Determination the optimum condition for production of superoxide dismutase (SOD) from a local isolate of staphylococcus aureus (original) (raw)
Related papers
2014
Results: SOD protein with high purity was obtained when imidazole concentrations of 100 mM, 200 mM and 250 mM were applied. The purified rSOD displayed specific activity of 1666.7 U mg-1when measured at 30oC and pH 7.8. The presence of conserved manganese-binding sites (H28, H83, D171, H175) and the inhibition of rSOD activity by NaN 3 but not by H 2O 2 or KCN and indicated that rSOD was Mn-dependent. The optimum temperature and pH were determined to be 40oC and 6.0, respectively. The Michaelis constant (Km), maximum velocity (V max), turnover number (k cat) and catalytic efficiency (kcat/K m) were found to be 371.2 µM, 1.738 µMS-1, 1.358 s-1, and 3.7x10-3 S-1µM-1 Conclusion: This is the first study to report the stability of the SOD of S. equorum against environmental factors. The SOD displays some thermostability, is active in wide pH, stable in the presence of denaturing and reducing agents, however it is relatively unstable to UVC exposure , respectively. The rSOD activity was s...
International Journal of Pharmacy and Pharmaceutical Sciences, 2014
Objective: Superoxide dismutase (SOD) (E. C: 1.15.1.1) from Staphylococcus equorum which catalyzes the dismutation of the superoxide anion (O2 .-) into molecular oxygen (O2) and hydrogen peroxide (H2O2 Methods: The protein was purified in a single-step purification using Ni-NTA affinity column with various imidazole concentrations. SOD activity was analyzed by colorimetric and activity staining using nitroblue tetrazolium (NBT). The purified rSOD was exposed to different temperatures and pHs, different concentrations of denaturing agents, reducing agents, and to UVC exposure.), is one of the most important classes of antioxidant enzymes and are used in pharmaceutical or cosmetic applications. SOD of S. equorum was purified from total protein into homogeneity and characterized to determine the unit activity, ion metal cofactor, optimum temperature and pH, kinetic parameters, and effect of denaturing and reducing agents and UVC exposure on the rSOD activity. Results: SOD protein with high purity was obtained when imidazole concentrations of 100 mM, 200 mM and 250 mM were applied. The purified rSOD displayed specific activity of 1666.7 U mg-1 when measured at 30 º C and pH 7.8. The presence of conserved manganese-binding sites (H28, H83, D171, H175) and the inhibition of rSOD activity by NaN3 but not by H2O2 or KCN and indicated that rSOD was Mn-dependent. The optimum temperature and pH were determined to be 40 º C and 6.0, respectively. The Michaelis constant (Km), maximum velocity (Vmax), turnover number (kcat) and catalytic efficiency (kcat/Km) were found to be 371.2 µM, 1.738 µMS-1 , 1.358 s-1 , and 3.7x10-3 S-1 µM-1 Conclusion: This is the first study to report the stability of the SOD of S. equorum against environmental factors. The SOD displays some thermostability, is active in wide pH, stable in the presence of denaturing and reducing agents, however it is relatively unstable to UVC exposure , respectively. The rSOD activity was slightly affected in the presence of detergents (0.5% SDS, 0.5% Triton-X 100), denaturing agents (6 M GdnHCl and 6 M urea) and reducing agent (5 mM βME). After exposure of rSOD by UVC for 45 min, it retained half of its activity.
Journal of Bacteriology, 1999
A Staphylococcus aureus mutant (SPW1) which is unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous to the sodA family of manganese-dependent superoxide dismutases (SOD). Whole-cell lysates of the parental 8325-4 strain demonstrated three zones of SOD activity by nondenaturing gel electrophoresis. The activities of two of these zones were dependent on manganese for activity and were absent in SPW1. The levels of SOD activity and sodA expression were growth-phase dependent, occurring most during postexponential phase. This response was also dependent on the level of aeration of the culture, with highest activity and expression occurring only under high aeration. Expression of sodA and, consequently, SOD activity could be induced by methyl viologen but only during the transition from exponential- to postexponential-phase growth. SPW1 was less able to survive amino acid limitation and acid stress but showed no alteration in pathogenic...
Inflammation Research, 2008
Background: Superoxide dismutase (SOD) and catalase are anti-oxidant enzymes potentially used by the bacteria to neutralize macrophage microbicidal molecules such as hydrogen peroxide (H2O2). Objective: To investigate contribution of bacterial anti-oxidant enzymes in intracellular survival of Staphylococcus aureus (S. aureus) within macrophages. Materials: Murine peritoneal macrophages and S. aureus (CMC-524, ICH-629 and ICH-757). Treatment: 106 colony forming units (CFU) of the 90 minutes (min) intracellularly viable S. aureus were administered (i.v.) per mouse through 0.1 ml saline. Methods: Anti-oxidant enzyme assay, phagocytic activity, H2O2 release, Zymography for catalase, serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) level were estimated. One-way Model I ANOVA and one tail Student’s t-test were performed. Results: Survival of S. aureus was least after 90 min of reincubation within macrophages. Maximum amount of bacterial anti-oxidant enzymes were released after 90 min of re-incubation. H2O2 released after 90 min of re-incubation with S. aureus was maximum. Higher activity of catalase and SOD by S. aureus occurred in response to the gradual production of H2O2. Serum IL-6 and TNF-α was also elevated 1h post infection. Conclusions: Bacterial catalase and SOD combat reactive oxygen species enabling S. aureus to persist within macrophages, inducing local inflammation, causing greater induction of serum TNF-α and IL-6.
The rising incidents of invasive infections due to multidrug resistant Staphylococcus aureus necessitate the exploration of newer targets for development of antibiotics. Pathogenicity of S. aureus is attributed to a wide range of virulence factors. The aim of this study was to screen the production of three virulence factors viz. extracellular protease, extracellular lipase and superoxide dismutase in human pathogenic strains of S. aureus for development of a test panel which could aid in screening of natural products of plant and microbial origin. 27 clinical isolates were compared for their enzyme expression profiles of which eight were finally selected. Sau G5 was the only protease producing organism selected in the test panel, while Sau G3 and Sau G9 were best SOD producers and Sau G16, Sau G18, Sau G22, Sau A5 and Sau A2 exhibited highest expression among different groups of clinical staphylococci.
Characterization of the Single Superoxide Dismutase of Staphylococcus xylosus
Applied and Environmental Microbiology, 2001
Staphylococcus xylosus is a facultative anaerobic bacterium used as a starter culture for fermented meat products. In an attempt to analyze the antioxidant capacities of this organism, the superoxide dismutase (SOD) was characterized.S. xylosus contains a single cytoplasmic SOD, which was not inhibited by H2O2. The SOD activity in crude extracts was completely lost upon metal depletion, but it could be recovered by manganese and very weakly by iron. It is therefore suggested that the S. xylosus SOD is a manganese-preferring enzyme. The corresponding gene, sod, was isolated from a genomic library of S. xylosus DNA and complemented the growth defect of an Escherichia coli SOD-deficient mutant. As deduced from the nucleotide sequence, sod encodes a protein of 199 amino acids with a molecular mass of 22.5 kDa. Two transcriptional start sites 25 and 120 bp upstream of thesod start codon were identified. A terminator-like structure downstream of the gene suggested a monocistronicsod mRNA....
Indian Journal of Microbiology, 2010
The present study was performed in order to carefully investigate the interaction of Staphylococcus aureus with murine macrophages and the contribution of catalase and superoxide dismutase in intracellular persistence of Staphylococcus aureus within murine macrophages during in vitro infection. We have reported that Staphylococcus aureus internalized by murine macrophages did not appear to be rapidly killed. Data indicating the contribution of a single catalase and superoxide dismutase in intracellular survival of Staphylococcus aureus were provided using established biochemical assays. The results of the present experiment suggest that the survival of Staphylococcus aureus within phagocytic cells is facilitated by its ability to resist oxidative products. Organisms in the log phase of growth clearly demonstrate a resistance to oxidative products.
Superoxide Dismutase in Bacteroides fragilis and Related Bacteroides Species
Superoxide dismutase (SOD) activity was demonstrated in cell-free extracts of Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides ovatus, and Bacteroides thetaiotaomicron. The strains were grown under. anaer-obic conditions in Trypticase soy broth, and the specific activity of SOD in the extracts was, in most strains, higher than in cell-free extracts ofEscherichia coli B grown under anaerobic conditions. Isoelectric focusing of the extracts in polyacrylamide gel demonstrated distinct forms of SOD in the different species.
Isolation and purification of superoxide dismutase from haloarcheal strain
2016
In this study, Haloarcheal strain RHA was isolated from water samples collected from salt pan. The strain was further treated with 2% pyrogallol and incubated in a water bath with shaker for 12 days. Supernatant was collected from early log phase and was used as enzyme source. The results showed that the optimum pH of SOD in crude extract was found to be at 9.0 and optimum temperature was found to be at 67 °C. In case of purified sample the activity of SOD was found to be increased at pH 9.8 and optimum temperature was noticed at 77oC. Activity of enzyme was increased in increasing concentration of enzyme source. The enzyme has great affinity for the substrate and it was calculated as 0.25mM by Michaelis menton plot. The activity of Superoxide dismutase from purified sample was also assayed in the presence of metal ions and the results obtained indicated that the activity was found to be increased significantly from 85.5 mM/mL and the activity was stable and constant up to 171 mM/mL...
Open Journal of Medical Microbiology, 2014
Background: Staphylococcus aureus is one of the most virulent gram positive bacteria. It produces a lot of toxins and enzymes, most of which are virulent factors. Among the enzyme that produces is the catalase which is very useful in differentiating staphylococci from streptococci [1]. Catalase is nearly ubiquitous among some of organisms that can grow in the presence of oxygen (air). It promotes the conversion of hydrogen peroxide, a powerful and potentially harmful oxidizing agent, to water and molecular oxygen; so the major function of catalase within cells is to prevent the accumulation of toxic levels of hydrogen peroxide formed as a by-product of metabolic processesprimarily that of the electron transport pathway. Objectives: The main aim of this study is to prove that human WBCs can produce H2O2. This H2O2 when reacting with catalase producing S. aureus can easily be degraded to H2O + O2. Methodology: In this study a total of 40 subjects were included. Aliquots of 2.5 ml of venous blood were collected by venous puncture after disinfecting the site of collection with 70% alcohol and the collected blood was drawn into EDITA containers (20 subject) and anticoagulant free containers (other 20 subject), centrifugation for 5 minute at 1500 RPM. The separated sera and plasma were converted to new sterile eppendrof tubes and freezing until used (we leaved the eppendrof tubes that contained sera and plasma at room temperature before using it for DE freezing). Standard catalase producing S. aureus were used by taking 1 colony from Macconkey media by using applicator wooden stick, and inserted in eppendrof tube, then air bubbles would appear to indicate occurrence of the reactions. Results: According to this study, it was proved that WBCs in human plasma or serum can produce H2O2; this H2O2 was reacted with catalase enzyme produce from colony of S. aureus to produce air bubbles and water. There were no differences between using H2O2 or human plasma/serum that contains WBCs to detect and identify S. aureus by both techniques. Conclusion: Based on the results of this study, we can use WBCs that are found in human plasma or serum to identify catalase producing S. aureus.