ISOLATION, SCREENING, AND PARTIAL CHARACTERIZATION OF OXALATE OXIDASE ENZYME FROM PSEUDOMONAS STRAIN UNDER INDUCED OXALATE STRESS CONDITION

the ability of OxO enzyme purified from Pseudomonas strain in the diagnosis of oxalate-related disorders and in the future could be used to prepare the diagnostic biosensors. Findings provide new directions for research in the study of Oxalate oxidase enzyme. Isolation and production of an oxalate degrading enzyme strains under salt stress conditions. Further research focused on the enzyme activity of isolated bacterial enzyme and optimization of methods for the determination of oxalate in the sample.

Identification of Pseudomonas culture by VITEK 2 system (software version VT2-08.01) Isolated bacterial culture was inoculated on a nutrient medium and incubated at 37ºC for 24 hrs. Suspensions were prepared by emulsifying bacterial isolates in 0.45% saline to the equivalent of a 0.5 McFarland turbidity standard, 1.5 × 10 7 CFU/ml. The same suspension was used for identification on the VITEK 2 system. The 0.5 McFarland bacterial suspension was diluted; cards were automatically filled, sealed, and loaded into the VITEK 2 instrument for incubation and reading (Garcia-Garrote et al., 2000).

Screening of medium for salt stress oxalic acid and potassium oxalate for isolated Pseudomonas strain
The growth medium was prepared in 250 ml flask composed of 1% glycerol, 1% yeast extract, 1% peptone, 0.1% dipotassium phosphate, 0.05% magnesium sulphate with 1% oxalic acid, and 2% potassium oxalate as a variant for the screening of suitable substrate for bacterial growth to produce oxalate enzyme under stress condition.

Process optimization for maximum bacterial biomass production by a various dose of potassium oxalate salt
The bacterial broth (PAF) was prepared in which content increase the concentration of potassium oxalate ranges from 1 to 12% were added, sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes. The isolated culture from 1.0% PO (Potassium oxalate) medium was inoculated in each flask and kept for observation for 24 hr at 37ºC. 200 µl sample was added in 96 well plates, and the absorbance was noted at 625 nm using Biotek ® H1-SYNERGY ELISA spectrophotometer.

Bulk production in suspension medium and harvesting of cells
Pseudomonas aeruginosa was grown in PAF medium broth (500 ml) with 2% Potassium oxalate supplemented in 1000 ml flask for 3-4 days at 37ºC. The heat cold shock method was used on obtained bacterial cell suspension. The sample was extracted in liquid nitrogen and resuspended in 20mM Tris-Cl pH (8) with 10mM Phenyl methyl sulphonyl fluoride (PMSF) (Alt et al., 1975;MacGregor, 1977).The 200 ml of obtained sample sonicated under the ice-cold condition operated for 1 hr cycle at 30% frequency with the pulse rate 5 seconds in ice-cold condition. Then sonicated mat samples centrifuged at 7000 rpm for 30 min and enzyme extracted (Anjum et al., 2015;Kanauchi et al., 2009).

Ammonium sulphate fractionation and dialysis
Ammonium sulphate fractionation was performed in two stages, at an initial concentration of 0-60% of ammonium sulphate was added slowly until it completely dissolved in cold condition at 4ºC for 5 hrs and later it was kept on stirring overnight a 4°C. After the precipitation process was completed, it was centrifuged at 4000 rpm, and the pellet was resuspended in buffer (Tris-Cl pH 8), and the supernatant was collected and used for the second stage of the 80% ammonium sulphate fractionation process. The 60% pellet was resuspended in buffer (Tris-Cl pH 8) subjected to dialysis using a dialysis sack mwco (110) Hi-Media dialysis membrane. The dialysis sack was attached to a float at one end and immersed in 100 times the volume of the sample and dialyzed against appropriate buffer (PBS) for 24 hr at 4ºC with continuous stirring, resuspended in buffer and stored at 4ºC. The appropriate dialysis buffer was determined by the subsequent purification procedure to be undertaken. After completion of 60% and 80%, dialysis samples were stored for further analysis and assay purposes (Hu & Guo, 2009).

Protein estimation and SDS gel electrophoresis after partial purification of oxalate oxidase from oxalate degrading bacteria
The unknown protein quantification was done by Biuret method, and SDS-PAGE was performed on 12% separating and 5% stacking polyacrylamide gels at 25°C using TARSON Mini Vertical Electrophoresis Assembly (7080). The gels loaded with the sample were run at 25/50/100 V till the dye front reached the end of separating gel. The protein bands were visualized by staining with Coomassie brilliant blue (CBB) G-250 (Laemmli, 1970).

Ion Exchange Column Chromatography (Fractionations) of Partial Purified Enzyme
Column chromatography with DEAE-cellulose chromatography Macro-Prep ®DEAE medium was used, which is an anion exchange medium. It was purchased from BioRad laboratories. The glass chromatography column was packed up to 15 cm height 50% slurry of the Macro-Prep ®DEAE medium. It was equilibrated with 20 mM tris CL buffer of pH 8 for the column chromatography process. Gradients of 50 ml were prepared to range between 200 to 800 mM NaCl in 20 mM tris-Cl buffer of pH 8. Further, 50 ml of 20 mM tris Cl buffer column was used to wash the column. Next, a 60% Ammonium sulphate precipitated sample (partially purified enzyme) was dialyzed, and it was loaded on the column. The column was washed with the same buffer to remove the unbound proteins, and the enzyme was eluted by applying a linear gradient of NaCl from 200 to 800 mM, and fractions of 2 ml were collected. Active fractions were pooled and stored for further analysis (Kotsira & Clonis, 1997).

Bacterial oxalate oxidase enzyme activity
MBTH (3-methyl benzathoizaline hydrazine) used to detect release of hydrogen peroxide in the enzymatic reaction of Barley oxidase enzyme upto 1mM. In the assay of oxalate oxidase, hydrogen peroxide is produced which is coupled with HRP which catalyses the MBTH-DMA (Kanauchi et al., 2009 ;Goodwin et al., 2017).

Biochemical characterization
Biochemical characterization tests were performed for isolated bacterial samples for the identification of Pseudomonas species. The test results are shown in Table  1. Figure 1A. Showed that no fluorescence on plates as compared to Figure 1B and Figure 1C. Yellow pigment like colonies on PAF plates in visible light conditions was observed, and under UV transilluminator (Figure 1B), it showed greenish fluorescence (Figure 1C). The Oxidase test was found to be positive for the isolated sample, as shown in Figure 2B (bacterial sample without PO) and Figure 2C ( bacterial sample with PO) compared to Figure 2A which was a negative control broth. Catalase test was also found to be positive, as a bacterial broth sample reacted to H2O2 to produce effervescence shown in Figure 3B with reference to negative control Figure 3A.   Identification of oxidase producing Bacterial culture by VITEK 2 system (software version VT2-08.01) The isolated colony was observed used for preparing a bacterial suspension of 0.5 McFarland. That sample was used for identification, and results were obtained after 6.78 hrs. The given Gram-negative bacteria Pseudomonas aeruginosa was identified using the VITEK 2 system.

Screening of medium for salt stress oxalic acid and potassium oxalate for isolated Pseudomonas strain
The increasing bacterial growth was observed in 3% Potassium oxalate medium compared to 2% oxalic acid medium. The Potassium oxalate supplemented medium showed increased growth shown in Table 2 and Figure4.

Process Optimization for Maximum Bacterial Biomass Production by different Concentration of Potassium Oxalate Salt
The cultured bacteria showed growth in salt stress range between 1 to 12% PO containing medium. The flask also showed bubbles formation, which are the indicators for the presnce of oxidase enzyme. The results are shown in Table 3 and Figure5. It is clearn that 3% of PO concentration have maximum bacterial growth. Hence 3% concentration of PO is selected in growth medium or is found to be more suitable for bulk production of oxalate enzyme. 0.120 + Figure 5 Effect on bacterial growth supplemented with increasing concentration of potassium oxalate salt (Induced stress condition)

Bulk production in suspension medium and harvesting of cells
Pseudomonas aeruginosa isolated and identified by VITEK 2 System, version 08.01 was further grown in PAF medium broth with 2% Potassium oxalate and incubated for 3-4 days at 37ºC. A thick slimy matlike biomass was observed at the end of incubation. The matt was pale yellow, very viscous in texture like egg yolk, and the bubble formation observed in culture. The mat was transferred and extracted in liquid nitrogen, the resultant extract was kept in a buffer having PMSF solution to avoid degradation of enzyme. The extracted sample was subjected to centrifugation. The collected supernatants of extract then proceeded for sonication under the cold condition for better recovery of enzyme and sample was stored for further analysis (Dashek, 2018).

Characterization of oxalate oxidase enzyme Ammonium sulphate fractionation and dialysis:
The partially purified samples were collected and subjected to protein estimation (Dashek, 2018;Neut et al., 2005).

Figure 6
Standard calibration graph for protein estimation using standard BSA (5 mg/ml) Protein estimation and SDS gel electrophoresis after partial purification of oxalate oxidase from oxalate degrading bacteria Bacterial oxidase enzyme was subjected to total protein content evaluation. It showed that protein content of crude bacterial oxidase enzyme is 0.086 mg/gm, 60% fractionated dialysis sample was 0.216 mg/gm and 80% fractionated dialysis sample was found to be 0.005 mg/gm. It was deduced by the BSA calibration graph (Figure 6 and Table 4). The SDS gel electrophoresis was performed for the crude enzyme, 60% and 80% dialyzed sample. The 60% dialyzed sample showed more prominent bands than crude and 80% dialyzed sample (Figure 4). The enzyme oxalate oxidase from Pseudomonas aeruginosa is observed to be the molecular weight between 45 Da to 95 Da in all the three samples (Sambrook & Russell, 2001;Steinberg, 2009).

Ion Exchange Column Chromatography (Fractionations) of Partial Purified Enzyme
The ion exchange Column chromatography was done using DEAE-cellulose chromatography Macro-Prep ® DEAE medium, and fractions were collected for further enzyme activity (Palanivelu, 2018;Zhang et al., 1996).

Bacterial oxalate oxidase enzyme activity
In the reaction, oxalate oxidase catalyzes the production of hydrogen peroxide from oxalate; the hydrogen peroxide further reacts with MBTH and DMA in the presence of peroxidase enzyme to form a purple color product (Indamine dye). The color of indamine dye was read at 600 nm using SYNERGY H1 microtiter plate reader from Biotek ® . The results indicate the increase in absorbance as the indamine concentration increases, which can be correlated with the activity of the oxalate oxidase enzyme. The standard linear curve was prepared using standard hydrogen peroxide. The activity can be defined as the amount of oxalate unit required to produce the 1 μmol of hydrogen peroxide at 55°C per minute. The H2O2 detection was performed using MBTH indamines dye measured using Result layout colorimetric scheme of MBTH based standard H2O2 detection using HRP of reaction samples shown in Figure 8. The activity was shown in Figure 9, it shows that a 60% dialyzed sample has more significant H2O2 release in the reaction compared to the standard OxO enzyme, 80% dialyzed sample, and crude enzyme (Goodwin et al., 2017; Goyal et al., 1999;Zhang et al., 1996).Some of the difficuties faced during study include the detection of H2O2 becomes unstable at higher temperature, thus the wstudy should be done at lower tempratures.

Figure 8
Result layout colorimetric scheme of MBTH based standard H2O2 detection using HRP Figure 9 Comparative activity of the crude enzyme, purified 60 and 80% dialyzed sample with standard OxO enzyme (BioVision)

CONCLUSION
Findings provide new directions for research in the study of Oxalate oxidase enzyme. Isolation and production of an oxalate degrading enzyme strains under salt stress conditions. Further research focused on the enzyme activity of isolated bacterial enzyme and optimization of methods for the determination of oxalate concentration in the sample. Hence the microbial oxalate enzyme can be used for widespread application in the enzyme industry and diagnostics.
Author contributions: This work was carried out in collboration between all authors. All authors have accepted the responsibility for the entire content of this submitted manuscript and approved the submission. Authors AP, RS and LD conceptulized and performed the experiments. AP and HP performed the formal analysis and investigation. AP, RS and HP also performed the writing, editing and reviewing of the article. All authors read and approved the manuscript.