STUDY ON THE PROPERTIES OF PURIFIED RECOMBINANT SUPEROXIDE DISMUTASE FROM STAPHYLOCOCCUS EQUORUM, A LOCAL ISOLATE FROM INDONESIA
Keywords:
Staphylococcus equorum, Manganese superoxide dismutase, Expression, His-tag purification, CharacterizationAbstract
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), 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.
Methods: The protein was purified in a single-step purification using Ni-NTA afï¬nity 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.
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 NaN3 but not by H2O2 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 (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-1µM-1, 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.
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
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