Partial Purification and Characterisation of Polyphenol Oxidase from Hibiscus rosa–sinensis L Ikram Madani Faculty of Science University of Khartoum Khartoum, Sudan Ikramahmed3@yahoo.com Pat M. Lee Materials Technology Program Universiti Teknologi MARA Shah Alam, Selangor, Malaysia patmlee.1@gmail.com Lee Kong Hung Department of Bitechnology, Malaysia University of Science and Technology Petaling Jaya, Selangor, Malaysia. konghunglee@hotmail.com Abstract— Polyphenol oxidase (PPO) from petals of Hibiscus rosa–sinensis L. (Chinese Rosa) was extracted and partially purified by ammonium sulphate precipitation. Its physical and chemical properties such as optimal pH and temperature, substrate specificity, and effects of inhibitors and activators on the enzymatic activity were investigated. The total protein contents of Hibiscus rosa–sinensis petals crude extract and partially purified extract were found to be 425 μg and 122 μg /100 g fresh petals respectively. The enzyme activity and its kinetic parameters, (K m and V max ) were determined using 4- methylcatechol and catechol as substrates. The optimum pH and temperature for Hibiscus PPO activities were 6.0 and 45 °C respectively. Ascorbic acid and ethylenediaminetetraacetic acid sodium salt were found to be potent inhibitors and copper sulphate was found to be activator for Hibiscus PPO. catechol, 4-methyl catechol , K m and V max I. INTRODUCTION Polyphenol oxidase (PPO, EC 1.14.18.1) is a common copper containing enzyme which is widely distributed in nature. In the presence of oxygen it catalyses two reactions: the hydroxylation of monophenols to o–diphenols and the oxidation of o–diphenols to o–quinones. This enzyme plays an important role in many plant metabolic processes. The oxidation of phenol compounds by PPO are polymerized to form dark–coloured compounds responsible for browning in fruits and vegetables. This is regarded as an undesirable sensory attribute of the produce and decreases its commercial and nutritive values. For this reason PPO has received increasing attention [1]. PPO enzyme has been isolated and characterized from produce of many commercially important plants and fruits such as lettuce [2], aubergine [3], Ferula sp. [4], strawberry [5], and cocoa beans [6]. The present investigation was designed to extract PPO from petals of Hibiscus rosa–sinensis (Chinese Rose) which is the national flower of Malaysia. The enzyme was partially purified from the aqueous extract of flower petals, and its substrate specificity, kinetic parameters, optimum conditions of pH and temperature, and effects of inhibitor and metallic compounds on the enzymatic activity were evaluated. The results of this study would provide an understanding of the browning of the hibiscus flower and means of prolonging the shelf flower. II. MATERIALS AND METHODS All chemicals used were of analytical grade. Polyvinylpyrrolidone from Acros Organics; ascorbic acid and copper sulphate from Fisher Scientific; monosodium phosphate from Fluka Biochemical; disodium phosphate and iron III chloride from Sigma Chemical Co; 4-methylcatechol and catechol from Merk–Schuchardt CHG; EDTA from Progma. A. Plant Materials Two hundred grams of fresh red flowers of Hibiscus rosa–sinensis, grown in Kuala Lumpur, Malaysia, were hand-picked from the plant and left to dry at room temperature. B. Enzyme Extraction and Partial Purification Ten grams of dried petals were homogenized in 80 mL of 0.1M phosphate buffer (pH 6.8) containing 10 mM ascorbic acid and 0.5% polyvinylpyrrolidone (PVP40) with the aid of a magnetic stirrer for 1h. The crude extract samples were centrifuged at 32000 g for 20 min at 4ºC. Solid ammonium sulphate (NH 4 ) 2 SO 4 was added to the supernatant to obtain 80% (NH 4 ) 2 SO 4 saturation. After 1 h, the precipitated proteins were separated by centrifugation at 32000 g for 30 min. The precipitate was re–dissolved in a small volume of distilled water and dialyzed at 4ºC against distilled water for 24 h with 4 changes of the water during dialysis. The dialyzed sample was lyophilized and this constitutes the partial purified PPO extract and was used as the Hibiscus PPO enzyme source. C. Determination of Hibiscus PPO Activity Hibiscus–PPO activity was determined by measuring the absorbance at 420 nm using a spectrophotometer (Pharmatech, Model UV–1700). To determine the best concentration of enzyme preparation corresponding to the highest enzyme activity, the activity was assayed in 3 mL of reaction mixture consisting of 0.5 mL substrate (0.02 M 4– methylcatechol and 0.02 M catechol separately) and different concentrations (0.025-0.5 mL) of the enzyme preparation (1mg/mL). This mixture was topped-up to 3.0 mL with the phosphate buffer (pH 6.8) in a 1 cm light path quartz cuvette. The blank consisted of 3.0 mL 0.1 M phosphate buffer (pH 6.8). Two controls were prepared: the cuvette of the first 110 Keywords Polyphenol oxidase, Hibiscus rosa–sinensis; - 2011 2nd International Conference on Biotechnology and Food Science IPCBEE vol.7 (2011) © (2011) IACSIT Press, Singapore