Purification and substrate specificity of peroxidase from sweet potato tubers J. Castillo Leon a , I.S. Alpeeva b , T.A. Chubar b , I.Yu. Galaev a , E. Csoregi a , I.Yu. Sakharov b,c, * a Department of Biotechnology, Lund University, P.O. Box 124, Lund S-221 00, Sweden b Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119992, Russia c Division of Chemistry, G.V. Plekhanov Russian Economic Academy, Stremyanny per. 28, 113054 Moscow, Russia Received 26 March 2002; received in revised form 8 July 2002; accepted 22 July 2002 Abstract Previously the screening of tropical plants demonstrated a high peroxidase activity in sweet potato (Ipomoea batatas ) tubers. The major peroxidase pool is localized in peel. Using peel of sweet potato as a source, the sweet potato peroxidase (SPP) has been isolated and purified to homogeneity. The enzyme purification included homogenization, extraction of colored compounds and consecutive chromatographies on Phenyl-Sepharose and DEAE-Toyopearl. The purified SPP had specific activity of 4900 U mg 1 protein, RZ (ratio of absorbances at 403 and 280 nm, respectively) 3.4, molecular mass of 37 kDa and isoelectric point of 3.5. The spectrum of peroxidase from sweet potato is typical for plant peroxidases with a Soret maximum at 401 nm and the maxima in the visible region at 497 and 638 nm, respectively. The substrate specificity of SPP is distinct from the specificity of other plant peroxidases, ferulic acid being the best substrate for SPP. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Peroxidase; Sweet potato; Purification; Substrate specificity 1. Introduction Peroxidase (EC 1.11.1.7; donor:hydrogen-peroxide oxidoreductase) catalyses the oxidation of various electron donor substrates (e.g. phenols, aromatic amines) by hydrogen peroxide [1]: E H 2 O 2 0 Cpd I H 2 O (1) Cpd I AH 2 0 Cpd II AH + (2) Cpd II AH 2 0 E AH + H 2 O (3) where E is the ferric enzyme, Cpd I and Cpd II are Compound I and Compound II, the oxidized inter- mediates of peroxidase; AH 2 and AH + are the electron donor substrate and the radical product of its one- electron oxidation, respectively. Peroxidase is one of the key enzymes controlling plant differentiation and development. It is known that this enzyme participates in the construction, rigidification and eventual lignification of cell walls, in the biosynth- esis of H 2 O 2 , in the protection of plant tissues from damage and infection by pathogenic microorganisms, and in wound healing [1  /3]. In vitro this enzyme is widely employed in microanalysis [4 /6]. Currently, peroxidases are used also in organic synthesis for the production of polymers and for the biotransformation of various drugs and chemicals [7,8]. Although peroxidases are ubiquitous in the plant kingdom, at present the major source of commercially available peroxidase is roots of horseradish (Armoracia rusticana ). However, the availability of peroxidases with elevated stability and different specificity allows the improvement of already existing analytical procedures and/or the development of new ones. Therefore, numer- Abbreviations: ABTS, 2,2?-azino-bis-(3-ethylbenzthiazoline-6- sulfonic acid); HRP, horseradish peroxidase; SPP, sweet potato peroxidase. * Corresponding author. Tel.:  /7-095-9393407; fax:  /7-095- 9392742 E-mail address: sakharov@enz.chem.msu.ru (I.Y. Sakharov). Plant Science 163 (2002) 1011  /1019 www.elsevier.com/locate/plantsci 0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII:S0168-9452(02)00275-3