Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.) Urszula Gawlik-Dziki * , Urszula Złotek, Michał S ´ wieca Department of Biochemistry and Food Chemistry, Agricultural University, Skromna 8, 20-704 Lublin, Poland Received 23 April 2007; received in revised form 23 July 2007; accepted 25 July 2007 Abstract Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical char- acteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K M and V max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min 1 for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min 1 for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a tem- perature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione  ascorbic acid > L-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Polyphenol oxidase; Purification; Lettuce; Lactuca sativa var. capitata L.; Characterization; Inhibitors 1. Introduction Polyphenol oxidase (PPO) is a common copper-contain- ing enzyme which is responsible for melanization in ani- mals and browning in plants. The enzyme catalyses two distinct reactions: the o-hydroxylation of monophenols to o-diphenols (acts like cresolase) (E.C. 1.14.18.1.) and the oxidation of o-diphenols to o-quinones (acts like catecho- lase) (E.C. 1.10.3.2.) (Rodrı ´ guez-Lo ´pez et al., 2001; van Gelder, Flurkey, & Wichers, 1997). Quinones are highly reactive electrophilic molecules that can polymerize, lead- ing to the formation of brown or black pigments. The role of PPO in plants is not yet clear. It is suggested that it may be involved in immunity reactions and in biosynthesis of plant components, and it also may play the role of a scav- enger of free radicals in photo-synthesizing tissues (Heim- dal, Larsen, & Poll, 1994). Enzymatic browning of fruit and vegetables after mini- mal processing is one of the most important causes of qual- ity losses, resulting in rejection by consumers. The quality and shelf life of lettuce varies depending on cultivars, matu- rity, processing and storage conditions, although the infor- mation on tissue browning susceptibility is limited (Castaner, Gil, & Ruiz, 1999). Activity of PPO has been studied in apples (Malus sp.) (Espin, Morales, Varon, Tudela, & Garcia-Carnovas, 1995), pears (Pyrus sp.) (Hwang, Yoon, & Kim, 1996) broad beans (Vicia faba L.) (Ganesa, Fox, & Flurkey, 1992), potatoes (Solanum tumerosum L.) (Chen et al., 1992), artichokes (Cynara scol- ymus L.) (Leoni & Palmeri, 1990), lettuce (Lectuca sativa L.) (Heimdal et al., 1994), plums (Prunus sp.) (Siddiq, Sin- ha, & Cash, 1992), banana (Musa cavendishii L.) (Galleazi, Sgarbieri, & Constantinides, 1981), peppermint (Mentha piperita L.) (Kavrayan & Aydemir, 2001), coffee (Coffea arabica L.) (Mazzafera & Robinson, 2000) and seeds of 0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.07.068 * Corresponding author. Tel.: +48 81 4623327; fax: +48 81 4623324. E-mail address: urszula.gawlik@ar.lublin.pl (U. Gawlik-Dziki). www.elsevier.com/locate/foodchem Available online at www.sciencedirect.com Food Chemistry 107 (2008) 129–135 Food Chemistry