Food Chemistry 157 (2014) 283–289

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Partial purification and characterization of polyphenol oxidase from persimmon José L. Navarro ⇑, Amparo Tárrega, Miguel A. Sentandreu, Enrique Sentandreu Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Avda. Agustín Escardino, 7, 46980 Paterna, Valencia, Spain

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Article history: Received 4 November 2013 Received in revised form 11 February 2014 Accepted 15 February 2014 Available online 24 February 2014 Keywords: Persimmons Diospyros kaki Polyphenol oxidase characterization Enzyme purification Polyacrylamide gel electrophoresis Enzyme isoforms

a b s t r a c t Activity of polyphenol oxidase (PPO) from ‘‘Rojo Brillante’’ persimmon (Diospyros kaki L.) fruits was characterized. Crude extracts were used for characterization of enzyme activity and stability at different temperatures (60, 70 and 80 °C), pHs (from 3.5 to 7.5) and substrate concentrations (catechol from 0 to 0.5 M). Maximum enzyme activity was reached at pH 5.5 and 55 °C. Enzyme stability was higher than PPO activities found in other natural sources, since above pH 5.5 the minimum time needed to achieve an enzyme inactivation of 90% was 70 min at 80 °C. However, at pH 4.0 the enzyme stability decreased, reaching inactivation levels above 90% after 10 min even at 60 °C. Thus it was concluded that acidification can circumvent browning problems caused by PPO activity. Moreover, polyacrylamide gel electrophoresis of the enriched extract revealed the presence of at least four bands with strong oxidase activity, suggesting the existence of different PPO isoforms. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Persimmon (Diospyros kaki L.) fruits were traditionally used in ancient China for medical purposes. It has been postulated that the healthy effects of persimmons come from their high content of phenolic compounds, mainly tannins (Gorinstein et al., 1994; Suzuki, Someya, Hu, & Tanokura, 2005), showing strong antioxidant and antiradical capacities (Chen, Fan, Yue, Wu, & Li, 2008; Kondo, Yoshikawa, & Katayama, 2004) that are useful against hypercholesterolemia (Rho et al., 2003), leukaemia (Achiwa, Hibasami, Katsuzaki, Imai, & Komiya, 1997), diabetes (Lee, Cho, Tanaka, & Yokozawa, 2007) or other diseases derived from cellular ageing (Lee, Cho, & Yokozawa, 2008). World persimmon production is led by China, with more than 3,000,000 tonnes, although during recent years it has been extended to other countries such as South Korea, Japan and Brazil, with 390, 189 and 154 thousand tonnes, respectively (FAO, 2010). Moreover, it is a promising crop in some Mediterranean countries such as Spain, Italy or Israel, with productions of 150, 50 and 30 thousand tonnes, respectively (FAO, 2010). In this context, it is particularly noteworthy that Spanish production has increased 11 fold during the last 15 years as a result of the popularity of the ‘‘Rojo Brillante’’ variety, which currently constitutes about 90% of total produced volume (Consejo Regulador de la Denominación de Origen ‘Caqui Ribera del Xuquer’). ⇑ Corresponding author. Tel.: +34 96 3900022; fax: +34 96 3636301. E-mail address: [email protected] (J.L. Navarro). http://dx.doi.org/10.1016/j.foodchem.2014.02.063 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

As in many other fruits, browning is a prominent problem for persimmon fruits and derived products. Polyphenol oxidase (PPO, a copper-containing enzyme) is one of the most common browning agents in nature, affecting fruits and vegetables during handling, storage and processing, as demonstrated previously in the cases of potato tubers (Marri, Frazzoli, Hochkoeppler, & Poggi, 2003), lettuce (Gawlik-Dziki, Złotek, & S´wieca, 2008) and green beans (Guo, Ma, Shi, & Xue, 2009). Very surprisingly, literature about purification and characterization of PPOs from persimmons is scarce and rather contradictory. Thus, Núñez-Delicado, Sojo, García-Carmona, and Sánchez-Ferrer (2003) stated that maximum PPO activity depended on adding sodium dodecyl sulphate (SDS) to the media, finding an optimum pH value below 3.5 (without SDS) or 5.5 (using SDS) when studying the Triumph variety. On the other hand, Özen, Colak, Dincer, and Güner (2004) found maximum PPO activity at pH 7.5 in persimmons cultivated in the region of Trabzon, (Turkey), Zhou, Wan, and Shen (2005) found a maximum at pH 6.6 in Luotian sweet persimmon, and Sung and Cho (1992) cited a maximum PPO activity at pH 7.0 in Korean persimmons. Similarly, reports about the influence of temperature on PPO activity of persimmons show different results, with Özen et al. (2004) and Zhou et al. (2005) reporting maximum PPO activity at 20 °C whereas Sung and Cho (1992) found a maximum at 50 °C. It seems evident that the activity features of PPO in persimmons vary greatly among varieties. To our knowledge, no data are currently available concerning the characterization of PPO activity from fruits of the ‘‘Rojo Brillante’’ cultivar.

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Furthermore, gelation is an added problem when persimmon fruits are processed to obtain purees as a basis for derived products. In a recent study, our group (Carbonell, Navarro, Sentandreu, & Sendra, 2011) patented a method for obtaining a non-gelified ‘‘Rojo Brillante’’ persimmon puree, mainly based on incubation of the puree with broad-spectrum carbohydrases. In this regard, Tárrega, Gurrea, Navarro, and Carbonell (2013) stated that gelation can be prevented by an enzymatic treatment with Viscozyme L at 25 °C for 30 min, although incubation produces browning as a collateral effect. This work aims to develop a method that allows purification and characterization of PPO from persimmon fruits belonging to the ‘‘Rojo Brillante’’ variety. 2. Materials and methods 2.1. Plant material and chemicals Persimmon (Diospyros kaki, cv. Rojo Brillante) fruits belonging to the ‘‘Kaki Ribera del Xúquer’’ denomination of origin were supplied by the ‘‘San Bernardo’’ cooperative (Carlet, Valencia, Spain). The fruits were treated with CO2 to remove astringency. Citric acid 1-hydrate, sodium di-hydrogen phosphate 1-hydrate, di-sodium hydrogen phosphate 12-hydrate, sodium acetate anhydrous and acetic acid were from Panreac (Panreac Química, S.A., Barcelona, Spain). Polyvinylpolypyrrolidone (PVPP), Triton X-100, catechol, Tris–HCl, glycerol bromophenol blue, ammonium sulphate, sodium dodecyl sulphate (SDS), dithiothreitol (DTT) and silver nitrate were from Sigma (Sigma–Aldrich Co., St. Louis, MO, USA). Polyacrylamide (Protogel) was from National Diagnostics (Atlanta, USA) The Bio-Rad protein assay was used for protein concentration, following the manufacturer’s instructions (cat. No. 5000006; Bio-Rad, Hercules, CA). 2.2. Preparation of the crude polyphenoloxidase (PPO) extract PPO crude extracts were prepared following the methodology proposed by Palma-Orozco, Sampedro, Ortiz-Moreno, and Nájera (2012), with some modifications. Briefly, fresh persimmon fruits were washed in tap water, their calyces were removed and the fruits were finely crushed using a domestic blender to obtain the puree for analyses. Fifteen grams of puree (with an original pH of 5.6) were extracted with 15 mL of 0.2 M phosphate buffer pH 7.0 containing 2 g/L of PVPP and 2 mL/L of Triton X-100. The mixture was homogenized (IKA DI 25 yellow line, IKA-Werke GmbH, Staufen, Germany) at 24,000 rpm for 1 min. The homogenate was incubated at 4 °C for 20 min and then centrifuged (at 4 °C in an Eppendorf 5804R centrifuge, Eppendorf Ibérica, Madrid, Spain) at 4500g for 25 min. Finally, the supernatant was filtered through Whatman No. 1 filter paper and then collected, constituting the crude enzyme extract. 2.3. Enzymatic assay and characterization of PPO from persimmon crude extracts 2.3.1. Enzyme activity Enzyme activity was determined as described by Sßener, Ümit Ünal, and Aksay (2011) with minimal modifications. Changes in absorbance were measured at 420 nm at room temperature (25 °C) using an Amersham Ultrospec 3300 Pro spectrophotometer (Amersham Pharmacia Biotech, Cambridge, UK). Reaction was initiated by adding (standard mixing conditions) 0.1 mL of the crude PPO extract to a cuvette containing 0.9 mL of 0.2 M catechol in 0.2 M sodium acetate buffer pH 5.5 (value at which maximum PPO activity is reached, see below). The blank contained 0.9 mL

of the substrate solution and 0.1 mL of sodium acetate buffer. PPO activity was determined from the slope of the initial linear part of experimental curves (absorbance vs. time) and expressed as absorbance increase per minute in 1 mL of the reaction mixture. All assays were performed in triplicate. To determine Michaelis–Menten parameters (Vmax and Km), PPO activity was calculated under the standard mixing conditions but using catechol solutions with concentrations ranging from 0 to 0.25 M. 2.3.2. Effect of pH on PPO activity PPO activity was determined in a pH range of 3.5–7.5 using 0.2 M acetate buffer for analyses between pH 3.5 and 5.5 whereas 0.2 M phosphate buffer was used in the range between pH 6.0 and 7.5. Assays were performed in triplicate using as substrate 0.2 M catechol solutions buffered at each respective analysis pH, keeping the aforementioned standard proportions. 2.3.3. Effect of temperature on PPO activity and stability PPO activity was determined at different reaction temperatures in the range of 15–65 °C using an Agilent 8453E UV–Vis spectrometer (Agilent Technologies GmbH, Karlsruhe, Germany) with a wavelength set at 420 nm and loading a thermostated cell holder with magnetic stirring. Analyses were performed in triplicate under the standard mixing conditions. On the other hand, thermal stability of PPO was determined by analysing in parallel heated and unheated buffered (at pH 4.0, 5.5 and 7.0) enzyme solutions for a period up to 75 min (unheated samples were stored at room temperature, 25 °C). As mentioned above, crude PPO extract was originally buffered at pH 7.0, then aliquots were separately adjusted to pH 5.5 and 4.0 with acetic acid. One millilitre of each buffered enzyme solution was poured into Eppendorf tubes and independently incubated in a water bath at 60, 70, and 80 °C. Every 10 min, heated aliquots (200 lL) were tempered until they reached room temperature, and PPO activity was measured using 0.2 M catechol solutions buffered at each respective pH. Determinations were performed in triplicate for every condition assayed (pH–time–temperature) and results were expressed as the relative percentage of PPO activity of heated crude enzyme solutions in relation to the activity from their respective unheated counterparts. 2.4. Purification of the PPO extract PPO was partially purified in two steps: the first one consisted in removing non-PPO proteins by an optimized heat treatment and the second step was based on (NH4)2SO4 precipitation using the procedure described by Palma-Orozco, Ortíz-Moreno, Dorantes-Alvarez, Sampedro, and Nájera (2011). The first stage was designed using the results obtained in the assays of thermal stability of persimmon PPO (see Fig. 3); the crude extract was heated at 60 °C for 1 h and then centrifuged (48,000g, 20 min) and filtered through Whatman No. 1 filter paper. In the second step the filtrate was subjected to selective (NH4)2SO4 precipitation (30–85% saturation) at 4 °C followed by centrifugation at 48,000g, 20 min. The precipitate was resuspended in a minimum volume of buffer (0.2 M sodium phosphate pH 7.0) and dialyzed overnight against three changes of the same buffer. The dialyzed solution was finally centrifuged at 48,000g, 20 min, and the supernatant was collected to obtain the PPO enriched extract (EE). 2.5. Protein concentration Protein concentration of the extracts obtained after each step of the PPO fractionation procedure was determined by the method of

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10% w/v SDS, 0.2 M DTT and 0.05% bromophenol blue, followed by heating at 95 °C for 4 min. The Precision Plus™ Protein Standards kit (Bio-Rad, Hercules, CA; cat. No. 161-0374) was used for estimating the molecular weight of the bands obtained. 3. Results and discussion 3.1. Effect of pH on the activity of the PPO crude extract

Fig. 1. Effect of pH on PPO activity of a crude extract of ‘‘Rojo Brillante’’ persimmon (temperature, 25 °C; catechol concentration, 20 mM).

Fig. 1 shows how, at room temperature (25 °C), PPO activity was greatly affected by pH. Very clearly, at pH values below 4 and at 7.5 PPO activity was rather low, while the maximum was found at a pH close to 5.5. A similar result was obtained by Núñez-Delicado et al. (2003) when they studied PPO activity in ‘‘Triumph’’ persimmon (but with the addition of 1 mM of SDS to the reaction mixture, since without this chemical maximum activity was reached at a pH below 3.5). However, studies performed in other persimmon varieties found maximum PPO activities at pH values higher than 5.5. For example, persimmon from Trabzon region in Turkey showed an optimum at pH 7.5 (Özen et al., 2004), for an unspecified Korean persimmon the optimum pH was 7.0 (Sung & Cho, 1992), while the value found for Luotian sweet persimmon (Zhou et al., 2005) and an unspecified Chinese persimmon (Zhan, Chen, Liu, & Zhang, 2007) was pH 6.6. These discrepancies seem to be quite normal in view of previous results regarding PPO activity determined in other plants. The maximum was found at pH 7.0 in banana root (Wuyts, De Waele, & Swennen, 2006), at pH 6.5 in banana pulp (Yang, Fujita, Ashrafuzzaman, Nakamura, & Hayashi, 2000), at pH 7.0–8.5 in apricot (Arslan, Temur, & Tozlu, 1998), at pH 5.5 in lettuce (Gawlik-Dziki et al., 2008) and at pH 5.6–6.5 in atemoya fruit (Rodrigues Chaves, de Souza, Aparecida, & Augusto, 2011). 3.2. Effect of temperature on activity and stability of the crude PPO extract

Fig. 2. Effect of temperature on PPO activity of a crude extract of ‘‘Rojo Brillante’’ persimmon (pH 5.5; catechol concentration, 20 mM).

Bradford (1976), using the Bio-Rad protein assay and bovine serum albumin as standard. 2.6. Polyacrylamide gel electrophoresis Non-denaturing electrophoresis (native PAGE) was carried out on preparative 10% polyacrylamide gels according to the method of Laemli (1970) in a Hoefer Mighty Small SE260 slab cell unit (San Francisco, CA). One hundred microlitres of each sample was mixed in equal volume with native sample buffer solution (0.5 M Tris pH 6.8, 50% v/v glycerol and 0.05% bromophenol blue). Thirty microlitres was loaded per sample lane in the gel used for determining the PPO enzyme activity, using 100 mM of catechol as substrate. For the gel revealed with silver nitrate, 15 lL was loaded for crude and heated samples, and 5 lL in the case of the enriched extract. Sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing and denaturing conditions (SDS–PAGE) was carried out in the same way as described for native PAGE but with different sample preparation. In this case, one hundred microlitres of each sample was mixed in equal volume with denaturing sample buffer solution containing 0.5 M Tris pH 6.8, 50% v/v glycerol,

Fig. 2 shows the influence of temperature on PPO activity determined in ‘‘Rojo Brillante’’ extracts under the conditions assayed (pH 5.5 and 20 mM catechol as substrate). As observed, maximum activity was reached at about 55 °C and, again, this result differed from those reported in the literature for other persimmon varieties. Thus, Özen et al. (2004) found 20 °C as the optimal temperature in persimmon from the Trazbon region in Turkey (which also showed an optimum pH of 7.5). It is worth mentioning that optimum temperatures for PPOs from other natural sources are generally lower than 55 °C. So, PPO from grape (Cash, Sistrunk, & Stutte, 1976), mamey fruit (Palma-Orozco et al., 2011), atemoya fruit (Rodrigues Chaves et al., 2011), goldnugget loquat (Sßener et al., 2011), hot pepper (Arnnok, Ruangviriyachai, Mahachai, Techawongstien, & Chanthai, 2010), butter lettuce (Gawlik-Dziki et al., 2008) and peach (Jen & Kahler, 1974) showed optimum temperatures at 25, 35–45, 28, 30, 30, 35, and 37 °C, respectively. Fig. 3 shows the relative enzymatic activity of crude PPO extracts at different pH values when heated at 60, 70 and 80 °C for a period of up to 75 min. The experimental data followed an exponential decay curve with marked temperature dependence. Very clearly, PPO activity at 60 °C was quite stable for a long period (for at least 75 min) at either pH 7.0 or pH 5.5, but the activity was sharply reduced at pH 4 even at the lowest temperature assayed (having a residual activity of about 10% after only 5 min of reaction). When the incubation temperature was increased to 70 °C, the relative PPO activities at pH 7.0 and 5.5 were respectively around 60% and 30% at the end of the time assayed, but at pH 4.0 the residual enzymatic activity was less than 10% after only 25 min. As expected, under the strongest thermal conditions

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Fig. 3. Thermal stability of crude PPO extract from the ‘‘Rojo Brillante’’ persimmon variety using 20 mM catechol as substrate at different pHs (results are expressed as the relative activity of heated PPO extracts regarding their respective unheated counterparts).

(80 °C) PPO activities were drastically affected, reaching, after 25 min of incubation, residual values of 50%, 20% and 0% for crude extracts treated at pH 7.0, 5.5 and 4.0, respectively. An explanation for the loss of enzymatic activity caused by heat can be found in the partial denaturation suffered by PPO, turning from its tertiary (folded) structure to a secondary (unfolded) structure, which leads to its complete inactivation (Dincer, Colak, Aydin, Kadioglu, & Güner, 2002). In any case, the thermal stability of crude PPO extract from the ‘‘Rojo Brillante’’ persimmon variety was quite high at either pH 7.0 or 5.5, in agreement with what was stated by Özen et al. (2004) when studying Turkish persimmon, whereas PPOs from other plant sources were reported to be much more thermosensitive (Dincer et al., 2002; Duangmal & Owusu Apenten, 1999). Similarly, the results clearly indicate that a slight acidification of persimmon

Table 1 Characteristics of PPO extracts.a Purification step

Total proteinb

PPO activity (units)c

Specific activity (units/ mg protein)

Crude extract Heat treatment (NH4)SO4 precipitation

0.221 ± 0.012 0.121 ± 0.008 0.018 ± 0.002

0.45 ± 0.04 0.38 ± 0.04 0.29 ± 0.02

2.04 3.14 16.11

a

Values obtained in triplicate. mg From 1 mL of crude extract. PPO activity units expressed as: DAbs420/min, with reference to 1 mL of crude extract. b c

puree, around pH 4.0, may strongly facilitate thermal inactivation of the enzyme.

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remained. The specific activity had increased about 8 times by the end of the purification procedure with respect to the original activity of the crude extract. 3.4. Effect of substrate concentration on the activity of the PPO enriched extract

Fig. 4. Michaelis–Menten curve corresponding to the enriched PPO persimmon extract at pH 5.5 and 25 °C.

Fig. 4 shows the variation in PPO activity determined in ‘‘Rojo Brillante’’ enriched extracts as a function of substrate (catechol) concentration. From the fitting to the Michaelis–Menten function, a Km constant value of 0.025 ± 0.005 M was obtained. These results cannot be accurately compared with those reported by other authors since, as mentioned above, persimmon PPO activity depends greatly on variety. In any case, this Km value is not far from those obtained (using catechol or methyl catechol as substrate) by Özen et al. (2004) in Turkish persimmons at pH 7.5 (Km = 0.012 M) and by Zhou et al. (2005) in Luotian sweet persimmon at pH 6.6 (Km = 0.01 M). 3.5. Polyacrylamide gel electrophoresis

3.3. Partial enzyme purification Table 1 shows the protein concentration and the PPO activity of crude and enriched extracts. As observed, in the first step of purification 45% of the protein was removed but the PPO activity only decreased by 15%. By the end of the second step more than 90% of the protein had been removed but 65% of PPO activity still

Non-denaturing polyacrylamide gel electrophoresis (native PAGE) was carried out for each step of the fractionation procedure developed in this work, allowing the characterization of PPO in persimmon extracts from the ‘‘Rojo Brillante’’ variety. The protein profile obtained at the end of each step is shown in Fig. 5A. Persimmon crude extract showed the presence of several faint bands above 75 kDa when revealed with silver staining. However, no

Fig. 5. 10% Native PAGE of the different extracts obtained during the fractionation procedure of ‘‘Rojo Brillante’’ persimmon polyphenol oxidase. CE: Crude Extract; S: Supernatant obtained after the heat treatment step; EE: Enriched and concentrated extract obtained after selective precipitation with ammonium sulphate; Std: Molecular weight standards. (A) Gel revealed with silver nitrate; (B) gel revealed by incubating the gel with 100 mM of 4-methyl catechol as a substrate of polyphenol oxidase.

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appreciable protein bands were revealed for the molecular range below 75 kDa (Fig. 5A, lane CE). When the crude extract was subjected to heat treatment under the conditions optimized for this fractionation procedure, the supernatant obtained (Fig. 5A, lane S) contained fewer protein bands than the crude extract, in accordance with the results obtained from the purification procedure (Table 1). It is worth noting that in this extract there is a group of protein bands around 150 kDa that were even better defined with respect to the crude extract, whereas the other bands decreased in intensity. These protein bands around 150 kDa are the major constituents of the extract obtained after selective precipitation of the supernatant with ammonium sulphate (Fig. 5A, lane EE). Interestingly, we can observe that in the region corresponding to these bands there is considerable oxidase activity when the gel is revealed with 100 mM of 4-methyl catechol (Fig. 5B). In this gel, only the lane corresponding to the extract obtained after ammonium sulphate precipitation yielded appreciable oxidase activity bands (Fig. 5B, lane EE). For the other two extracts (Lanes CE and S in Fig. 5B), the protein amount in the PPO bands may have been too low to observe appreciable catechol oxidase activity. In the case of the EE extract, we can observe up to four different bands yielding oxidase activity, most probably corresponding to different PPO isoforms of the ‘‘Rojo Brillante’’ variety. It must be emphasized that, while Özen et al. (2004) found two PPO isoenzymes in persimmon cultivars from Trabzon (Turkey), Núñez-Delicado et al. (2003) found only one activity band. However, in our case we were able to observe a minimum of four different activity bands, probably corresponding to different isoforms of PPO enzyme (Fig. 5B, lane EE). The same samples corresponding to each of the ‘‘Rojo Brillante’’ PPO purification steps were analysed by polyacrylamide gel electrophoresis under reducing and denaturing conditions (SDS–PAGE). The results obtained are shown in Fig. 6. In contrast to the native PAGE results (Fig. 5A), this time the crude extract showed the presence of many protein bands below 75 kDa (Fig. 6, lane CE). After the heat treatment step, we can observe that most of these protein

bands have been successfully reduced or eliminated, with the exception of three main bands in the region around 22–28 kDa, thus confirming the efficacy of this purification step. These three main protein bands are also the main constituents of the extract obtained after selective precipitation with ammonium sulphate (Fig. 6, lane EE), suggesting that they are protein subunits conforming the various ‘‘Rojo Brillante’’ PPO isoforms having higher Mr values (Fig. 5A, lane EE). Consequently, to conduct a complete study about the differences and similarities of PPO activity from different persimmon varieties or between persimmons and other fruits, further actions (i.e., based on gel electrophoresis for separation of isoenzymes that would subsequently be identified by mass spectrometry analysis) to determine their isoenzyme profile would be necessary. Moreover, as usual for plants, the isoenzyme profile of persimmons is probably influenced by the ripening stage of the fruit and the particular characteristics provided by the growing area. 4. Conclusions Polyphenol oxidase activity from extracts of the ‘‘Rojo Brillante’’ persimmon variety has been characterized for the first time. Optimum temperature for maximum enzyme activity was 55 °C, which is much higher than the values commonly reported for PPOs from other persimmon cultivars. Moreover, its thermal stability is also higher than that shown by most PPOs found in nature and depends strongly on pH, temperature and incubation time. To achieve a significant reduction of the enzyme activity at pH 5.5 and 7.0, it was necessary to subject crude PPO extract to severe heating conditions of 80 °C for at least 70 min. However, PPO was drastically inactivated at pH 4.0 even when mild heat treatments of 60 °C for 10 min were used, and shorter incubation times were required at higher temperatures to stop enzymatic activity completely. Hence, acidification of persimmon puree is recommended to promote PPO inactivation and to prevent browning, which would make it easier to obtain innovative and profitable persimmon-derived products. In addition, four activity bands were detected in the ‘‘Rojo Brillante’’ extracts assayed by gel electrophoresis, suggesting for the first time the presence of at least four PPO isoenzymes in persimmon fruits. Further research is needed for a more detailed characterization of the various PPO isoenzymes during ripening and postharvest treatments. Acknowledgements This research was supported by the Spanish Government (Ministerio de Ciencia e Innovación, MICINN, Project AGL200911805ALI and Juan de la Cierva programme for Tárrega’s contract). The authors acknowledge the critical review of the manuscript by Dr. J. V. Carbonell. References

Fig. 6. Denaturing 10% SDS–PAGE of the extracts obtained after each step of the fractionation procedure presented here for polyphenol oxidase. CE: Crude extract; S: Supernatant obtained after heat treatment; EE: Enriched and concentrated extract obtained after selective precipitation with ammonium sulphate. Std: Molecular weight standards. The gel was revealed with silver nitrate staining.

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