Abstract
Cu, Zn-superoxide dismutase was isolated from Aspergillus niger mycelia, harvested at the mid-logarithmic growth phase. The purification scheme aimed at the optimization of the ethanol/chloroform extraction (Tsuchihashi extraction) through response surface methodology. Upon optimum extraction conditions, it was possible to obtain electrophoretically pure enzyme preparations, by the application of one step anion exchange chromatography. The enzyme yield of this simple purification procedure was above 75% while the specific activity of the final preparation was among the highest reported for eucariotic microorganisms. The purified enzyme exhibited similar physicochemical characteristics with other Aspergillus sp. superoxide dismutases revealing an apparent tetrameric structure with a subunit molecular weight of 19 kDa, and a pl of 5.95.
Similar content being viewed by others
References
Arai K, Iizuka S, Makita A, Oikawa K and Taniguchi N (1986) Purification of Cu,Zn-superoxide dismutase from human erythrocytes by immunoaffinity chromatography. J. Immunol. Methods 91: 139–143.
Arnold LD and Lepock JR (1982) Reversibility of the thermal denaturation of yeast superoxide dismutase. FEBS Lett. 146: 302–306.
Asayama K and Burr IM (1984) Joint purification of mangano and cuprozinc superoxide dismutases from a single source-a simplified method. Anal. Biochem. 136: 336–339.
Bannister JV and Bannister WH (1987) Aspects of the structure, function and applications of superoxide dismutase. Cr. Rev. Biochem. 22: 111–180.
Bettarello YM, Cabrera-Crespo J, Ho PL, Carvalho A and Raw I (1993) Purification of superoxide dismutase fiom placental haemolysate blood: A simple and efficient method. Biochem. Mol. Biol. Int. 30: 45–51.
Box GEP, Hunter WG and Hunter JS (1978) Responce Surface Methods. In: Statistics for Experimenters-An Introduction to Design, Data Analysis and Model Building, pp 510–539. John Wiley & Sons Inc.
Bradford MB (1976) A rapid and sensitive method for quantification of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Callahan HL, Crouch RK and James ER (1991) Dirofilaria immitissuperoxide dismutase: Purification and characterization. Mol. Biochem. Parasitology 49: 245–252.
Eldred GE and Hoffert JR (1981) A test of endogenous interferences in superoxide dismutase assays. Anal. Biochem. 110: 137–143.
Eriksson CE and Na A (1995) Antioxidant enzymes in raw materials and processed foods. Biochem. Soc. Symp. 61: 221–34.
Fridovich I (1986) Superoxide dismutases. Adv. Enzymol. 58: 61–97.
Geller BL and Winge DR (1983) A method for distinguishing Cu,Zn-and Mn-containing superoxide dismutases. Anal. Biochem. 128: 86–92.
Goscin SA and Fridovich I (1972) The purification and properties of superoxide dismutase from Saccharomyces cerevisiae. Biochim. Biophys. Acta 289: 276–283.
Gralla EB and Kosman DJ (1992) Molecular genetics of superoxide dismutases in yeasts and related fungi. Adv. Gen. 30: 251–319.
Grunow M and Schoop W (1992) Purification of pro-and eucariotic superoxide dismutases by charge-controlled hydrophobic chromatography. J. Chromatogr. 590: 247–253.
Hassan HM and Fridovich I (1977) Regulation of superoxide dismutase synthesis in Escherichia coli: glucose effect. J. Bacteriol. 132: 505–510.
Hatzinikolaou DG and Macris BJ (1995) Factors regulating production of glucose oxidase by Aspergillus niger. Enz. Microb. Technol. 17: 530–534.
Holdom MD, Hay RJ and Hamilton AJ (l995) Purification, Nterminal aminoacid sequence and partial characterization of a Cu,Zn superoxide dismutase from the pathogenic fungus Aspergillus fumigatus. Free Rad. Res. 22: 519–531.
Holdom Nm, Hay RJ and Hamilton AJ (1996) The Cu,Zn superoxide dismutases of Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus: Purification and biochemical comparison with the Aspergillus fumigatusCu,Zn superoxide dismutase. Infect. and Immun. 64: 3326–3332.
Inouye K, Nakamura K, Mitoma Y, Matsumoto M and Igarashi T (1985) Application of a new ion exchanger TSK-gel DEAESPW, to the purification of Cu,Zn-superoxide dismutase of bovine erythrocytes. J. Chromatogr. 327: 301–311.
Kafarov V (1976) Development of mathematical models by experiment and statistics. Statistical optinzization. In: Cybernetic Methods in Chemistry and Chemical Engineering, pp 167–223. MIR Publishers, Moscow, USSR.
Lavelle F and Michelson AM (1975) Purification and study of two superoxide dismutases of the mushroom Pleurotus olearius. Biochimie 57: 375–381.
Lee F-J and Hassan HM (1985) Biosynthesis of superoxide dismutase in Saccharomyces cerevisiae: Effects of paraquat and copper. J. Free Rad. Biol. Chem. 1: 319–325.
Lingnert H, Akesson G and Eriksson CE (1989) Antioxidative effect of superoxide dismutase from Saccharomyces cerevisiaein model systems. J, Agric. Food Chem. 37: 23–28.
McCord JM and Fridovich I (1969) Superoxide dismutase. Enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244: 6049–6055.
Meyer AS, Rorbaek K and Adlen-Nissen J (1994) Critical assessment of the applicability of superoxide dismutase as an antioxidant in lipid foods. Food Chem. 51: 171–178.
Misra HP and Fridovich I (1972) The purification and properties of superoxide dismutase from Neurospora crassa. J. Biol. Chem. 247: 3410–3414.
Nice DJ and Robinson DS (1992) Inhibition of lipid autoxidation by bovine superoxide dismutase. Food Chem. 45: 99–105.
Oda T, Akaike T, Hamamoto T, Suzuki F, Hirano T and Maeda H (1989) Oxygen radicats in influenza-induced pathogenesis and treatment with pyran polymer-conjugated SOD. Science 244: 974–976.
Padiglia A, Medda R, Cruciani E, Lorrai A and Floris G (1996) Purification and properties of Oryza sativaCu-Zn superoxide dismutase. Prep. Biochem. Biotechnol. 26: 135–142.
Schimina ME, Bossa F, Lania A, Capo CR, Carlini P and Calabrese L (1993) The primary structure of turtle Cu,Zn superoxide dismutase. Structural and functional irrelevance of an insert conferring proteolytic susceptibility. Eur. J. Biochem. 211: 843–849.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hatzinikolaou, D.G., Tsoukia, C., Kekos, D. et al. An efficient and optimized purification procedure for the superoxide dismutase from Aspergillus niger. Partial characterization of the purified enzyme. Bioseparation 7, 39–46 (1998). https://doi.org/10.1023/A:1007986510153
Issue Date:
DOI: https://doi.org/10.1023/A:1007986510153