ENZYME AND MICROBIAL TECHNOLOGY, vol.25, pp.392-399, 1999 (SCI-Expanded)
The superoxide dismutase that protects against oxidative stress of superoxide radicals in living cells was isolated and purified from Phanerochaete chrysosporium and partially characterized. Cells cultivated under optimized growth conditions were distrupted by grinding with glass beads in a mixer-mill. Partial protein precipitation in crude extract was affected by using (NH4)(2)SO4, polyethylene glycol, and methanol methods. Fractionation of superoxide dismutase was performed by O-diethylaminoethyl-cellulose ion-exchange chromatography, followed by Sephadex G-100 gel-filtration chromatography. Purified enzyme has a molecular weight of 44 000 +/- 800 and is comprised of two equal sized subunits each having an Mn element. The optimum pH of purified MnSOD was obtained as 8.8. Enzyme remained stable at pH 7.0-8.8, 25 degrees C and up to 45 degrees C at pH 7.4 for 1 h incubation. The enzyme was insensitive to cyanide, hydrogen peroxide, ditiothreitol, sodium azide, Triton X-100, and beta-mercaptoethanol and was sensitive to sodium dodecyl sulfate. Phenylmethylsulfonyl fluoride (2 mM) and iodoacetamide showed only a 15% inhibition effect on the enzyme. Up to 50% activity reduction was observed for 100 mM ionic strength of phosphate and chloride. (C) 1999 Elsevier Science Inc. All rights reserved.