Purification and characterization of Opuntia peroxidase

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Abstract-Wide applications of peroxidase in different areas of clinical biochemistry, biotechnology, food industry etc. enhances the interest for further study on the enzyme. The distribution of peroxidase activity and the kinetic parameters of three plant species [( ...

Applied Biochemistry and Biotechnology, 2012

BACKGROUND: Avocado (Persea americana Mill, cv. Hass) fruit ranks tenth in terms of the most important products for Mexico. Avocado products are quite unstable due to the presence of oxidative enzymes such as polyphenol oxidase and peroxidase. The present study is to characterize the activity of purified avocado peroxidase from avocado in order to ascertain the biochemical and kinetic properties and their inhibition conditions. RESULTS: Purification was performed by Sephacryl S 200 HR gel filtration chromatography and its estimated molecular weight was 40 kDa. The zymogram showed an isoelectric point of 4.7. Six substrates were tested in order to ascertain the affinity of the enzyme for these substrates. The purified peroxidase was found to have low K m (0.296 mM) and high catalytic efficiency (2688 mM −1 s −1) using 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), optimum activity being reached at 51 • C, pH 3.8. The addition of dithiothreitol, β-mercaptoethanol, ascorbic acid, sodium azide, L-cysteine and Tween-20 had high inhibitory effects, while metals ions such as Cu + , Fe 2+ and Mn 2+ had weak inhibitory activity on purified avocado peroxidase. CONCLUSION: The purified avocado peroxidase exhibits high inhibition (K i = 0.37 µM) with 1.97 µM n-propyl gallate using ABTS as substrate at 51 • C, pH 3.8 for 10 min.

Journal of Molecular Catalysis B-enzymatic, 2011

Peroxidase was purified to homogeneity from a tree legume Leucaena leucocephala. On SDS-PAGE the purified enzyme exhibited two distinct subunits each of 66 and 58kDa. Determination of native molecular weight of the purified peroxidase revealed a size of ∼200kDa suggesting a heterotrimeric structure (consisting of two subunits of 66kDa and one subunit of 58kDa) for native peroxidase. Purified peroxidase was

2011

A peroxidase (PD-cP; 0.47 mg/100 g leaves) was purified from autumn leaves of Phytolacca dioica L. and characterized. PD-cP was obtained by acid precipitation followed by gel-filtration and cation exchange chromatography. Amino acid composition and N-terminal sequence of PD-cP up to residue 15 were similar to that of Spinacia oleracea (N-terminal pairwise comparison showing four amino acid differences). PD-cP showed a molecular mass of approx. 36 kDa by SDS-PAGE, pH and temperature optima at 3.0 and 50.0 o C, respectively and seasonal variation. The Michaelis-Menten constant (KM) for H2O2 was 5.27 mM, and the velocity maximum (Vmax) 1.31 nmol min -1 , while the enzyme turnover was 0.148 s -1 .

J. Agric. Food Chem, 1998

Thermal characteristics and substrate specificity of peroxidase (POX) from okra were determined. Crude POX was separated into eight fractions (P1−P8) by DEAE−cellulose chromatography. Heat inactivation of POX was biphasic and fitted to a first-order kinetic ...

Scientia Horticulturae, 2005

Peroxidases (EC 1.11.1.7) were extracted from fresh cladodes harvested from nine ecotypes of the cactus species Opuntia ficus indica Mill. growing as a collection, in Marrakech (South Morocco). Two enzyme fractions were obtained by a progressive solubility method leading to soluble peroxidases (S) and ionically wall-bound peroxidases (I). The preferred substrate for cladode peroxidases was determined to be o-dianisidine over 4-chloro-1-naphtol and guaiacol. Contrarily to roots, no guaiacol-based activity was found in cladodes. The late ecotypes 1'Haddaouia' and 'Moussa' showed a relatively high peroxidase ratio S/I (units g À1 fresh weight). When subjected to electrophoresis on polyacrylamide gels, S and I peroxidase fractions each exhibited two enzyme forms based on their electric charges in basic and acidic gel media. Acidic peroxidase forms, well separated on basic gels, showed two principal migration zones with great differences in their enzyme activities depending on the fraction types S and I. Acidic ionically wall-bound peroxidases exhibited fast, highly active isoforms with R f values 0.42-0.58. Basic forms, represented essentially in fractions S and resolved on acidic gels, were typified by slow and fast isoforms with a double banded pattern in most ecotypes. Opuntia ecotypes collected from localities surrounding Marrakech exhibited fast basic soluble isoforms, while distant ecotypes from Marrakech, including late ones, were typified by a fast acidic ionically bound isoperoxidase of R f 0.58. Possible roles of O. f. indica peroxidases in growth and their evaluation as markers in this cactus species are discussed in this study.

International Journal of Food Properties, 2011

Peroxidase (EC 1.11.1.7; donor: hydrogen peroxide oxidoreductase) is an oxidoreductase enzyme found in many fruits and vegetables. This enzyme was purified from sweet gourd (Cucurbita moschata Lam. Poiret) by ammonium sulphate precipitation and CM-Sephadex ion-exchange chromatography. Furthermore, optimum pH, optimum temperature, optimum ionic strength, stable pH, and stable temperature conditions were determined as 7.2, 50°C, 0.4 M, 8.0, and 40°C, respectively. The molecular weight (MW) of the enzyme was estimated to be 85 kDa by SDS-PAGE method. The values of Km and Vmax were calculated from the Lineweaver-Burk graph for guaiacol/H2O2 substrate patterns.

Applied Biochemistry and Biotechnology, 2008

The major pool of peroxidase activity is present in the peel of some Egyptian citrus species and cultivars compared to the juice and pulp. Citrus jambhiri cv. Adalia had the highest peroxidase activity among the examined species. Four anionic and one cationic peroxidase isoenzymes from C. jambhiri were detected using the purification procedure including ammonium sulfate precipitation, chromatography on diethylaminoethanolcellulose, carboxymethyl-cellulose, and Sephacryl S-200 columns. Cationic peroxidase POII is proved to be pure, and its molecular weight was 56 kDa. A study of substrate specificity identified the physiological role of POII, which catalyzed the oxidation of some phenolic substrates in the order of o-phenylenediamine>guaiacol>o-dianisidine>pyrogal-lol>catechol. The kinetic parameters (K m , V max , and V max /K m) of POII for hydrolysis toward H 2 O 2 and electron donor substrates were studied. The enzyme had pH and temperature optima at 5.5 and 40°C, respectively. POII was stable at 10-40°C and unstable above 50°C. The thermal inactivation profile of POII is biphasic and characterized by a rapid decline in activity on exposure to heat. The most of POII activity (70-80%) was lost at 50, 60, and 70°C after 15, 10, and 5 min of incubation, respectively. Most of the examined metal ions had a very slight effect on POII except of Li + , Zn 2+ , and Hg 2+ , which had partial inhibitory effects. In the present study, the instability of peroxidase above 50°C makes the high temperature short time treatment very efficient for the inactivation of peel peroxidase contaminated in orange juice to avoid the formation of off-flavors.

Biochemistry (Moscow), 2011

Peroxidases (EC 1.11.1.X) are enzymes of the oxi doreductase class that catalyze the oxidation of a wide range of substrates using hydrogen peroxide. Their molecular weight ranges from 17 to 84 kDa and the polypeptide chain from 153 to 753 amino acids. The pro teins usually include 10 11 α helices, while β sheets are absent or rare. Most peroxidases consist of two structural domains with a heme, ferriprotoporphyrin IX prosthetic group, in a hydrophobic pocket (Fig. 1) [1]. The generally accepted classification proposed in 1992 [2] divides peroxidases into two large superfamilies, animal peroxidases and plant peroxidases. The superfam ily of plant peroxidases is further divided into three class es. This system initially based on amino acid sequence comparison was later confirmed by X ray data obtained for representatives of different peroxidase classes. The first class of plant peroxidases includes intracel lular enzymes such as ascorbate peroxidase, yeast

PLANT PHYSIOLOGY, 1984

Two peroxidases, one anionic and one cationic, have been purified from the proteins secreted by peanut (Arachis hypogaea L. var Virginia 56R) cells in suspension culture. These two peroxidases apparently have identical catalytic properties. www.plant.org on May 17, 2016 -Published by www.plantphysiol.org Downloaded from