6 Cellular and Molecular Biology Original Research Puriication and characterization of polyphenol oxidase from corn tassel R. Gul Guven¹ * , N. Aslan¹, K. Guven², F. Matpan Bekler 3 , O. Acer² 1 Science Teaching Section, Education Faculty, Dicle University, Diyarbakir, Turkey 2 Molecular Biology and Genetics Departement, Science Faculty, Dicle University, Diyarbakir, Turkey 3 Biology Departement, Science Faculty, Dicle University, Diyarbakir, Turkey Abstract: In this study, polyphenol oxidase (PPO) from corn tassel was extracted and partially puriied through (NH 4 ) 2 SO 4 precipitation and gel iltration chromatography. Optimal temperatures for subsrates catechol and 4-methyl catechol were 40 °C and 30 °C, respectively. The optimal pH values were 8.0 for catechol and 6.0 for 4-methyl catechol. Catechol was the most suitible substrate (Km: 3.48 mM, Vmax: 1.0 Abs./ min.). The moleculer mass of PPO was determined as 158 kDa. In this work, sodium azide, ethylenediaminetetraacetic acid (EDTA) and sodium dode- cyl sulfate (SDS) were found to inhibit the enzyme activity as 26.6 %, 22.2 % and 12.2 % ratio, respectively. Besides, the effects of carbohydrates such as sucrose, fructose, ribose and glucose on PPO activity were investigated. The enzyme was found to be activated 17 % by fructose and ribose, 16 % by glucose and 4 % by sucrose. Key words: Corn tassel , polyphenol oxidase,characterisation, substrate speciity, inhibition. Introduction Polyphenol oxidases (PPO), belong to a set of cop- per containing metalloenzymes that are members of oxidoreductases that catalyze the oxidation of a wide range of phenolic compounds by utilizing molecular oxygen (1). Polyphenol oxidase is widely distributed among higher plants, which is an enzyme responsible for hydroxylation of monophenols to form o-diphenols and their further oxidation to colored and highly reac- tive o-quinones. PPO is well known to be the principal enzyme involved in enzymatic browning. Particularly, when a plant gets a bruise, cut or damage, the enzyme leads to the oxidation of typical phenolic compounds to form a polymer structure, resulting in protection of the plant against microorganisms or insects (2). This pro- cess causes enzymatic browning of fruits and vegetables which is undesirable in food technology as it results in loss of quality (3, 4, 5). Corn (Zea mays L.) is one of the most widely planted crops over the world and the world production was es- timated to be 471 million tons (6). Corn tassels are a waste part of the plant, which can be processed to pro- duce valuable products such as volatile oils (7), the la- vonol glycosides of kaempferol, isorhamnetin and quer- cetin (8) and lipids (9). Corn tassel (CT) is mainly seen as waste by corn processing industry. Since it contains a variety of active ingredients, such as lavonoid, alkaloid, allantoin, pentosan, inositol, polysaccharide, vitamin K, vitamin C, several kinds of organic acids, and sterols, CT may well serve as an inexpensive herbal drug (10). Therfore, it is of importance to characterise the enzymes such as PPO leading to loss of quality in corn tassel. PPO has been isolated and puriied from various sources, such as Turkish tea leaf (11), strawberry (12) ,ispir sugar bean (13), persimmon (14), and Jackfruit (15), Ataulfo mango (16) and its properties have been extensively studied. To our best knowledge, there is no data in literature on the puriication and characteriza- tion of PPO from corn tassel. The main objectives of the present study are to characterize PPO puriied from corn tassel, to determine its kinetic and characteristic properties, as well as investigating the effect of some activators and inhibitors on the enzyme activity to elu- cidate the mechanism of its inhibition by selected che- mical compounds. Materials and Methods Materials and reagents The corn tassel was obtained from a local market Diyarbakir City, Turkey and stored in 4°C until used. Catechol was purchased from Merck (Darmstadt, Ger- many). Ammonium sulphate, 4-methylcatechol, Se- phadex G-100 gel iltration resin, polyethylene glycol (PEG), citric acid and all chemicals for electrophoresis studies were purchased from Sigma Chem. Co. All che- micals used in this study were of analytical grade. Preparation and extraction of polyphenoloxidase (PPO) from corn tassel Corn tassel (14.50 grams) obtained from the local market was homogenized in the extraction solution 100 ml of 0.1 M phosphate buffer containing 4 % PEG at pH 6.5 and 10 mM ascorbic acid by using a blender. The crude extract samples were centrifuged at 10.000 g for 20 min at 4 ºC. The homogenate was iltered through double layered ilter paper. Solid ammonium sulphate (NH 4 ) 2 SO 4 was slowly added to the supernatant to get 80 % (NH 4 ) 2 SO 4 saturation under cold conditions. After 1 h, centrifugation was carried out at 15.000 g for 30 min to separate the precipitated proteins. The precipi- Received June 28, 2016; Accepted November 03, 2016; Published November 30, 2016 * Corresponding author: Assoc. Prof. Reyhan Gul Guven, Dicle University, Education Faculty, Science Teaching Section, Diyarbakir, Turkey. Email: rgguven@dicle.edu.tr Copyright: © 2016 by the C.M.B. Association. All rights reserved. Gul Guven et al. Cell. Mol. Biol.2016, 62 (13): 6-11 ISSN: 1165-158X doi: 10.14715/cmb/2016.62.13.2