Received date: August 29, 2013; Accepted date: November 05, 2013; Published date: November 11, 2013
Citation: Petkar Medha B, Pillai Meena M, Kulkarni Amarja A, Bondre Sushma H, Rao KRSS (2013) Purification and Characterization of Superoxide Dismutase Isolated From Sewage Isolated E. coli. J Microb Biochem Technol 5:102-106. doi: 10.4172/1948-5948.1000109
Copyright: © 2013 Petkar Medha B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Superoxide dismutase (SOD), which plays a very important role in protecting organisms from oxygen toxicity, has therapeutic importance. It was purified from sewage isolated E. coli and characterized. Eukaryotic cells also produce SOD but culturing and maintenance of eukaryotic cells for production of SOD is costly as well as difficult. Using prokaryotic cells i.e. bacteria, production cost can be reduced. A rich bacterial source was identified. Bacterial membrane was ruptured in the presence of lysozyme and glass bead. Following ammonium sulphate precipitation, SOD-containing solution was applied to DEAE-cellulose and then Sephadex G- 75 gel columns. SOD was purified 63.91-fold with a specific activity of 3835U/ mg. The molecular weight was estimated to be 35.713 kDa by SDSPAGE gel. Maximum SOD activity was observed between pH 7.0 to 7.5 at temperature range 37–50ºC. This enzyme has fair thermal stability. The enzyme was found to be stable in presence of 1% salt only. The activity found to be gradually reduced approximately 50% at higher concentrations. It was totally inactivated above 9% salt concentration.
SOD, purification, characterization, sewage isolate E. coli
The superoxide radical is an intermediate reduction product of oxygen produced in a variety of biological reactions. The superoxide radical (O2·−) and other reactive derivatives in cells have received recent attention as agents of oxygen toxicity. Most organisms produce defense systems, such as metallo-enzymes, to protect themselves from reactive oxygen species. Metallo-enzymes that catalyze the dismutation of superoxide free radicals (O2·−) to hydrogen peroxide (H2O2) and oxygen (O2), are known as superoxide dismutases (SODs). SODs play an important role to protect the cells from the oxidative damage of superoxide radicals. Cell damage may also be due to the superoxide itself or, indirectly, even more reactive oxygen species, such as hydroxyl radicals (·OH), formation of which, via the Fenton reaction, is favored by excess superoxide [1-5]. SODs are divided into three classes on the basis of their active site metals: copper and zinc (Cu/Zn-SOD), manganese (Mn-SOD)/ iron (Fe-SOD) and in some bacteria one more variety is found named as Ni-SOD . CuZn-SOD was found widely in the cytoplasm and certainly in the mitochondrial intermembrane space of the eukaryotic cells and chloroplasts of plants. It is also reported in bacteria like S. aureus, Pseudomonas spp., E. coli etc [7-10] Mn-SOD are located in prokaryotes and in the mitochondria of eukaryotes , while Fe-SOD has been found in bacteria, blue-green algae and protozoa [12,13]. Recent reports also indicated that the enzyme was present in higher land plants .
Pure /reference SOD (Sigma) Coomassie brilliant blue G, gel filtration and electrophoresis molecular weight marker kits, Sephadex G-75 was purchased from Sigma and DEAE-cellulose from Himedia. Pure /reference SOD isolated from E. coli was purchased from sigma. All other chemical reagents used were of analytical grade. Optical measurements were achieved with a spectrophotometer (UV- 1800, Shimadzu, Japan) at 560 nm and 650 nm. Electrophoresis was performed with Edvotek M-36 during experimental studies.
Isolation and purification of superoxide dismutase
Scientist showed the presence of SOD in S. aureus  and P. aeruginosa . It was decided to choose these bacteria for production of SOD. Total 64 samples were used for SOD testing. Urine  and pus , suspected for S. aureus were collected from various pathological laboratories and hospitals (CPR Hospital, Computek laboratory, Micropath lab, Shivtej laboratory etc. in Kolhapur and confirmed by morphological and biochemical testing) 10 non pathological samples were also used . Total 26 samples Pseudomonas spp were tested. Urine  and pus , suspected for Pseudomonas spp were collected from various pathological laboratories and hospitals (further confirmed by morphological and biochemical testing). 8 non pathological spp were also used. Among these 8 samples of S. aureus and 12 samples of P. aeruginosa showed presence of SOD. Sewage samples were also used for analysis. From 6 different sites around Kolhapur, sewage samples were collected. A loopful of each sample was streaked on SRBM (Sulphate reducing bacterial medium) as well as on Nutrient agar using four quadrant method. SRBM incubated anaerobically to obtain sulphur reducing bacteria, while nutrient agar incubated aerobically for 24 hr at 37ºC to get aerobic flora. 12 samples of sewage isolate were selected for SOD assay.
Assay for estimation of SOD activity 
The method is based on comparison of yellow colored nitroblue tetrazolium to blue colored farmazone by O2. In this method O2 is generated by addition of riboflavin. Also the reaction is initiated by addition of riboflavin. Illumination of solutions containing riboflavin, methionine, and NBT resulted in a linear accumulation of the blue formazan, whether oxygen was present or not. In the absence of oxygen, superoxide dismutase was without influence whereas, under aerobic conditions, superoxide dismutase inhibited the reduction of NBT. Thus, illumination of reaction mixtures which contained 0.4 ml riboflavin (60 μM), 1.2 ml methionine (130 mM), 0.6 ml NBT (750 μM), and 3.4 ml phosphate buffer (0.2 M pH7.8), 0.3 ml EDTA (10 mM) and 0.1 ml suspension of respective bacteria or enzyme solution caused an increase in absorbance at 560 nm. Control tube was wrapped with black cloth. The assay tubes were exposed to UV light (30 cm length, 2.5 cm diameter) for 30 min. The non-irradiated mixture was served as blank. The cell suspensions were prepared from all test samples having same absorption value at 620 nm. Assay run in triplicate and average of three was considered for results.
Purification of SOD
After SOD assay, it was found that sewage isolates produces more SOD than non sewage isolates as per Table 1. Among them highest producer RJ-2-1 was selected for further work i. e. production and partial purification of SOD. This bacterium was showing characteristics like E. coli 4157 confirmed by biochemical tests and 16s r-RNA analysis. Selected bacterium was cultured on Nutrient agar plates and suspended in 0.1 M phosphate buffer (pH 7.5) by scrapping them with the help of sterile lancet. Cells were collected by centrifugation (10000 g, 10 min), then washed twice with 0.1 M phosphate buffer (pH 7.5). The suspension was ground in mortar with glass beads and vortexed for 5 min. in presence of lysozyme. The disrupted product was centrifuged (10000 g, 10 min) and supernatant was re-centrifuged (25000 g, 30 min). Then supernatant was 30% saturated with solid ammonium sulfate and resulting precipitate was discarded. More ammonium sulfate was added to the supernatant to reach 80% saturation. The precipitate was dissolved in 0.1 M phosphate buffer (pH 7.5) and dialyzed against the same buffer overnight. Then content of dialysis bags was centrifuged (25000 g, 20 min). Supernatant containing SOD activity was applied to a DEAE-cellulose column (40×1.6 cm), which was equilibrated with 0.05 M phosphate buffer (pH 7.4) at 400ºC. Elution of the enzyme was achieved by establishing a linear gradient with a 0.06-0.140 M phosphate buffer (pH 7.4) at a flow rate of 0.5 ml/ min. Fractions containing SOD activity were loaded onto a Sephadex G-75 (90×1.6 cm) column equilibrated with 0.05 M phosphate buffer (pH 7.4) and eluted with the same buffer at a flow rate of 0.2 ml/min.
|O.D. at 550 nm|
|Sr. No||S. aureus||P. aeruginosa||Sewage isolate|
Abbreviations used - CPR Hospital –CPR; Sanjivani Hospital – SANJ; Saraswati Hospital – SAR; Computek laboratory- COMP; Micropath laboratory – MPL; Shivtej laboratory – SVT; Rajendra nagar – RJ; Ratnappa Kumbhar nagar – RK; Sarnaubatwadi – SRW; Bapat camp – BP; Urine sample – U; Pus sample- P; Non pathological sample – NP
Table 1: SOD activity/ml of bacterial culture.
Molecular weight determination
The apparent molecular weight of the purified SOD was determined by SDS-PAGE. The molecular weight of purified SOD was estimated using the calibration curve. SDS-polyacrylamide gel electrophoresis was performed according to the Westermeier method using a horizontal slab gel apparatus. The following proteins were used as SDSPAGE electrophoresis molecular weight determination.
Standards: BSA (66 kDa), Trypsin (MW- 23.3 KD ) Pure /reference SOD (MW- 35.713 KD ) and isolated SOD were dissolved in 0.0625 M Tris HCl buffer (pH 6.8) containing 5% β-mercaptoethanol and denatured by 4 min incubation in boiling water.
The 40 μl marker proteins were loaded in first, third and fourth well and the sample was loaded in the second well. Electrophoresis was performed according to discontinuous method with 12.50% separating and 2.50% stacking polyacrylamide gels at room temperature at 30 mA for 4 h. Gel was stained with Coomassie brilliant blue R-250 dye reagent overnight.
Determination of type of SOD
Type of SOD was confirmed by performing inhibition assay. In this experiment standard assay was run in presence of specific SOD inhibitors. Blue color of farmazone is formed after activity of O2.-, SOD is dismutating O2.- so in presence of SOD blue color is not formed but if SOD is inhibited by specific inhibitor blue color i.e. farmazone is not formed
During the purification steps, protein levels were determined spectrophotometrically according to the Folin Lowry method, using bovine serum albumin as the standard. Protein amounts in column fractions were observed via absorbance variations at 650 nm.
The effect of pH on SOD activity
The pH activity profile of SOD was studied at standard assay conditions by using 0.05 M phosphate buffer having pH values between 6.0 and 8.0 and incubated for 2 h at 250ºC. After incubation, the activity of enzyme preparations was measured under standard assay conditions. pH stability being expressed as mean enzyme activity.
Estimation of temperature stability
The optimum temperature for enzyme activity was measured by incubating the enzyme at different temperatures. Enzyme was incubated for 2 h in 0.05 M phosphate buffer (pH 7.4) at temperature values between 15 and 700ºC. After incubation, the activity of enzyme preparations was measured under standard assay conditions. Temperature stability was expressed as mean enzyme activity.
Estimation of NaCl conc. on SOD activity
Reaction mixture was incubated in presence of 2 ml of respective salt concentration. After incubation, the activity of enzyme preparations was measured under standard assay conditions.
The purified SOD enzyme (70 μg/ml) was stored in 0.0 M phosphate buffer (pH 7.4) at 40ºC. No loss of activity was observed over 3 months.
The isolation of SOD from bacterial culture was performed by the disintegration of the membrane by mechanical, enzyme solubilization and freeze-thaw methods. 5 min grinding was applied with mortal and pastel. The disrupted product was centrifuged (10000 g, 10 min). The supernatant was re-centrifuged (25000 g, 30 min) and the activity of SOD in supernatant was measured 60 U/mg. while the specific activity of SOD with cell suspension was found to be 69.61 U/mg. As SOD is membrane bound enzyme, activity may be decreased after the disintegration of the cell membrane.
The first purification step for obtaining SOD from the crude extract was achieved by fractional precipitation of proteins by using ammonium sulphate (30-80%). The best precipitation yield was obtained by adding four volumes of ethanol per volume of sample in the bottom precipitate phase. The precipitate having the highest SOD activity was resuspended in 0.05 M potassium phosphate buffer, pH 7.4. The samples were stirred for 15 min and the insoluble material removed by centrifugation at 4,000 g for 15 min. Activity of SOD at this stage was found 580 U/mg.
Using DEAE cellulose column the unbound proteins were eluted by equilibration buffer. The bound proteins having SOD, were eluted via a linear gradient method by using 0.06-0.140 M phosphate buffers at pH 7.4 having specific activity 1976 U/mg.
Fractions having SOD were applied to a Sephadex G-75 gel chromatography column for further purification. SOD activity was eluted between fractions 9−13. A 63.91-fold purification of the enzyme with a 3835 U/mg specific activity was achieved. The results of the purification procedure are summarized in Table 2.
|Purification steps||Total proteins
|Specific activity (U/mg)||Fold purification||% yield|
|Sephadex G-75 chromatography||70.86||271748||3835||63.91||75.18|
Table 2: Purification steps of SOD from sewage isolate bacteria.
SDS PAGE analysis
The apparent molecular weight of enzyme was 35.71 kDa by SDS PAGE gel electrophoresis (Figure 1)
Type of SOD
In inhibition assay blue color was formed in presence of H2O2. H2O2 is Cu/Zn- SOD inhibitor. It indicates that type of isolated purified SOD was Cu/Zn- SOD.
The effect of pH, temperature, salt concentration on SOD activity
The effect of pH on enzyme stability was determined under standard assay conditions after 2 h pre-incubation using 0.05 M phosphate buffer, (pH 6.0-8.0) at 250ºC (Table 3). The purified SOD was found to be stable at pH 7.5 under the conditions studied (Figure 2). Activity loss was more than 40% at pH 6 and 8, while 11% and 26% at pH 7 and 6.5 respectively. The thermal stability of the SOD was also investigated between 15-700ºC for 2 h incubation period in 0.05 M phosphate buffer of pH 7.4 (Table 4). This SOD showed rather fair thermal stability. Activity loss was only 8% at 500ºC but 80% at 700ºC after 2 h incubation (Figure 3). The enzyme was found to be stable in presence of 1% salt only (Table 5). The activity found to be gradually reduced approximately 50% at higher concentrations (Figure 4). The purified SOD enzyme (70 μg/ml) was stored in 0.05 M phosphate buffer (pH 7.4) at 40ºC. No loss of activity was observed over 3 months.
|pH||Mean enzyme activity (U/ml)|
|6||6.738 ± 0.371915|
|6.5||8.530 ± 0.36680|
|7||10.30 ± 0.529739|
|7.5||11.499 ± 0.463334|
|8||6.634 ± 0.357867|
Table 3: The effect of pH on SOD activity.
|Temperature °C||Mean enzyme activity (U/ml)|
|15||5.789 ± 0.492239|
|25||7.56 ± 0.62038|
|37||12.76 ± 0.579137|
|50||11.08 ± 0.522814|
|70||2.522 ± 0.534557|
Table 4: The effect of temperature on SOD activity.
|Salt concentration (%)||Mean enzyme activity (U/ml)|
Table 5: The effect of salt concentration on SOD activity.
Sewage isolated bacterium showed maximum amount of SOD in suspension form. Among them E. coli was higher producer. SOD purified from sewage isolated E. coli has optimum conditions at temperature 370ºC and pH 7.5 same as standard/reference SOD (Sigma). But specific activity found to be different, isolated enzyme has 3835U/mg specific activity at 63.91-fold purification while standard/ reference SOD has 4285 U/mg specific activity. Our enzyme has 28% more specific activity than standard/reference SOD. In E. coli 474B5, 17 U/mg of SOD is reported  and in E. coli LE392, 12 U/ mg of SOD  is reported in suspension form while our bacterium was showing presence of 69.61 U/mg of SOD in suspension form. Molecular wt. of Mn SOD from Escherichia coli B was reported 39000 kD .While Mn SOD isolated from K12 strain have molecular wt 24 kD and pH 6.88 . SOD isolated from Aspergillus species showed (3,294-3,531 U/mg) activity . Superoxide dismutase purified from the photoautotrophic cyanobacterium Gloeocapsa spp. has a molar mass of 38 kDa, as estimated by gel filtration . SOD separated from Thermothrix spp isolated from thermal spa water in Serbia, showed high superoxide dismutase activity. The enzyme molecular weight determined by gel chromatography is 37 kD. While the optimum pH for enzyme activity was in the range of 8 to 10. The optimum temperature for SOD activity was 600ºC . SOD purified and partially characterized from chicken erythrocytes. The apparent molecular weight of the chicken erythrocyte SOD was found to be 30.6 kDa by gel filtration on a Sephadex G-75 column and 15 kDa by SDS slab gel electrophoresis. Maximum activity was observed between pH 7.0 to 9.0 at 250ºC . SOD of Neurosporo crassa , and yeast [14,25] had a molecular weight of about 32 kDa. Generally, Cu- Zn SODs are stable at neutral pH [11,14] The pH dependence of the Fe- SOD stability of Aerobacter aerogenesis, remained comparatively stable at alkaline pH 7.0-11.0, but was rapidly inactivated below pH 7.0 . The activity of Cu-Zn SODs is dependent on ionic strength and alkaline pH in a way that typically reflects the functional role of charged amino acid residues, in particular lysine [20,27]. At neutral pH, however, the radical exists mainly as O2.−, and the dismutation reaction is relatively slow due to electrostatic repulsion of negatively charged radicals . In similar conditions, the thermal, pH and storage stability of our SOD is better than that of Cu-Zn SOD purified from the venom of the H. fulvipes scorpion , Aerobacter aerogenesis  and cabbage .
We thank to Kolhapur Institute of Technology’s College of Engineering for financial support.
There are no any potential sources of conflict of interest.