Purification  and  partial  characterization  of  whiteradish  (Raphanus  sativus  L.  var.  Long  white) peroxidase  from  cell  suspension  culture  extract

Andi Tenri  Adjeong Karossi,; Sri  Pudjiraharti,

Purification and partial characterization of whiteradish (Raphanus sativus L. var. Long white) peroxidase from cell suspension culture extract

Sri Pudjiraharti | Andi Tenri Adjeong Karossi

Abstract:

Peroxidase, particularly Horseradish Peroxidase (HRP), has been widely used as a component in clinical diagnostic reagents for the Enzyme-Linked Immunosorbent Assay (ELISA) technique. White radish (Raphanus sativus L.) has been identified as another potential source of peroxidase. In this study, white radish was used for the production of peroxidase using a cell suspension culture technique. The enzyme was isolated through ammonium sulfate precipitation, followed by purification using DEAE-cellulose column chromatography, eluted with 0.01 M phosphate buffer (pH 7.5) and a 0–0.5 M NaCl gradient. A major protein peak showing the highest activity and 25-fold purity compared to the crude enzyme was observed. This protein was partially characterized. SDS-polyacrylamide gel electrophoresis revealed one main band with a molecular weight of 47,000 Da. This white radish peroxidase (WRP) demonstrated high efficiency, with maximum activity at 55°C and pH 7.5, a Km of 76.6 µg/mL, and a Vmax of 275 µg/mL/minute using hydrogen peroxide as the substrate and pyrogallol as the hydrogen donor.

References:

ANDRADE, J.L., M. S. LEMOS, A. S. SANTOS, O. A C. ANTUNES and N. PEREIRA, JR. Peroxidase production and application in the isosafrole biotransformation into piperonal. www.eere.energy.gov./biomass/biotech_symposium/docs/lb-62.doc.
ANONYMOUS. 1991. Ion exchange chromatography. Principles and Methods. Sweden: Pharmacia Fine Chemicals.
CHEN, HUEY-LING, CHANG, MEI-HWEI, NI, YEN-HSUAN, HSU, HONG-YUAN, ΚΑΟ, JIA-HORNG, and CHEN, PEI-JER. 1997. Hepatitis G virus infection in normal and prospectively followed posttransfusion children. Pediatric Research 42(6): 784-787.
COLOWICK, SP. and N. O. KAPLAN. 1957. Methods in Enzymology, Vol III, Acad. Press. Inc., New York.
FIELDING, J. L. and A. HALL. 1978. Biochemical and cytochemical study of peroxidase activity in root of Pisum sativum. J. Exp. Bot. 29:969-981.
ISKANDAR, Y.M., S. PUDJIRAHARTI and A. T. KAROSSI. 1996. Production of peroxidase from Armorachia lapathifolia (Horseradish) callus. In: Proceeding of Pemaparan Hasil Litbang Ilmu Pengetahuan Teknik. Bandung, 14-26 October, 1996.
JOON-SUPYEOM, JAE-BEOM LEE, SEUNG-HO RYU, HEE-JUNG KANG, SUNHIL KIM, YOUNG-A KIM, SUN-YOUNG AHN, JE-EUN CHA and JAE-WON PARK. 2006. Evaluation of a new third-generation ELISA for the detection of HIV infection. Annals of Clinical & Laboratory Science 36:73-78.
KOTWAL, GJ., B. M. BAROUDY, Ι. Κ. KURAMOTOT, F. F. MCDONALD, G M. SCHIFF, P. V. HOLLANDT and J. B. ZELDIS. 1992. Detection of acute hepatitis C virus infection by ELISA using a synthetic peptide comprising a structural epitope (early diagnosis/blood screening). Proc. Nati. Acad. Sci. 89: 4486-4489.
LAEMLI, U.K. 1970. Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature 227: 680-685.
LEE, T. T. 1973. Extraction and quantitation of plant peroxidase isoenzyme. Plant. Physiol. 29:198-203
MURASHIGE, K. 1974. Plant Propagation through tissue culture. Ann Rev. Plant. Physiol. 25: 35-36.
PUDJIRAHARTI S., T. A. BUDIWATI and A. T KAROSSI. Peroxidase production through cell suspension culture technique of Raphanus sativus L.var.long white. In: Proceedings of Indonesian Chemistry Society Seminar: Jakarta 22 February, 2006
SAUNDERS, B.C., A. G. HOLMES SIEDLE and B. P. STARK. 1974. Peroxidase. Butterworth Co. Ltd, London.
VITALI A, B. BOTTA, G D. MONACHE, S. ZAPPITELLI, P. RICCIARDI, S. MELINO, R. PETRUZELLI and B. GIARDINA. 1998. Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies. Biochem J., 331: 513-519.
WILKINSON, M.L., J. M. GRIFFITHS and R. R. ALEXANDER. 1985. Basic Biochemical Methods. John Willey and Sons, New York.
YAMADA, Y., K. SETSUO, K. WATANAKA, and H. UZO. 1987. Production of Horse Radish Peroxidase by Plant Cell. J. Chem. Tech. Biotech. 38:31-39