Elsevier

Process Biochemistry

Volume 48, Issue 10, October 2013, Pages 1516-1523
Process Biochemistry

Cassava (Manihot esculenta Crantz of cv. KU50) peroxidase and its potential for the detection of some thiol compounds based on the inhibitory effect of 3,3′,5,5′-tetramethylbenzidine oxidation

https://doi.org/10.1016/j.procbio.2013.07.014Get rights and content

Highlights

  • Peroxidase was first isolated from cassava leave, an agricultural waste in Thailand.

  • CSP had a potential for the detection of thiosemicarbazide, thiourea and thiophanate methyl.

  • This can be an advantage to the applicable use of the CSP in chemical analysis.

Abstract

Cassava peroxidase (CSP) isolated from cassava leaves (Manihot esculenta Crantz of cv. KU50) was purified by DEAE and concanavalin-A column chromatography. CSP was a haem-containing cationic glycoprotein with the molecular weight of 38 and 44 kDa determined by MALDI-TOF-MS. Its kinetic catalysis in the presence and absence of some thiols were investigated and compared to those of horseradish and soybean peroxidases by using urea hydrogen peroxide (UHP) and 3,3′,5,5′-tetramethylbenzidine (TMB) as substrates. Inhibitory effects of some pesticides containing thiol groups such as thiosemicabazide, thiourea and thiophanate-methyl on TMB oxidation were graphically demonstrated their ability to be either competitive or noncompetitive inhibitors, depending on their properties. The degrees of inhibition were expressed as Ki and IC50 values. The applicable range for the detection of thiosemicabazide in solution was found to be in the range of 10–100 μM, whereas those of thiourea and thiophanate-methyl were 40–400 μM and 10–460 μM, respectively. On the contrary, the inhibitory effects of some phenolic pollutants; 4-nitrophenol, 4-phenylphenol and pentachlorophenol on TMB oxidation were not significantly observed and the phenomenon was discussed.

Introduction

Peroxidases are classified as oxidoreductases (EC 1.11.1.7) which belong to a large family of enzymes that catalyze the oxidation of various organic and inorganic substances by hydrogen peroxide or related compounds. They are commonly found in a variety of isomers in plants and play an important role in maintenance of reduced state molecules in cell and in defense mechanism [1], [2]. These isoenzymes were differentially expressed in various tissues and organs depending on environmental conditions [3], [4]. Several oxidoreductases have been explored for both analytical and industrial applications. However, the well-known classical plant peroxidase is horseradish peroxidase (HRP) due to its relative high activity and stability with a wide range of substrates available. It has been found to be well suited in many analytical applications such as immunohistology and enzyme amplified immunoassay system for the quantitative determination of trace substances based on ELISA (enzyme-linked immunosorbent assay) as a valuable tool in biotechnology [5]. In electrochemical analysis, HRP is the most widely used enzyme for the construction of biosensors for the determination of hydrogen peroxide [6], [7] as well as the detection of peroxidase inhibitors such as sulfides [8] and thiols [9]. In addition, the HRP catalyzed reaction also has a potential application in the removal of phenolic pollutants from waste water [10], [11]. However, the main drawback for HRP application is its low stability with respect to hydrogen peroxide and acid concentration [12]. Therefore, alternative plant peroxidases with higher stability and/or different specificity have been investigated to improve their utilization in analytical kits and in the construction of biosensors. Peroxidase from various plant sources such as soybean peroxidase (SBP) from seed coat is commercially available and has been used as an alternative source of available peroxidase [13]. Furthermore peroxidase activities were also screened in a wild endemic hemi-parasitic plant (Viscum angulatum) [14], artichoke leaves [15] and tree legume stem [16].

Cassava (Manihot esculenta Crantz) is an economic crop of Thailand which is the major supplier of cassava for feed, food, and industrial uses to the world market [17]. There have been some reports describing that peroxidase and ATPase have a role in deterioration of cassava tuber during postharvest [18]. Peroxidases could be found in many parts of cassava such as root, stem, petiole and leaf which were considered as agricultural waste after cassava root harvest. Therefore, our work focused on the extraction and purification of peroxidase from cassava leaves and the influence of some thiol pesticides and phenolic pollutants toward its catalytic activity was investigated and compared to those of HRP and SBP. The possible use of cassava peroxidase (CSP) in analytical application for the detection of some thiol pesticides and phenolic pollutants based on the inhibition of 3,3′,5,5′-tetramethylbenzidine oxidation was also explored.

Section snippets

Materials

Cassava leaves of cultivar KU50 were collected from Rayong field crops research center at Rayong province, Thailand. Horseradish peroxidase (Type II) and soybean peroxidase were commercial peroxidases purchased from Sigma (U.S.A.). Concanavalin-A, 3,3′-diaminebenzidine (DAB), DEAE-cellulose, sepharose 4B, 3,3′,5,5′-tetramethylbenzidine (TMB) and urea hydrogen peroxide (UHP) were from Sigma (U.S.A.) and ammonium sulfate was from Fluka (Buchs, Switzerland). Protein molecular weight and pI markers

Preparation and characteristics of CSP

Peroxidase was successfully extracted from cassava leaves as crude with approximately 190 units/g of leaf and the purification table of CSP is given in Table 1. After ammonium sulfate fractionation and DEAE-cellulose column chromatography, the unbound portion with peroxidase activity was observed as a major protein with the specific activity of 1200 units/mg protein. It was further purified by Concanavalin-A affinity column which specifically binds molecules containing α-d-mannopyranosyl and α-d

Conclusions

Peroxidase from cassava leaves, an agricultural waste in Thailand, could be an alternative source of cationic haem peroxidase. Its catalytic ability for urea hydrogen peroxide and TMB oxidation was in the pH range of 5–8. CSP-catalyzed reaction under experimentally designed condition had a potential for the detection of thiosemicarbazide, thiourea and thiophanate methyl as its reversible inhibitors. The catalytic activity of CSP was particularly influenced by some thiol pesticides and phenolic

Acknowledgments

The authors would like to thank Dr. Jaran Jainhuknan for the technical assistance of the molecular weight determination by MADI-TOF and gratefully acknowledge the laboratory facilities and financial support provided by Department of Biochemistry, Program in Biotechnology, Faculty of Science and Graduate School of Chulalongkorn University.

References (43)

  • J. Cheng et al.

    Horseradish peroxidase immobilized on aluminum pillared interlayered clay for the catalytic oxidation of phenolic wastewater

    Water Research

    (2006)
  • J.K.A. Kamal1 et al.

    Kinetic stabilities of soybean and horseradish peroxidases

    Biochemical Engineering Journal

    (2008)
  • M.K. Das et al.

    A novel cationic peroxidase (VanPrx) from a hemi-parasitic plant (Viscum angulatum) of Western Ghats (India): purification, characterization and kinetic properties

    Journal of Molecular Catalysis B: Enzymatic

    (2011)
  • A. Cardinali et al.

    Purification, biochemical characterization and cloning of a new cationic peroxidase isoenzyme from artichoke

    Plant Physiology and Biochemistry

    (2011)
  • V.P. Pandey et al.

    Purification and characterization of peroxidase from Leucaena leucocephala, a tree legume

    Journal of Molecular Catalysis B: Enzymatic

    (2011)
  • C.F. Fernandes et al.

    Induction of an anionic peroxidase in cowpea leaves by exogenous salicylic acid

    Journal of Plant Physiology

    (2006)
  • M.M. Bradford

    A rapid and sensitive method for the quantitation of micrograms quantities for proteins utilizing the principle of protein-dye binding

    Analytical Biochemistry

    (1976)
  • M.V. Miranda et al.

    Study of variables involved in horseradish and soybean peroxidase purification by affinity chromatography on concanavalin-A-agrarose

    Process Biochemistry

    (2002)
  • M.K. Das et al.

    A novel cationic peroxidase (VanPrx) from a hemi-parasitic plant (Viscum angulatum) of Western Ghats (India): purification, characterization and kinetic properties

    Journal of Molecular Catalysis B: Enzymatic

    (2011)
  • K.G. Welinder et al.

    Covalent structure of soybean seed coat peroxidase

    Biochimica et Biophysica Acta

    (2004)
  • Y. Misono et al.

    Resonance Raman and adsorption spectroscopic studies on the electronchemical oxidation processes of 3,3′,5,5′-tetramethylbenzidine

    Journal of Electroanalytical Chemistry

    (1997)
  • Cited by (5)

    • A peroxidase purified from cowpea roots possesses high thermal stability and displays antifungal activity against Colletotrichum gloeosporioides and Fusarium oxysporum

      2022, Biocatalysis and Agricultural Biotechnology
      Citation Excerpt :

      Vu-RPOX presented optimal enzyme activity at pH 6.0 (Fig. 4a). Many other plant peroxidases have optimal pH peroxidase activity at pHs varying from 4 to 7 (Pandey et al., 2017), at slight acidic conditions such as the leaf peroxidase from Vigna unguiculata (pH 6.0) (Fernandes et al., 2006), cassava and soybean peroxidase (SBP) (pH 6.0) (Jongmevasna et al., 2013), Armoracia rusticana (HPR) horseradish peroxidase (pH 5.5), Vigna mungo peroxidase (pH 5.5) (Ajila, and Prasada, 2009), leaf peroxidases from rosemary (Rosmarinus officinalis L.) (Aghelan and Shariat, 2015), Citrus medica (Mall et al., 2013), and Eruca vesicaria sbsp. Sativa (Nadaroglu et al., 2013), and Beta vulgaris peroxidase (pH 5.0) (Rudrappa et al., 2007).

    View full text