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
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.
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