PARTIAL PURIFICATION AND PRELIMINARY CHARACTERIZATION OF PEROXIDASE FROM TELFAIRA OCCIDENTALIS USING SODIUM MALONATE BUFFER

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ABSTRACT
This study had set out to carry out the extraction, partial purification and preliminary characterization of peroxidases from Telfairia occidentalis. Peroxidases (POD) were extracted from the leaves of Telfairia occidentalis, using sodium malonate buffer pH 7.0. During the preliminary characterization of Telfairia occidentalis peroxidases, it was shown that the optimum per cent relative activity was evident at the temperature of 40oC, wavelength of 350nm and pH 4.5. The 40oC optimum temperature shows that peroxidase from Telfairia occidentalis is moderately thermostable.

CHAPTER ONE
INTRODUCTION

1.1 Background of Study
Peroxidase (POD) is a heme protein, which is a member of oxidoreductases [E.C.1.11.1.7] and catalyses the oxidation of a wide variety of organic and inorganic substrates using hydrogen peroxide as the electron acceptor (Banci, 1997; Yemenicioğlu et al., 1998). Peroxidase, a biotechnologically important enzyme is a ubiquitous enzyme which belongs to the oxidoreductase class of enzyme and generally catalyzes a reaction between hydrogen peroxide as electron acceptor and many kinds of substrates by means of oxygen liberation (Castillo Ferapontova, Hushpulian, Tasca & Tishkov, 2006). It is a heat stable, intracellular enzyme widely distributed in nature and is found in plant kingdom, micro-organisms and animals where it catalyze the reduction of hydrogen peroxide to water, rendering it harmless (Choi, Lim, Kim, Min & Lee, 2001). Plants are the rich sources of peroxidase and primarily found in roots and sprouts of higher plants.
The Plant Peroxidase (POD) superfamily comprises heme-containing glycoproteins that differ in their structure and catalytic properties. The enzyme is present naturally in plants like potato tuber, horse radish, beet, soybean, tomato, banana, papaya, carrot, turnip, wheat, dates, beats and strawberry. Peroxidases are widely distributed in living organisms including microorganisms, plants and animals. POD is mainly located in the cell wall (Chen et al., 2002) and it is one of the key enzymes controlling plant growth and development. It takes place in various cellular processes including construction, rigidification and eventual lignifications of cell walls (Quiroga et al., 2000), suberization (Bernards et al., 1999), auxin oxidation (Gazaryan and Lagrimini, 1996), senescence (Santos et al., 2001), organogenesis (Lee et al., 2002) phenol oxidation (Lagrimini, 1991), cross-linking of cell wall proteins (Schnabelrauch et al., 1996), salt stress tolerance (Hiraga et al., 2001) and protection of tissue from damage and infection by pathogenic microorganisms (Sakharov et al., 2000; Gülçin and Yildirim, 2005; Sat, 2008). POD and catalase are two major systems for the enzymatic removal of H2O2 and peroxidative damage of cell walls is controlled by the potency of antioxidative peroxidase enzyme system (Velikova et al., 2000). However, the role that peroxidase plays in metabolism is not clear because of the large number of reactions it catalyzes and the considerable number of isoenzymic species (Kim and Lee, 2005). Therefore, POD is also widely used for clinical diagnosis and microanalytical immunoassays because of its high sensitivity. New applications for POD have been suggested in the medicinal, chemical and food industries (Kwak et al., 1996). Other applications include synthesis of various aromatic compounds and removal of peroxide from foodstuff and industrial wastes (Aruna and Lali, 2001). Concerning the physiological role of peroxidases, it has been shown that the enzyme participates in the formation of lignins in the secondary cell walls during normal growth (Pedreno et al., 1995) and in the formation of phenolic polymers such as lignins, suberins, etc. when plants are infected or wounded (Köksal and Gülçin, 2008). It was reported that peroxidase had been used for biotransformation of organic molecules (Gülçin and Yildirim, 2005). Because of its broader catalytic activity, a wide range of chemicals can be modified using POD. Also, it can be used for the applications such as synthesis of various aromatic compounds, removal of phenolics from waste waters and the removal of peroxides from foodstuffs, beverages and industrial wastes (Torres et al., 1997). POD is also related to quality of plant commodities, particularly the flavor, in both raw and processed foods. POD activity is also correlated to fruit ripening as shown in a number of cases and it is also involved in enzymatic browning, either or together with polyphenol oxidase activity. A more precise understanding of the implication of POD in these mechanisms is an essential step towards a more efficient control of these undesirable reactions, particularly in heat-processed products, which frequently contain residual peroxidase activity (Cardinali et al., 2007; Köksal and Gülçin, 2008). Although, peroxidases are widely distributed in the plant kingdom, the major source of commercially available peroxidase is roots of horseradish. On the other hand, availability of peroxidases with different specificity would promote the development of new analytical methods and potential industrial processes (Köksal and Gülçin, 2008). Peroxidase has been isolated and characterized from a large number of plant sources like fruits, leaves, tubers and grains.

1.2 Justification of the study
Multiple peroxidases are widely distributed in plants, microbes, and animal tissues and have been used in a great number of analytical applications such as clinical diagnosis (blood sugar and cholesterol), immunoassays, biosensor construction, food processing and food storage, treatment of waste water containing phenols and aromatic amines, bio-bleaching processes, lignin degradation in fuel, production of dimeric alkaloids, oxidations, and bio- transformation of organic compounds. In involvement in various metabolic processes such as ethylene biogenesis, cell development and membrane integrity, defense mechanism toward pathogens and various abiotic stresses, including metal ions and UV stress, salt, air pollution damage, cold tolerance, control of cell elongation, polymerization of extension, generation of reactive oxygen species, hydrogen peroxide scavenging has been long reported. Since these enzymes can participate in a number of oxidation and biodegradation reactions associated with changes of flavour, color, texture, and the nutritional quality of food. The control of the activity of Peroxidase (POD) and polyphenoloxidase (POP) is of great importance in the processing of fruits, vegetables, and its products. Hence, studying the peroxidases of Telfairia occidentalis could possibly give insights into understanding the various therapeutic uses and other medicinal applications of this plant.

1.3 Aim and Objectives of Study
The aim of this study is the extraction, partial purification and preliminary characterization of from peroxidase Telfairia occidentalis using sodium malonate buffer. The following specific objectives were set out to actualize the main aim which includes;
To extract and partially purify peroxidase enzyme from Telfairia occidentalis
To identify peroxidase in Telfairia occidentalis by determining
Optimum pH
Optimum Temperature
Optimum wavelength
Optimum metal ion (Cu2+, Zn2+, Co2+ and Mn2+) sensitivity

PARTIAL PURIFICATION AND PRELIMINARY CHARACTERIZATION OF PEROXIDASE FROM TELFAIRA OCCIDENTALIS USING SODIUM MALONATE BUFFER