Unless specified all the chemicals and reagents are obtained from HiMedia laboratories (Mumbai).
Immunoinformatics analysis for antigenicity prediction
Sequence and structural data: The amino acid sequence of PfADI (416 amino acids) and its modeled 3-D structure were used for immunoinformatics analysis. MhADI amino acid sequence was retrieved from UniProtKB (https://www.uniprot.org/uniprot/P41141) and used for comparative studies.
Prediction of antigenicity and allergenicity: The antigenicity was predicted using VaxiJen server (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html). VexiJen uses alignment independent approach to predict the antigenicity of the whole protein  . The allergenicity prediction was performed using AlgPred (http://crdd.osdd.net/raghava/algpred/) server  . The antigenicity and allergenicity of PfADI were compared with MhADI.
T-cell epitope prediction: The MHC-II binding T cell epitopes were predicted using Immune Epitope Database (IEDB) server (http://tools.iedb.org/mhcii/). The reference alleles were chosen on the basis of their wide global frequency. A total of 8 HLA-DRB1 alleles (*01:01,*03:01, *04:01, *07:01, *08:01, *11:01,*13:01, and *15:01) were used to predict the T cell epitopes [22, 23] . The epitope density was obtained by calculating the relative frequency of the predicted epitopes. Relative frequency (fi)= ni/N , where ni is the number of epitopes within the threshold (percentile values ≤ 10) and N represents the total number of predicted epitopes.
B-cell epitope prediction: Linear or continuous B cell epitopes were predicted using BepiPred server (http://tools.iedb.org/bcell/). Conformational B cell epitopes were predicted using modeled PfADI 3D structure. BEpro (previously known as PEPITO) (http://pepito.proteomics.ics.uci.edu/) and DiscoTope2.0 tool (http://www.cbs.dtu.dk/services/DiscoTope/) were employed for the prediction of conformational epitopes [24, 25]. While using BEpro residues with epitope score ≥1 were regarded as conformational B cell epitopes, a threshold of -3.7 was used for prediction by DiscoTope 2.0 server.
Cloning of ADI and sequence analysis of P. furukawaii arcA gene:
The arcA gene coding for ADI enzyme in P. furukawaii cells was amplified by polymerase chain reaction (PCR). Primers (Forward primer- 5’ATATCCATGGCGATGTCCAAAGTCAAACTCGG3’ and reverse primer- 5’ATTACTCGAGGTAGTCGATCGGATCGCGG3’) were designed and the PCR amplification was carried out using Phusion® (Thermo Scientific) and 2% DMSO in the MJ Mini thermal cycler (Bio-Rad). The reaction conditions were as follow: Initial denaturation at 98˚C for 5 minutes followed by 30 cycles of denaturation at 95˚C for 30 seconds, annealing at 58˚C for 45 seconds, extension at 72˚C for 45 seconds with a final extension at 72˚C for 5 minutes.
The amplified fragment was cloned in pET-28a (+) vector at NcoI-XhoI restriction sites using standard cloning procedures. Fast digest NcoI and XhoI were obtained from Thermo Scientific (Waltham, MA, USA). The resultant recombinant plasmids were transformed in competent Escherichia coli DH5α cells. Colony PCR was performed to screen the colonies with the construct. The positive clones were also confirmed by double digestion of the plasmid isolated from the colonies. The cloning was further confirmed by automated dideoxy DNA sequencing and the homologies of the nucleotide sequences was analyzed using NCBI BLAST.
Construction of phylogenetic tree:
Phylogenetic tree of ADI sequences (retrieved from UniProt https://www.uniprot.org/) of 19 Pseudomonas spp. and 2 Mycoplasma spp. (Table S1) was constructed by UPGMA method  using MEGA X software . The distances were calculated by the Poisson correction method .
Expression of PfADI in heterologous host:
pET-arcA construct was transformed into E. coli BL21 cultured at 37 ⁰C in Luria Bertani broth containing kanamycin (50 μg mL-1). The induction conditions for the recombinant PfADI expression were optimized and performed with 1 mM isopropyl β-D-thio-galactopyranoside (IPTG) at O.D.600nm ~ 0.6. The induction was carried out for 6h. The expression of recombinant PfADI was checked on SDS-PAGE and later confirmed by western blotting.
Purification of recombinant PfADI:
Recombinant PfADI was over-expressed as inclusion bodies in the cytoplasm which was purified with the help of Ni2+-NTA affinity chromatography using the manufacturer’s guidelines (Qiagen, Germany). The E. coli cells with induced recombinant PfADI were harvested and the pellet was dissolved in 8M urea and incubated at room temperature for 2-3 hours till the solution became clear, this step was followed by centrifugation. The pellet was discarded, and the supernatant was incubated with Ni2+-NTA slurry previously equilibrated with a lysis buffer overnight for binding. The bound slurry was passed through the column and the flow through was collected. On column renaturation of protein was performed by decreasing gradient of urea (8M to 0M). The purification steps following renaturation were carried out at 4°C. Later the protein was washed with 20mM and 40mM imidazole and eluted at 200 mM-500mM imidazole concentration in the elution buffer.
In order to determine PfADI activity, the enzyme assay was performed using the method described by De Angelis and coworkers with certain modifications . The reaction mixture was prepared by adding 150 µl of purified recombinant PfADI into 150 µl of 50 mM substrate (L-arginine) and 1.85 ml of 50 mM acetate buffer. The mixture was then placed in a water bath at 37 °C for a duration of 1 h. The enzymatic reaction was stopped at the end of 1h by adding 2N HCl (500µl)). After the completion of the reaction, the mixture was centrifuged and 100µl of supernatant was taken for the next step i.e. color development. The development of color determines the amount of product (L-citrulline) formation. Color is developed using DAMO-TSC (diacetyl monoxime-thiosemicarbazide) method . In the color development step, 100µl of the previously mentioned supernatant was added in a test tube containing 2 ml acid-ferric solution and 1 ml of DAMO-TSC solution. The mixture was vortexed thoroughly and kept at 100 °C for 10 min. The developed color was analyzed using spectrophotometer by measuring absorbance at 520 nm. 1 U of ADI is defined as the the amount of enzyme required to catalyze the conversion of one micro mole of substrate (L-arginine) into one micro mole of product (L-citrulline) in one minute under the standardized conditions. The amount of protein was quantified using Bradford’s assay and the specific enzyme activity was evaluated.
Characterization of recombinant ADI from P. furukawaii
Effect of pH on the purified ADI
Effect of pH on the purified enzyme was used to determine optimum pH for ADI activity and pH stability of the enzyme. In order to determine the pH optima; ADI activity was calculated by the use of different buffers in the reaction mixture. Acetate buffer (50 mM, pH 5.5), citrate buffer (50 mM, pH 4.3), potassium phosphate buffer (50 mM, pH 7), TrisHCl (50 mM, pH 8.8) were used. The highest ADI activity was set as 100% and relative activity was determined.
To observe the pH stability of the enzyme, ADI was pre-incubated in Acetate buffer with the pH ranging from 4 to 8 at 4 °C for 12 hours. The residual enzyme activity at different pH was evaluated using standard assay conditions. All these experiments were carried out in triplicates and the average value was recorded.
Effect of temperature on ADI
The optimum temperature and the thermostability of the recombinant purified ADI was assessed. The optimum temperature was determined by incubating ADI with L-arginine in different temperatures (37 °C -100 °C). The ADI activity was calculated. Highest ADI activity was set as 100% and relative enzyme activity was calculated.
The thermostability of recombinant ADI was determined by incubating it at various temperatures (4 °C, 37 °C, 60 °C, 100 °C ) for different time intervals.
Determination of kinetic parameters of recombinant PfADI
The kinetic parameters Km and Vmax of recombinant PfADI were evaluated using the Lineweaver-Burk plot. To calculate these parameters the enzyme activity was calculated in the presence of increasing substrate (L-arginine) concentration. The arginine concentration in the reaction mixture varied from 0.1 to 50 mM. Keeping the other conditions standard and uniform, the experiments were carried out in triplicates and mean value was used to plot the graph. The linear regression equation obtained from the double reciprocal plot was used to calculate the Km and Vmax values. The obtained regression equation was compared with the Michaelis–Menten equation.
Protein structure prediction of PfADI:
SWISS-MODEL server (https://swissmodel.expasy.org/) was used to predict the 3D structure of PfADI. To predict the structure, the amino acid sequence corresponding to the cloned arcA gene (GenBank Accession MK318561, deposited by the authors) of P furukawaii was used. The sequence length of PfADI consisted of 416 amino acids. The crystal structure of ADI from Pseudomonas aeruginosa (PDB code_2ACI) was used as a template for building the model. The quality of the predicted 3D structure of recombinant ADI was validated by Ramachandran plot obtained for the model (URL: https://swissmodel.expasy.org/assess).
In vitro anticancer activity:
Recombinant PfADI was tested for its anticancer activity on HCC cell lines HepG2. HepG2 cells were procured from ATCC. The cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (fetal bovine serum from invitrogen), 1% antibiotic solution (PenStrep from invitrogen ) in 5% CO2 atmosphere at 37 ⁰C. Trypsin-EDTA solution was used to dissociate the cells. The previously cultured cells were adjusted to a cell count of 1.0 x 105 cells/ml using DMEM supplemented with 2% FBS. 1 X 104 cells/well were seeded in 96 well microtiter plate and was incubated for 24h for the formation of a partial monolayer of cells. The media was removed after 24 h and 100 µl of different concentrations of rPfADI was dispensed in the wells of the microtiter plates followed by an incubation for 72h at 37 oC in 5% CO2 atmosphere. After 72 h, the recombinant PfADI solutions from each well was discarded and 100 µl of MTT solution was added. The plates were again incubated for 4 h. After incubation the supernatant was flicked off and 100 µl of DMSO was added to each well and a gentle shake was given to solubilize the formazan. The absorbance at 590 nm was determined using a microplate reader. The % growth inhibition was evaluated using the given formula and IC50 value of recombinant PfADI for the inhibition of HepG2 cells was determined from the dose-response curve computed using GraphPad prism 9. Doxorubicin was used as the control drug.
% Inhibition = (OD590 of Control – OD590 of sample)/OD590 of Control) x 100.