Abstract
Growing peroxidase utilisation in different industries encourages the search for high benefit/cost ratio purification methods such as aqueous two-phase partition. In this way, the partitioning behaviour of peroxidase from Armoracia rusticanaroots in polyvinylpirrolidone/Reppal and polyvinylpirrolidone/salt aqueous two-phase systems was investigated. Based on these results, a two-step purification process was developed. In the first system (polyvinylpyrrolidone K30/Reppal PES 200, pH 7.0), cell debris and some contaminating proteins were shifted to the bottom phase while peroxidase concentrated in the top phase. After discarding the bottom phase, the second step involved addition of magnesium sulphate thus forming a second aqueous two-phase system. At this step, the enzyme was extracted into the salt-rich bottom phase. The overall yield was 75% and the purification factor 7.3.
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References
Alberti BN and Klibanov AM (1982) Peroxidase for removal of hazardous aromatics from industrial wastewaters. In: Exemer JH (ed), Detoxification of Hazardous Wastes (pp. 349–56). Ann Arbor Science Publishers, Ann Arbor.
Albertsson PA (1986) Partition of Cells and Macromolecules, 3rd ed., Ch. 15 (pp. 529–587). John Wiley and Sons, New York.
Arseguel D and Baboulène M (1994) Removal of phenol from coupling of talc and peroxidase. Application for depollution of waste water containing phenolic compounds. J. Chem. Tech. Biotechnol. 61: 331–335.
Behn I, Hellthaler G, Fiebig H and Appelt S (1985) DDR Patent 228824.
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Casl MT and Malnar M(1986) Determination of the optimal capacity for purification of horseradish peroxidase on concanavalin A. Jugoslav. Med. Biokem. 4: 33–38.
Casl MT and Kostrencic C (1987) Isolation and purification of horseradish peroxidase. Ann. Clin. Biochem. 24 suppl. 2: 271.
Chaga G, Andersson L, Ersson B and Berg M (1992) Use of immobilized metal ions as a negative adsorbent for purification of enzymes: application to phosphoglycerate mutase from chicken muscle extract and horseradish peroxidase. Biomed. Chromatog. 6: 172–176.
Chavez C and Flurkey WH (1984) Biospecific adsorption of peroxidase. J. Chromatog. 298: 169–171.
Giuliano KA (1991) Aqueous two-phase protein partitioning using textile dyes as affinity ligands. Analyt. Biochem. 197: 333–339.
Grodecka B, Kowalski S and Szymanska E (1985) Purification of peroxidase from aqueous horseradish extracts. Pol. Patent PL 133652.
Lascu I, Abrudan I, Muresan L, Presecan E, Vonica A and Proinov I (1986) Salting-out chromatography on unsubstituted Sepharose CL-6B as a convenient method for purifying proteins from dilute crude extracts. Application to horseradish peroxidase. J. Chromatogr. 357: 436–439.
Kula MR (1990) Trends and future prospects in aqueous two-phase extraction. Bioseparation 1: 181–189.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
Miranda MV and Cascone O (1994) Partition behaviour of horseradish peroxidase isoenzymes in aqueous two-phase poly(ethyleneglycol)/phosphate systems. Biotechnol. Tech. 8: 275–280.
Miranda MV, Fernández-Lahore HM and Cascone O (1995) Horseradish peroxidase extraction and purification by aqueous two-phase partition. Appl. Biochem. Biotechnol. 53: 147–154.
Müller, W (1990) Liquid-liquid partition chromatography of biopolymers in aqueous two-phase systems. Bioseparation 1: 265–282.
Nicell JA, Saadi KW and Buchanan ID (1995) Phenol polymerization and precipitation by horseradish peroxidase enzyme and additive. Biores. Tech. 54: 5–16.
Shannon LM, Kay E and Lew LY (1966) Peroxidase isozymes from horseradish roots. J. Biol. Chem. 241: 2166–2171.
Suh PG, Lee YH, Lee JH, Chung JH, Kim SR, Tchai BS and Chung HK (1989) Purification of peroxidase by using monoclonal antibody to horseradish peroxidase and its application to enzyme immunoassay. Korean J. Biochem. 21: 33–40.
Tjissen P (1985) In: Burdon RH and van Knippenberg PH (eds) Practice and Theory of Enzyme Immunoassays, Ch.10 (pp. 187–188). Elsevier, New York.
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Miranda, M.V., Cascone, O. Partition of horseradish peroxidase in aqueous two-phase systems containing polyvinylpyrrolidone. Bioseparation 7, 25–30 (1998). https://doi.org/10.1023/A:1007913300427
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DOI: https://doi.org/10.1023/A:1007913300427