Purification of superoxide dismutase from bacterial isolate

The cells were harvested from bacterial culture isolate by centrifugation and were suspended in Tris-HCl buffer of pH 8.0. The optical density (O.D.) of cells was measured at 600nm and each time cells of 21.5 optical densities (O.D.) were disrupted and used for the purification of the enzyme. Purification of superoxide dismutase from SPB-13. Ammonium sulphate precipitation method was used for concentrating the proteins. After this, the enzyme was further purified by DEAE-Sepharose column. The DEAE-sepharose column was loaded with 3ml of concentrated protein.The enzyme activity was only recorded in fractions ranging from 22, 23, 24, 25, 26, 27 and 28. These fractions were polled together. The polled fractions along with crude enzyme were checked for purity by SDS-PAGE.The maximum activity of superoxide dismutase of SPB-13 was found to be in Tris-HCl buffer, 60mM molarity, pH 8.0, incubation time was found to be 2.0 minutes, incubation temperature 450C. An enzyme concentration of 10µg was found to be optimum. Thermostability of superoxide dismutase was checked and enzyme was found to be thermostable upto 550C for 60 minutes.