u003eDPPH\u003c/em\u003e radical scavenging assay using \u003cem\u003edot-blot\u003c/em\u003e. With a few minor adjustments, the Soler-Rivas et al., 2000 approach was used to qualitatively evaluate \u003cem\u003eADI\u003c/em\u003e extracts for anti-oxidant activity using \u003cem\u003eDPPH\u003c/em\u003e radical. In summary, a small amount(5 \u0026micro;l) of every \u003cem\u003eADI\u003c/em\u003e extract dilution and standard antioxidant was meticulously placed onto a thin-layer chromatography( \u003cem\u003eTMC\u003c/em\u003e) plate( \u003cem\u003eSilica gel 60 F254\u003c/em\u003e, \u003cem\u003eMerck\u003c/em\u003e) and left to air dry. After that, the sheets were sprayed with \u003cem\u003eDPPH\u003c/em\u003e( \u003cem\u003e0.2% W/V\u003c/em\u003e in \u003cem\u003eADI\u003c/em\u003e 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.\u003csup\u003e27\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of apoptosis induced via\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003cem\u003eCaspase 3 assay\u003c/em\u003e was utilized for this purpose. The procedure was performed as described before according to \u003cem\u003eKim RH et al, 2009\u003c/em\u003e.\u003csup\u003e28\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eProduction of\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e \u003cb\u003evia bacterial recombinant\u003c/b\u003e \u003cb\u003eDNA\u003c/b\u003e \u003cb\u003etechnology\u003c/b\u003e:\u003c/p\u003e \u003cp\u003e \u003cb\u003eForward primer\u003c/b\u003e: \u003cem\u003e5\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-TGACCCAATGCCAAACCTAT\u0026minus;3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eReverse primer\u003c/b\u003e: \u003cem\u003e5\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-CGTCTCCACCTTCGATTGAT\u0026minus;3\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe QIAGEN Multiplex \u003cem\u003ePCR Kit\u003c/em\u003e, Catalog number 206143 was purchased from QIAGEN,USA. \u003cem\u003epET-26b(+)\u003c/em\u003e, which was acquired from \u003cem\u003eNovagene in the USA\u003c/em\u003e, served as the expression vector. \u003cem\u003eGenSmart\u0026trade; Design software\u003c/em\u003e was used to create the expression vector. The tagged protein, \u003cem\u003e6x histidine\u003c/em\u003e attached to the \u003cem\u003eC terminus\u003c/em\u003e of an \u003cem\u003eADI molecule\u003c/em\u003e, was \u003cem\u003epromotor T7 Lac\u003c/em\u003e. As the expression host, \u003cem\u003eEscherichia coli BL21[DE3] polys S\u003c/em\u003e was used. They were all employed in the bacterial recombinant \u003cem\u003eDNA\u003c/em\u003e method to produce \u003cem\u003eADI\u003c/em\u003e. The inducer of the transcription process was \u003cem\u003eIPTG\u003c/em\u003e. \u003cem\u003eEscherichia coli DH5\u003c/em\u003e, which was procured from \u003cem\u003eStratagene Corporation in the USA\u003c/em\u003e, served as the primary host for plasmid manufacture and replication. Using \u003cem\u003erestriction endonuclease type II enzymes\u003c/em\u003e( \u003cem\u003eDNA cutting enzymes\u003c/em\u003e) \u003cem\u003eXbal\u003c/em\u003e and \u003cem\u003eACCI\u003c/em\u003e, the predominant bacterial isolate found in various soil conditions in Egypt was subjected to genomic \u003cem\u003eDNA\u003c/em\u003e isolation. The German company \u003cem\u003eSigma-Aldrich\u003c/em\u003e supplied these cutting enzymes. Additionally, using the same restriction endonuclease \u003cem\u003etype II enzymes\u003c/em\u003e that were used to extract genomic \u003cem\u003eDNA\u003c/em\u003e from the primary bacterial isolates, genomic \u003cem\u003eDNA\u003c/em\u003e was amplified using the polymerase chain reaction( \u003cem\u003ePCR\u003c/em\u003e) technique and then sub-cloned to the prokaryotic expression vector \u003cem\u003ePET26b(+)\u003c/em\u003e. After then, \u003cem\u003eEscherichia coli BL21[DE3] poly S\u003c/em\u003e was transformed from \u003cem\u003ePET26b(+)\u003c/em\u003e. Gene expression was started when \u003cem\u003eIPTG\u003c/em\u003e was added because it increased transcription at the promotor site \u003cem\u003eT7 Lac\u003c/em\u003e. For standard bacterial culture, \u003cem\u003eLB\u003c/em\u003e and \u003cem\u003eLA broth\u003c/em\u003e were employed, and the incubation temperature was set at \u003cem\u003e37\u0026deg;C\u003c/em\u003e for \u003cem\u003e24 hours\u003c/em\u003e. In accordance with the manufacturer's instructions, \u003cem\u003eAmpicillin\u003c/em\u003e and/or \u003cem\u003eKanamycin\u003c/em\u003e were added to the culture medium. \u003cem\u003eIon exchange column chromatography\u003c/em\u003e, dialysis, and \u003cem\u003e70% Ammonium Sulphate\u003c/em\u003e were used to purify the recombinant protein.\u003csup\u003e29\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eKinetic properties of the purified\u003c/b\u003e \u003cb\u003eADI\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eA \u003cem\u003epH\u003c/em\u003e range of \u003cem\u003e5.0 to 13.0\u003c/em\u003e was used to establish the ideal \u003cem\u003epH\u003c/em\u003e for \u003cem\u003eADI\u003c/em\u003e activity. Enzyme preparations at different \u003cem\u003epH\u003c/em\u003e values were incubated for one hour to conduct \u003cem\u003epH stability\u003c/em\u003e tests. Following this, the relative activity was measured using conventional assay procedures. By testing \u003cem\u003eADI\u003c/em\u003e activity at various temperatures between \u003cem\u003e30\u003c/em\u003e and \u003cem\u003e100\u0026deg;C\u003c/em\u003e, the optimum temperature for enzyme activity was found. Furthermore, the enzyme's thermal stability was assessed by assaying \u003cem\u003eADI\u003c/em\u003e activity after the lyophilized enzyme was incubated for one hour at the same temperatures. With concentrations ranging from \u003cem\u003e0.0 to 0.1M\u003c/em\u003e, \u003cem\u003eL-arginine\u003c/em\u003e was used as a substrate to calculate the kinetic parameters( \u003cem\u003eKm and Vmax\u003c/em\u003e) for \u003cem\u003eADI\u003c/em\u003e.Using the equation obtained from the \u003cem\u003elinear-regression analysis\u003c/em\u003e of the curve, the \u003cem\u003eMichaelis-Menten\u003c/em\u003e parameters were ascertained from \u003cem\u003eLineaweaver-Burk plots\u003c/em\u003e.\u003csup\u003e30\u003c/sup\u003e\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eSTATISTICAL ANALYSIS\u003c/h2\u003e \u003cp\u003eThe average and standard deviation of each experiment were reported. Each experiment was carried out in triplicate. \u003cem\u003eMinitab software\u003c/em\u003e was utilized for conducting statistical analyses, which involved one-way analysis of variance( \u003cem\u003eANOVA\u003c/em\u003e) with a \u003cem\u003ep-value\u003c/em\u003e of less than \u003cem\u003e0.05\u003c/em\u003e.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eBacterial isolates were grown on \u003cem\u003eMAA\u003c/em\u003e plates with characterized Pink zones due to \u003cem\u003eArginine deiminase\u003c/em\u003e activity. Maximum \u003cem\u003eArginine deiminase\u003c/em\u003e production was achieved using an incubation period of \u003cem\u003e24 hours\u003c/em\u003e, optimum \u003cem\u003epH 8\u003c/em\u003e and temperature \u003cem\u003e40℃\u003c/em\u003e and agitation rate \u003cem\u003e200 rpm\u003c/em\u003e. On the other hand, \u003cem\u003eMannitol\u003c/em\u003e and \u003cem\u003ePeptone\u003c/em\u003e were used as the sole metabolic carbon and nitrogen sources, respectively which contributed to the highest production of \u003cem\u003eADI\u003c/em\u003e. The purified \u003cem\u003eADI\u003c/em\u003e demonstrated final specific activity of approximately \u003cem\u003e36.07 U/mg\u003c/em\u003e and molecular mass was detected to be \u003cem\u003e36.18 kDa\u003c/em\u003e using \u003cem\u003eSDS-PAGE gel filtration technique\u003c/em\u003e. The maximum activity and stability of the purified enzyme occurred at \u003cem\u003epH 9\u003c/em\u003e and \u003cem\u003e8\u003c/em\u003e, respectively as well as temperatures \u003cem\u003e40℃\u003c/em\u003e and \u003cem\u003e37℃\u003c/em\u003e.Complete thermal stability was confirmed at \u003cem\u003e70℃\u003c/em\u003e for \u003cem\u003eone hour\u003c/em\u003e. The \u003cem\u003eKm\u003c/em\u003e and \u003cem\u003eVmax\u003c/em\u003e of \u003cem\u003eADI\u003c/em\u003e were \u003cem\u003e0.05871 M\u003c/em\u003e and \u003cem\u003e40.36 \u0026micro;mol/ml/min\u003c/em\u003e, respectively; while, \u003cem\u003eKcat\u003c/em\u003e reached about \u003cem\u003e5.014min\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e. The anticancer activity was determined through \u003cem\u003eMTT\u003c/em\u003e assay. \u003cem\u003eADI\u003c/em\u003e demonstrated significant cytotoxic actions versus \u003cem\u003erenal\u003c/em\u003e( \u003cem\u003eCaki-1\u003c/em\u003e) and \u003cem\u003eleukemic\u003c/em\u003e( \u003cem\u003eK-562\u003c/em\u003e) cancer cell lines( \u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e10.31 \u0026micro;g/ml\u003c/em\u003e and \u003cem\u003e16.08 \u0026micro;g/ml\u003c/em\u003e, respectively). Negligible \u003cem\u003eADI\u003c/em\u003e cytotoxicity effect on \u003cem\u003eVero CCL-81\u0026trade;\u003c/em\u003e cell lines were observed during \u003cem\u003eMTT\u003c/em\u003e assay. \u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e of standard \u003cem\u003eDoxorubicin\u003c/em\u003e anticancer drug against \u003cem\u003erenal\u003c/em\u003e( \u003cem\u003eCaki-1\u003c/em\u003e) and \u003cem\u003eleukemic\u003c/em\u003e( \u003cem\u003eK-562\u003c/em\u003e) cancer cell lines( \u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e9.87 \u0026micro;g/ml\u003c/em\u003e and 7\u003cem\u003e.33 \u0026micro;g/ml\u003c/em\u003e, respectively). Standard L-asparaginase demonstrated only \u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;~\u0026thinsp;5 \u003cem\u003e\u0026micro;g\u003c/em\u003e/ml against \u003cem\u003eleukemic\u003c/em\u003e( \u003cem\u003eK-562\u003c/em\u003e) cancer cell line but failed to exert significant cytotoxicity against \u003cem\u003eVero\u003c/em\u003e and renal cancer cell lines. \u003cem\u003eDPPH\u003c/em\u003e scavenging free radical assay using \u003cem\u003eAscorbic acid\u003c/em\u003e as a standard drug revealed that \u003cem\u003eADI\u003c/em\u003e possess no antioxidant activities. \u003cem\u003e5.68 mg\u003c/em\u003e total protein was produced during the initial culture of \u003cem\u003eADI\u003c/em\u003e on \u003cem\u003eMAA\u003c/em\u003e medium as estimated via \u003cem\u003eBradford\u003c/em\u003e assay utilizing \u003cem\u003eBovine Albumin\u003c/em\u003e as a standard agent. \u003cem\u003eIn vitro\u003c/em\u003e resoluteness of biological half life of \u003cem\u003eADI\u003c/em\u003e exploiting nesslerization assay was ascertained to be closely \u003cem\u003e300 min\u003c/em\u003e. Only \u003cem\u003e29\u003c/em\u003e bacterial isolates producing different arginine degrading enzymes were detected to be grown on \u003cem\u003eMAA\u003c/em\u003e, from which \u003cem\u003e17\u003c/em\u003e bacterial isolates showed the highest production of \u003cem\u003eADI\u003c/em\u003e. Morphological, biochemical reactions detected that \u003cem\u003eBacillus subtilis\u003c/em\u003e was the major bacterial species producing \u003cem\u003eADI\u003c/em\u003e. \u003cem\u003e16S rRNA\u003c/em\u003e gene sequencing technique using \u003cem\u003eBLASTn\u003c/em\u003e demonstrated that \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e was the predominant Bacillus secreting ADI in the present study. The specific activity of recombinant \u003cem\u003eADI\u003c/em\u003e was \u003cem\u003e140.09 U/mg\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe biochemical processes profile of \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e is shown in Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e. Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e presents a \u003cem\u003eBLASTn\u003c/em\u003e analysis of \u003cem\u003e16S rRNA\u003c/em\u003e sequencing. Aeration impacts on ADI production are seen in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The carbon and nitrogen growth variables that impact ADI production are displayed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The effects of metal cations on \u003cem\u003eADI\u003c/em\u003e activity and production are shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. It illustrates how varying substrate concentrations affect the productivity of \u003cem\u003eADI\u003c/em\u003e. The effects of \u003cem\u003epH\u003c/em\u003e and temperature on \u003cem\u003eADI\u003c/em\u003e production are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The effects of \u003cem\u003epH\u003c/em\u003e and temperature on \u003cem\u003eADI\u003c/em\u003e activity are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e isolates that produce ADI under a standard microscope are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e14\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the \u003cem\u003eADI\u003c/em\u003e monomer \u003cem\u003e3D structure\u003c/em\u003e using the \u003cem\u003eSWISS-MODEL\u003c/em\u003e software. There are 418 amino acids in ADI. The evaluation of \u003cem\u003eADI-induced\u003c/em\u003e apoptosis is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e using colorimetric \u003cem\u003eCaspase 3\u003c/em\u003e activity detection. The \u003cem\u003eMichaelis Menten plot\u003c/em\u003e of \u003cem\u003eADI\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e. The \u003cem\u003eBradford\u003c/em\u003e test is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e12\u003c/span\u003e and is used to measure the protein concentration in \u003cem\u003eADI\u003c/em\u003e productivity studies. The absorbance of \u003cem\u003eADI\u003c/em\u003e at \u003cem\u003e480 nm\u003c/em\u003e wavelength caused by ammonia leakage is seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e. More \u003cem\u003eADI\u003c/em\u003e activities were seen in response to \u003cem\u003eAmmonia\u003c/em\u003e releases. The optical density of \u003cem\u003eADI\u003c/em\u003e activity at 530 nm is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As the \u003cem\u003eADI\u003c/em\u003e concentration grew, absorbance increased as well. The test \u003cem\u003eADI\u003c/em\u003e's phylogenetic guide tree, created by Clustal Omega software, is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e13\u003c/span\u003e. The \u003cem\u003eMTT\u003c/em\u003e test of \u003cem\u003eADI\u003c/em\u003e on a leukemic cancer cell line is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows how to determine whether \u003cem\u003eADI\u003c/em\u003e-induced apoptosis occurred by looking for \u003cem\u003eCaspase 3\u003c/em\u003e activity at \u003cem\u003e450 nm\u003c/em\u003e. The \u003cem\u003eMTT\u003c/em\u003e test of \u003cem\u003eADI\u003c/em\u003e on a kidney cancer cell line is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Using \u003cem\u003eCB-DOCk2\u003c/em\u003e software, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the strong attraction of the \u003cem\u003eMn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u0026thinsp;2\u003c/em\u003e\u003c/sup\u003e metal cation to \u003cem\u003eADI\u003c/em\u003e. The optical density of \u003cem\u003eADI\u003c/em\u003e activity at \u003cem\u003e530 nm\u003c/em\u003e is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As the \u003cem\u003eADI\u003c/em\u003e concentration grew, absorbance increased as well. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e illustrates how \u003cem\u003eADI\u003c/em\u003e is docked with \u003cem\u003eCB-DOCK 2\u003c/em\u003e software. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the purification of \u003cem\u003eADI\u003c/em\u003e produced by \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e using \u003cem\u003eSDS-PAGE\u003c/em\u003e. Nearly \u003cem\u003e85%\u003c/em\u003e of the sample was pure.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eADI catalyzing the hydrolysis of L-arginine to L-citrulline and Ammonia was found to be an enzyme of therapeutic importance in the management of cancer, especially auxotrophic cancers for L-arginine such as Melanoma and renal cell carcinoma. The present study described the recombinant production, properties and anticancer potential of the enzyme from \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e. About \u003cem\u003e58%\u003c/em\u003e of bacterial isolates grown on \u003cem\u003eMAA\u003c/em\u003e showed high productivity of arginine degrading enzymes, from which \u003cem\u003e34%\u003c/em\u003e bacterial isolates produced \u003cem\u003eADI\u003c/em\u003e. Morphological characterization and biochemical reactions; besides \u003cem\u003e16S rRNA\u003c/em\u003e sequencing technique revealed that \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e bacterial isolates were the potent ADI producers. A \u003cem\u003e1230\u003c/em\u003e base pair gene encoding 410 aminoacids was PCR amplified and cloned into \u003cem\u003eBL21(DE3) poly sS strain\u003c/em\u003e of \u003cem\u003eE.col\u003c/em\u003ei expression host using \u003cem\u003epET26b(+) plasmid\u003c/em\u003e which was the expression vector. The creation of the recombinant \u003cem\u003eADI\u003c/em\u003e was induced under the influence of \u003cem\u003e0.5 mM IPTG\u003c/em\u003e. On the other hand, \u003cem\u003eADI\u003c/em\u003e was purified via \u003cem\u003e70%\u003c/em\u003e ammonium sulfate salting out from the supernatant of the centrifuge tube. Thereafter, \u003cem\u003eADI\u003c/em\u003e was extracted as a refined enzyme( \u003cem\u003e85% purity\u003c/em\u003e) using cation exchange chromatography and gel filtration through \u003cem\u003eSDS-PAGE\u003c/em\u003e. Purified enzyme was analyzed for the kinetics in Silico studies and experimentally. Through \u003cem\u003eMTT\u003c/em\u003e assay, \u003cem\u003eADI\u003c/em\u003e showed anticancer potential against auxotrophic cancers for \u003cem\u003eL-arginine\u003c/em\u003e such as renal cancer; as well as It demonstrated significant antileukaemic activities. The recombinant \u003cem\u003eADI\u003c/em\u003e enzyme showed a molecular mass of \u003cem\u003e36.18 kDa\u003c/em\u003e, specific activity of \u003cem\u003e140.09 U/mg which was fourth fold more than the initial culture\u003c/em\u003e. The \u003cem\u003eKm\u003c/em\u003e and \u003cem\u003eVmax\u003c/em\u003e of \u003cem\u003eADI\u003c/em\u003e were \u003cem\u003e0.05871 M\u003c/em\u003e and \u003cem\u003e40.36 \u0026micro;mol/ml/min\u003c/em\u003e, respectively; while, \u003cem\u003eKcat\u003c/em\u003e reached about \u003cem\u003e5.014min\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;\u0026thinsp;1\u003c/em\u003e\u003c/sup\u003e reflecting high affinity of the enzyme to L-arginine substrate. Optimum temperature and pH were \u003cem\u003e37\u0026ndash;40 ℃\u003c/em\u003e and 6\u0026ndash;10, respectively. \u003cem\u003eADI\u003c/em\u003e demonstrated the highest activity at \u003cem\u003e37 ℃\u003c/em\u003e and \u003cem\u003epH 9\u003c/em\u003e. On the other hand, \u003cem\u003eADI\u003c/em\u003e showed thermostability at 70 ℃ for \u003cem\u003e1 hour\u003c/em\u003e.During the initial culture on \u003cem\u003eMAA\u003c/em\u003e plates, the productivity and the activity were detected to be increased in presence of \u003cem\u003eCo\u003c/em\u003e\u0026thinsp;\u003csup\u003e\u003cem\u003e+\u0026thinsp;2\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eNi\u003c/em\u003e\u0026thinsp;\u003csup\u003e\u003cem\u003e+\u0026thinsp;2\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eMn\u003c/em\u003e\u0026thinsp;\u003csup\u003e\u003cem\u003e+\u0026thinsp;2\u003c/em\u003e\u003c/sup\u003e metal ions; while they were diminished significantly in presence of \u003cem\u003eFe\u003c/em\u003e\u0026thinsp;\u003csup\u003e\u003cem\u003e+\u0026thinsp;3\u003c/em\u003e\u003c/sup\u003e metal ions. No detectable enzyme activity was detected when either \u003cem\u003eL- glutamine\u003c/em\u003e or \u003cem\u003eL-asparagine\u003c/em\u003e was used as the substrate for \u003cem\u003eADI\u003c/em\u003e. Peptone and Mannitol were the optimal activators for \u003cem\u003eADI\u003c/em\u003e producing bacterial isolates. Ebrahimi N et al study, 2016 demonstrated that \u003cem\u003eADI\u003c/em\u003e was produced via \u003cem\u003eEnterbacter sp. Sgn 1\u003c/em\u003e as a potential anticancer agent;3\u003csup\u003e1\u003c/sup\u003e whereas in the present study \u003cem\u003eADI\u003c/em\u003e was produced from Bacillus \u003cem\u003esubtilis DE111\u003c/em\u003e. In Silico studies confirmed that \u003cem\u003eADI\u003c/em\u003e existed as a monomer having \u003cem\u003eArg 9\u003c/em\u003e, \u003cem\u003eALa 85\u003c/em\u003e, \u003cem\u003eThr 107\u003c/em\u003e, \u003cem\u003eHis 108\u003c/em\u003e, \u003cem\u003eGln 139\u003c/em\u003e, \u003cem\u003eLeu 161\u003c/em\u003e and \u003cem\u003eLys 253\u003c/em\u003e being the putative active site residues. The free energy change calculated via molecular docking studies of the enzyme and the substrate was observed to be \u003cem\u003e∆G\u0026minus;5.1 KJ/mole\u003c/em\u003e indicating the optimum affinity of \u003cem\u003eADI\u003c/em\u003e with \u003cem\u003eL-arginine substrate\u003c/em\u003e. The optimal shaking speed for the production of \u003cem\u003eADI\u003c/em\u003e was noticed at \u003cem\u003e200 rpm\u003c/em\u003e after \u003cem\u003e24 hours\u003c/em\u003e incubation time. The more the concentration of \u003cem\u003eL-arginine\u003c/em\u003e as a substrate the more \u003cem\u003eADI\u003c/em\u003e was produced during the initial culture on \u003cem\u003eMAA\u003c/em\u003e plates. The significant rise in \u003cem\u003eCaspase 3 activity\u003c/em\u003e was demonstrated using \u003cem\u003eCaspase 3 activity assay\u003c/em\u003e suggesting that \u003cem\u003eADI\u003c/em\u003e induced programmed cell death of cancer cells( apoptosis) selectively. \u003cem\u003eADI\u003c/em\u003e chiefly destroyed the renal cancer cell line through its inhibition of L-arginine from the external media surrounding these auxotrophic cancer cells for \u003cem\u003eL-arginine\u003c/em\u003e. On the other hand the leukemic cancer cell line which was not auxotrophic for \u003cem\u003eL-arginine( due to abundance of Arginosuccinate synthetase I\u003c/em\u003e was broke down through apoptosis induced via recombinant \u003cem\u003eADI. ADI\u003c/em\u003e exhibited \u003cem\u003e68%\u003c/em\u003e and \u003cem\u003e51%\u003c/em\u003e growth inhibition of leukemic and renal cancer cell lines, respectively as indicated using \u003cem\u003eMTT\u003c/em\u003e assay. In the present study, \u003cem\u003eMTT\u003c/em\u003e assay revealed that \u003cem\u003eADI\u003c/em\u003e ascertained comparable efficacy to standard \u003cem\u003eL-asparaginase\u003c/em\u003e and \u003cem\u003eDoxorubicin\u003c/em\u003e against leukemic cancer cell line and renal cancer cell line. Neither \u003cem\u003eADI\u003c/em\u003e antioxidant nor scavenging free radical activities were detected via \u003cem\u003eDPPH\u003c/em\u003e assay.\u003c/p\u003e "},{"header":"CONCLUSION","content":"\u003cp\u003eThe present work was a promising study due to the evolution of novel bacterial recombinant \u003cem\u003eADI\u003c/em\u003e from different soil environments in Egypt as a potential anti-neoplastic mediator versus renal auxotrophic cancer cells for \u003cem\u003eL-arginine\u003c/em\u003e as well as leukaemia cancers.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eThere is no conflict of interest.\u003c/p\u003e \u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe present study was funded by me.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eThakker DP, Narayanan R. Arginine deiminase produced by lactic acid bacteria as a potent anti-cancer drug. Med Oncol. 2023 May 12;40(6):175. doi: 10.1007/s12032-023-02043-4. PMID: 37171497.\u003c/li\u003e\n\u003cli\u003eXiong L, Teng JL, Botelho MG, Lo RC, Lau SK, Woo PC. Arginine Metabolism in Bacterial Pathogenesis and Cancer Therapy. Int J Mol Sci. 2016 Mar 11;17(3):363. doi: 10.3390/ijms17030363. PMID: 26978353; PMCID: PMC4813224.\u003c/li\u003e\n\u003cli\u003eFeun LG, Kuo MT, Savaraj N. Arginine deprivation in cancer therapy. Curr Opin Clin Nutr Metab Care. 2015 Jan;18(1):78-82. doi: 10.1097/MCO.0000000000000122. PMID: 25474015.\u003c/li\u003e\n\u003cli\u003eKuo MT, Savaraj N, Feun LG. Targeted cellular metabolism for cancer chemotherapy with recombinant arginine-degrading enzymes. Oncotarget. 2010 Aug;1(4):246-51. doi: 10.18632/oncotarget.135. PMID: 21152246; PMCID: PMC2998341.\u003c/li\u003e\n\u003cli\u003eTsai WB, Aiba I, Lee SY, Feun L, Savaraj N, Kuo MT. Resistance to arginine deiminase treatment in melanoma cells is associated with induced argininosuccinate synthetase expression involving c-Myc/HIF-1alpha/Sp4. Mol Cancer Ther. 2009 Dec;8(12):3223-33. doi: 10.1158/1535-7163.MCT-09-0794. PMID: 19934275; PMCID: PMC2795119.\u003c/li\u003e\n\u003cli\u003eLong Y, Tsai WB, Chang JT, Estecio M, Wangpaichitr M, Savaraj N, Feun LG, Chen HH, Kuo MT. Cisplatin-induced synthetic lethality to arginine-starvation therapy by transcriptional suppression of ASS1 is regulated by DEC1, HIF-1\u0026alpha;, and c-Myc transcription network and is independent of ASS1 promoter DNA methylation. Oncotarget. 2016 Dec 13;7(50):82658-82670. doi: 10.18632/oncotarget.12308. PMID: 27765932; PMCID: PMC5347722.\u003c/li\u003e\n\u003cli\u003eFeun L, You M, Wu CJ, Kuo MT, Wangpaichitr M, Spector S, Savaraj N. Arginine deprivation as a targeted therapy for cancer. Curr Pharm Des. 2008;14(11):1049-57. doi: 10.2174/138161208784246199. PMID: 18473854; PMCID: PMC3096551.\u003c/li\u003e\n\u003cli\u003eThongkum A, Wu C, Li YY, Wangpaichitr M, Navasumrit P, Parnlob V, Sricharunrat T, Bhudhisawasdi V, Ruchirawat M, Savaraj N. The Combination of Arginine Deprivation and 5-Fluorouracil Improves Therapeutic Efficacy in Argininosuccinate Synthetase Negative Hepatocellular Carcinoma. Int J Mol Sci. 2017 Jun 1;18(6):1175. doi: 10.3390/ijms18061175. PMID: 28587170; PMCID: PMC5485998.\u003c/li\u003e\n\u003cli\u003eSavaraj N, Wu C, Li YY, Wangpaichitr M, You M, Bomalaski J, He W, Kuo MT, Feun LG. Targeting argininosuccinate synthetase negative melanomas using combination of arginine degrading enzyme and cisplatin. Oncotarget. 2015 Mar 20;6(8):6295-309. doi: 10.18632/oncotarget.3370. PMID: 25749046; PMCID: PMC4467438.\u003c/li\u003e\n\u003cli\u003eKim RH, Coates JM, Bowles TL, McNerney GP, Sutcliffe J, Jung JU, Gandour-Edwards R, Chuang FY, Bold RJ, Kung HJ. Arginine deiminase as a novel therapy for prostate cancer induces autophagy and caspase-independent apoptosis. Cancer Res. 2009 Jan 15;69(2):700-8. doi: 10.1158/0008-5472.CAN-08-3157. PMID: 19147587; PMCID: PMC2629384.\u003c/li\u003e\n\u003cli\u003eBowles TL, Kim R, Galante J, Parsons CM, Virudachalam S, Kung HJ, Bold RJ. Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase. Int J Cancer. 2008 Oct 15;123(8):1950-5. doi: 10.1002/ijc.23723. PMID: 18661517; PMCID: PMC4294549.\u003c/li\u003e\n\u003cli\u003eEnsor CM, Holtsberg FW, Bomalaski JS, Clark MA. Pegylated arginine deiminase (ADI-SS PEG20,000 mw) inhibits human melanomas and hepatocellular carcinomas in vitro and in vivo. Cancer Res. 2002 Oct 1;62(19):5443-50. PMID: 12359751.\u003c/li\u003e\n\u003cli\u003eNi Y, Schwaneberg U, Sun ZH. Arginine deiminase, a potential anti-tumor drug. Cancer Lett. 2008 Mar 8;261(1):1-11. doi: 10.1016/j.canlet.2007.11.038. Epub 2008 Jan 7. PMID: 18179862.\u003c/li\u003e\n\u003cli\u003eShen LJ, Shen WC. Drug evaluation: ADI-PEG-20--a PEGylated arginine deiminase for arginine-auxotrophic cancers. Curr Opin Mol Ther. 2006 Jun;8(3):240-8. PMID: 16774044.\u003c/li\u003e\n\u003cli\u003eAscierto PA, Scala S, Castello G, Daponte A, Simeone E, Ottaiano A, Beneduce G, De Rosa V, Izzo F, Melucci MT, Ensor CM, Prestayko AW, Holtsberg FW, Bomalaski JS, Clark MA, Savaraj N, Feun LG, Logan TF. Pegylated arginine deiminase treatment of patients with metastatic melanoma: results from phase I and II studies. J Clin Oncol. 2005 Oct 20;23(30):7660-8. doi: 10.1200/JCO.2005.02.0933. Erratum in: J Clin Oncol. 2006 Aug 20;24(24):4047. PMID: 16234528.\u003c/li\u003e\n\u003cli\u003eIzzo F, Marra P, Beneduce G, Castello G, Vallone P, De Rosa V, Cremona F, Ensor CM, Holtsberg FW, Bomalaski JS, Clark MA, Ng C, Curley SA. Pegylated arginine deiminase treatment of patients with unresectable hepatocellular carcinoma: results from phase I/II studies. J Clin Oncol. 2004 May 15;22(10):1815-22. doi: 10.1200/JCO.2004.11.120. PMID: 15143074.\u003c/li\u003e\n\u003cli\u003eGlazer ES, Piccirillo M, Albino V, Di Giacomo R, Palaia R, Mastro AA, Beneduce G, Castello G, De Rosa V, Petrillo A, Ascierto PA, Curley SA, Izzo F. Phase II study of pegylated arginine deiminase for nonresectable and metastatic hepatocellular carcinoma. J Clin Oncol. 2010 May 1;28(13):2220-6. doi: 10.1200/JCO.2009.26.7765. Epub 2010 Mar 29. PMID: 20351325.\u003c/li\u003e\n\u003cli\u003eCurley SA, Bomalaski JS, Ensor CM, Holtsberg FW, Clark MA. Regression of hepatocellular cancer in a patient treated with arginine deiminase. Hepatogastroenterology. 2003 Sep-Oct;50(53):1214-6. PMID: 14571701.\u003c/li\u003e\n\u003cli\u003eChu YD, Lai MW, Yeh CT. Unlocking the Potential of Arginine Deprivation Therapy: Recent Breakthroughs and Promising Future for Cancer Treatment. Int J Mol Sci. 2023 Jun 26;24(13):10668. doi: 10.3390/ijms241310668. PMID: 37445845; PMCID: PMC10341449.\u003c/li\u003e\n\u003cli\u003eWang W, Li M, Miao M, Zhang T. Characterization of a recombinant arginine deiminase from Halothermothrix orenii and its application in citrulline production. Biotechnol Appl Biochem. 2023 Apr;70(2):526-536. doi: 10.1002/bab.2375. Epub 2022 Jun 27. PMID: 35761421.\u003c/li\u003e\n\u003cli\u003eRashad YM, Hafez M, Rashad M. Diazotrophic Azotobacter salinestris YRNF3: a probable calcite-solubilizing bio-agent for improving the calcareous soil properties. Sci Rep. 2023 Nov 23;13(1):20621. doi: 10.1038/s41598-023-47924-w. PMID: 37996572; PMCID: PMC10667278.\u003c/li\u003e\n\u003cli\u003eWei X, Chow HY, Chong HC, Leung SL, Ho MK, Lee MY, Leung YC. Arginine Is a Novel Drug Target for Arginine Decarboxylase in Human Colorectal Cancer Cells. Int J Mol Sci. 2023 Sep 6;24(18):13741. doi: 10.3390/ijms241813741. PMID: 37762044; PMCID: PMC10531272.\u003c/li\u003e\n\u003cli\u003eZhao MM, Du SS, Li QH, Chen T, Qiu H, Wu Q, Chen SS, Zhou Y, Zhang Y, Hu Y, Su YL, Shen L, Zhang F, Weng D, Li HP. High throughput 16SrRNA gene sequencing reveals the correlation between Propionibacterium acnes and sarcoidosis. Respir Res. 2017 Feb 1;18(1):28. doi: 10.1186/s12931-017-0515-z. PMID: 28143482; PMCID: PMC5286795.\u003c/li\u003e\n\u003cli\u003eAlrumman SA, Mostafa YS, Al-Izran KA, Alfaifi MY, Taha TH, Elbehairi SE. Production and Anticancer Activity of an L-Asparaginase from Bacillus licheniformis Isolated from the Red Sea, Saudi Arabia. Sci Rep. 2019 Mar 6;9(1):3756. doi: 10.1038/s41598-019-40512-x. PMID: 30842557; PMCID: PMC6403232.\u003c/li\u003e\n\u003cli\u003eKielkopf CL, Bauer W, Urbatsch IL. Bradford Assay for Determining Protein Concentration. Cold Spring Harb Protoc. 2020 Apr 1;2020(4):102269. doi: 10.1101/pdb.prot102269. PMID: 32238597.\u003c/li\u003e\n\u003cli\u003eBuranaamnuay K. The MTT assay application to measure the viability of spermatozoa: A variety of the assay protocols. Open Vet J. 2021 Apr-Jun;11(2):251-269. doi: 10.5455/OVJ.2021.v11.i2.9. Epub 2021 May 8. PMID: 34307082; PMCID: PMC8288735. \u003c/li\u003e\n\u003cli\u003eNile SH, Khobragade CN, Park SW. Optimized and comparative antioxidant assays and its applications in herbal and synthetic drug analysis as an antioxidants. Mini Rev Med Chem. 2012 Sep 1;12(10):1007-14. doi: 10.2174/138955712802762310. PMID: 22625419.\u003c/li\u003e\n\u003cli\u003eKim RH, Coates JM, Bowles TL, McNerney GP, Sutcliffe J, Jung JU, Gandour-Edwards R, Chuang FY, Bold RJ, Kung HJ. Arginine deiminase as a novel therapy for prostate cancer induces autophagy and caspase-independent apoptosis. Cancer Res. 2009 Jan 15;69(2):700-8. doi: 10.1158/0008-5472.CAN-08-3157. PMID: 19147587; PMCID: PMC2629384.\u003c/li\u003e\n\u003cli\u003eJeong SJ, Heo K, Park JY, Lee KW, Park JY, Joo SH, Kim JH. Characterization of AprE176, a fibrinolytic enzyme from Bacillus subtilis HK176. J Microbiol Biotechnol. 2015 Jan;25(1):89-97. doi: 10.4014/jmb.1409.09087. PMID: 25315053.\u003c/li\u003e\n\u003cli\u003eShrivastava A, Khan AA, Shrivastav A, Jain SK, Singhal PK. Kinetic studies of L-asparaginase from Penicillium digitatum. Prep Biochem Biotechnol. 2012;42(6):574-81. doi: 10.1080/10826068.2012.672943. PMID: 23030468.\u003c/li\u003e\n\u003cli\u003eEbrahimi N, Gharibi S, Ghoshoon MB, Karimi Z, Gholami A, Nezafat N, Mohkam M, Ghasemi Y. Selective Isolation and Identification of Arginine Degrading Bacteria; the Optimized Arginine Deaminase Production by Enterobacter sp. sgn1 as a New Source of This Potentially Anti-Tumor Enzyme. J App Pharm Sci, 2016; 6 (09): 093-101.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIt shows \u003cem\u003eCarbon\u003c/em\u003e and \u003cem\u003eNitrogen\u003c/em\u003e growth factors affecting \u003cem\u003eADI\u003c/em\u003e productivity:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCarbon\u003c/em\u003e source( 2%W/V)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eNitrogen\u003c/em\u003e source( 1% W/V)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eADI\u003c/em\u003e productivity( U/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYeast\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMannitol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePeptone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e193\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL-arginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNa Nitrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e127\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoluble Starch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmmonium Sulfate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e155\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIt represents the effects of temperature and \u003cem\u003epH\u003c/em\u003e on \u003cem\u003eADI\u003c/em\u003e productivity:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature( \u003cem\u003e℃\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003epH\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eADI\u003c/em\u003e productivity( \u003cem\u003eU/ml\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e191\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIt demonstrates impacts of \u003cem\u003epH\u003c/em\u003e and temperature on \u003cem\u003eADI\u003c/em\u003e activity:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature( \u003cem\u003e℃\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003epH\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eADI\u003c/em\u003e activity( \u003cem\u003eU/ml\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e179\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e191\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e187\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e140\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIt demonstrates influences of metal cations on \u003cem\u003eADI\u003c/em\u003e productivity \u003cem\u003eand\u003c/em\u003e activity:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetal Cation( \u003cem\u003e5mM\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eADI\u003c/em\u003e productivity( \u003cem\u003eU/ml\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMetal Cation( \u003cem\u003e10 mM\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eADI\u003c/em\u003e activity( \u003cem\u003eU/ml\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNa\u0026thinsp;\u003csup\u003e+\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFe\u0026thinsp;\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eK\u0026thinsp;\u003csup\u003e+\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK\u0026thinsp;\u003csup\u003e+\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCo\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNa\u0026thinsp;\u003csup\u003e+\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e151\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNi\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNi\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMn\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCo\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e193\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMg\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMn\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e185\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFe\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMg\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e173\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFe\u0026thinsp;\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFe\u0026thinsp;\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e145\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIt demonstrates the influence of different substrate concentrations on \u003cem\u003eADI\u003c/em\u003e productivity:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSubstrate concentration( \u003cem\u003emM\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eADI\u003c/em\u003e productivity( U/ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e139\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIt shows aeration effects on \u003cem\u003eADI\u003c/em\u003e productivity:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShaking speed( \u003cem\u003erpm\u003c/em\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eADI\u003c/em\u003e productivity( U/ ml)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e117\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIt indicates \u003cem\u003e16S rRNA\u003c/em\u003e sequencing analysis using \u003cem\u003eBLASTn\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDescription\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMax Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal Score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQuery Cover\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eE value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePer. identity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis strain DE111\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1530\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus sp. (in: Bacteria) strain HA15-23\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis strain KMB31\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis strain LXA7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis strain bacs2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus sp. (in: Bacteria) strain ITI09\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus sp. (in: Bacteria) strain AU3b4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus subtilis strain MRHB1-205\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1519\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBacillus sp. (in: Bacteria) strain BGS3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e99.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIt represents \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e biochemical reactions profile:\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiochemical reaction\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFindings\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCitrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCatalase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlagella\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGelatin hydrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGas\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGram staining\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMotility\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOxidase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNitrate reduction\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl red( MR)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVogues proskaeur( VP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpore\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRods\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUrease\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePigment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFructose fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArabinose fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlucose fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInositol fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaltose fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMannitol fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStarch fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRhamanose fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e+ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArabitol fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDulcitol fermentation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-ve\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Faculty of Pharmacy","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cancer, Arginine, Purification, 16S rRNA, degrading enzymes","lastPublishedDoi":"10.21203/rs.3.rs-4126905/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4126905/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuxotrophic cancers for \u003cem\u003eArginine\u003c/em\u003e are a leading cause of death worldwide. The manufacture of novel arginine degrading enzymes such as \u003cem\u003eArginine deiminase enzyme\u003c/em\u003e is mandatory due to this crisis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAim of the study:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince certain tumor cells are auxotrophic for \u003cem\u003eArginine\u003c/em\u003e, the depletion of the extracellular \u003cem\u003eArginine\u003c/em\u003e by means of \u003cem\u003eArginine\u003c/em\u003e \u003cem\u003edeiminase enzyme\u003c/em\u003e was exploited in the present study to target such tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Selective recovery of some bacterial isolates from different environmental sites in Egypt and assessment their capabilities for \u003cem\u003eArginine deiminase\u003c/em\u003e production. Studying environmental and physiological factors affecting \u003cem\u003eArginine deiminase \u003c/em\u003eproduction by some selected isolates. Characterization of of activity \u003cem\u003eArginine deiminase \u003c/em\u003eproduced by certain selected isolates as well as its production through bacterial recombinant \u003cem\u003eDNA\u003c/em\u003e technology. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe major bacterial isolates grown on \u003cem\u003emineral Arginine agar( MAA) plates\u003c/em\u003e producing \u003cem\u003eADI \u003c/em\u003ewere further identified as \u003cem\u003eBacillus subtilis DE 111 using 16S rRNA sequencing technique\u003c/em\u003e. \u003cem\u003eThe Arginine deiminase\u003c/em\u003e production and activity were optimal at \u003cem\u003e40℃\u003c/em\u003e and alkaline \u003cem\u003epH\u003c/em\u003e. \u003cem\u003eMn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+2\u003c/em\u003e\u003c/sup\u003e,\u003cem\u003eNi\u003c/em\u003e\u003csup\u003e\u003cem\u003e+2\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eCo\u003c/em\u003e\u003csup\u003e\u003cem\u003e+2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e metal ions as cofactors\u003c/em\u003e were optimum activators for production and activity of \u003cem\u003eADI\u003c/em\u003e. The results showed that the potent cytotoxic consequences of \u003cem\u003eADI\u003c/em\u003e were exerted on the renal and leukemic cancer cell lines. \u003cem\u003eADI\u003c/em\u003e produced via bacterial recombinant \u003cem\u003eDNA\u003c/em\u003e technology showed efficacious\u003cem\u003e IC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e \u003cem\u003e10.31\u003c/em\u003e± \u003cem\u003e0.2\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eand \u003cem\u003e16.08\u003c/em\u003e± \u003cem\u003e0.3 µg/ml\u003c/em\u003e against \u003cem\u003erenal\u003c/em\u003e(\u003cem\u003e Caki-1\u003c/em\u003e) and \u003cem\u003eleukemic\u003c/em\u003e(\u003cem\u003e K-562\u003c/em\u003e) cancer cell lines, respectively. The purified\u0026nbsp; monomeric \u003cem\u003eADI\u003c/em\u003e was \u003cem\u003e36.18 KDa\u003c/em\u003e molecular mass as determined using \u003cem\u003eSDS-PAGE\u003c/em\u003e, the specific activity reached \u003cem\u003e36.07 U/mg\u003c/em\u003e.\u003cem\u003e Km\u003c/em\u003e,\u003cem\u003eVmax\u003c/em\u003e and\u003cem\u003e Kcat\u003c/em\u003e were \u003cem\u003e0.05871 M,\u003c/em\u003e \u003cem\u003e40.36 µmol/ml/min and 5.014min\u003c/em\u003e\u003csup\u003e\u003cem\u003e-1\u003c/em\u003e\u003c/sup\u003e\u003cem\u003erespectively\u003c/em\u003e. Optimum\u003cem\u003e pH\u003c/em\u003e and temperature for productivity and activity ranged from \u003cem\u003e6-10\u003c/em\u003e and \u003cem\u003e37-70℃\u003c/em\u003e,respectively. Total protein estimation using \u003cem\u003eBar-ford \u003c/em\u003eassay was determined to be \u003cem\u003e5.68 mg\u003c/em\u003e during the initial culture. \u003cem\u003eADI\u003c/em\u003e purification was achieved using \u003cem\u003e70% Ammonium Sulfate\u003c/em\u003e followed by\u003cem\u003e Ni\u003c/em\u003e\u003csup\u003e\u003cem\u003e+2\u003c/em\u003e\u003c/sup\u003e-immobilized affinity column chromatography with a final purification fold of \u003cem\u003e15.03\u003c/em\u003e.\u003cem\u003eIn vitro\u003c/em\u003e determination of biological\u0026nbsp; half life of\u003cem\u003e ADI\u003c/em\u003e using nesslerization assay was observed to be nearly \u003cem\u003e300 min\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eADI\u003c/em\u003e produced from \u003cem\u003eBacillus subtilis DE111\u003c/em\u003e demonstrated efficacious anticancer activities against leukemic(\u003cem\u003e K-562\u003c/em\u003e) and renal(\u003cem\u003e Caki-1\u003c/em\u003e) auxotrophic cancers for \u003cem\u003eArginine \u003c/em\u003edue to the depletion of \u003cem\u003eL-arginine\u003c/em\u003e from the external surrounding environments.\u003c/p\u003e","manuscriptTitle":"Isolation and identification of bacterial isolates producing Arginine deiminase from assorted soil environments in Egypt using16S rRNA sequencing technique","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-20 09:17:43","doi":"10.21203/rs.3.rs-4126905/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d0031d8d-af9b-4f9a-9f8f-6c976e6b9940","owner":[],"postedDate":"March 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":29605118,"name":"Applied \u0026 Industrial Microbiology"}],"tags":[],"updatedAt":"2024-03-20T09:17:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-20 09:17:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4126905","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4126905","identity":"rs-4126905","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}