Article pubs.acs.org/JAFC
Purification and Partial Biochemical Characterization of Polyphenol Oxidase from Mango (Mangifera indica cv. Manila) Gisela Palma-Orozco,†,‡ Norma A. Marrufo-Hernández,‡ José G. Sampedro,§ and Hugo Nájera*,‡ †
Unidad Profesional Interdisciplinaria de Biotecnologı ́a, Departamento de Bioingenierı ́a, Instituto Politécnico Nacional, Acueducto s/n, Col. La Laguna Ticoman, Delegación Gustavo A. Madero, C.P. 07340, México, D.F., Mexico § Instituto de Fı ́sica, Universidad Autónoma de San Luis Potosı ́, Av. Manuel Nava 6, Zona Universitaria, C.P. 78290, San Luis Potosı ́, SLP, Mexico ‡ Departamento de Ciencias Naturales, Universidad Autónoma Metropolitana − Cuajimalpa, Av. Vasco de Quiroga 4871, colonia Santa Fe Cuajimalpa, delegación Cuajimalpa de Morelos, C.P. 05300, México, D.F., Mexico ABSTRACT: Polyphenol oxidase (PPO) is an enzyme widely distributed in the plant kingdom that has been detected in most fruits and vegetables. PPO was extracted and purified from Manila mango (Mangifera indica), and its biochemical properties were studied. PPO was purified 216-fold by hydrophobic interaction and ion exchange chromatography. PPO was purified to homogeneity, and the estimated PPO molecular weight (MW) by SDS-PAGE was ≈31.5 kDa. However, a MW of 65 kDa was determined by gel filtration, indicating a dimeric structure for the native PPO. The isolated PPO showed the highest affinity to pyrogallol (Km = 2.77 mM) followed by 4-methylcatechol (Km = 3.14 mM) and catechol (Km = 15.14 mM). The optimum pH for activity was 6.0. PPO was stable in the temperature range of 20−70 °C. PPO activity was completely inhibited by tropolone, ascorbic acid, sodium metabisulfite, and kojic acid at 0.1 mM. KEYWORDS: Manila mango (Mangifera indica), purification, enzymatic browning, inhibitors, polyphenol oxidase
■
INTRODUCTION Mango is a tropical and subtropical fruit belonging to the Anacardiaceae family, genus Mangifera, species indica; 150 cultivars of mangoes are produced around the world. Manila mango is native to The Philippines and early in the 19th century was taken to the west coast of Mexico. In Mexico, Manila mango was crossed with other different mangoes that arrived later to the country, thus resulting in the Manila mango known in Mexico.1 In Mexico, Manila mango is mainly produced in the states of Guerrero and Nayarit.2 Mango fruit matures at 4−5 months after flowering. Mango fruit is a climacteric fruit and therefore is harvested at physiological ripeness and subsequently ripens at room temperature (25 °C). After ripening, the fruit may be stored at room temperature for 1 week or for 2 weeks if it is refrigerated. Mango fruit is long (reaching on average a weight of 270 g) and has a single flat seed surrounded by yellow flesh with a sweet flavor. Mango fruit is a rich source of dietary fiber and contains compounds with antioxidant activity and health benefits; some of these molecules were identified as phenolic compounds, carotenoids, and vitamins A and C.3 Mango fruit is highly accepted and therefore consumed worldwide. Mango flesh is used in processed products such as puree, nectar, canned slices, syrup, and jam.4 However, mango fruit is highly susceptible to oxidative damage during handling or when processed, displaying enzymatic browning due mainly to oxidation of phenolic compounds by the enzyme polyphenol oxidase (PPO).5 Enzymatic browning of mango fruit leads to the development of unpleasant color and flavor and even sometimes to the loss of nutrients, thus decreasing consumer acceptance.6 © XXXX American Chemical Society
PPO is a dinuclear copper-containing enzyme that catalyzes two different reactions in the presence of molecular oxygen using phenolic compounds as substrates, namely, the hydroxylation of monophenols to their corresponding odiphenols (monophenolase or tyrosinase activity) and the oxidation of o-diphenols to o-quinones (diphenolase or catechol oxidase activity). The o-quinones are highly reactive molecules that undergo complex nonenzymatic changes, that is, they either polymerize or react with structural constituents of fruit cells to form molecularly complex brown, red, or black pigments.7 PPO has been isolated and studied from different fruits and vegetables such as banana pulp,8 Bramley’s Seedling apple,9 and mamey;10 some mango varieties have been partially purified and characterized including Haden mango,11 Tainong mango,12 Badami mango,13 and Chokanan mango.14 In this work PPO from Manila mango was completely purified and its bio- and physicochemical characterizations were performed. Substrate specificity, effect of inhibitors, activity dependence on pH, thermal stability, enzyme kinetic parameters, and molecular weight were determined.
■
MATERIALS AND METHODS
Raw Materials and Chemicals. The Manila mango fruit in ripened stage was obtained from a market in Mexico City. The fruit was processed immediately once the crude extract was obtained. Phenyl-Sepharose 4-fast flow, Mono Q HR 10/10, Superdex 200 10/ 300 GL, and XK 16/20 columns were from GE Healthcare (New Received: December 6, 2013 Revised: September 8, 2014 Accepted: September 11, 2014
A
dx.doi.org/10.1021/jf5029784 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Table 1. PPO Purification from Manila Mango Fruit (Mangifera indica cv. Manila)a
a
purification step
volume (mL)
activity (units/mL)
total activity (units)
protein content (mg/mL)
specific activity (units/mg protein)
purification (fold)
yield (%)
crude extract (NH4)2SO4 precipitation 30−85% phenyl-Sepharose ion exchange
920 800 20 16
779 804 4366 1399
717048 643200 87320 22384
19.10 4.95 1.03 0.15
41 162 4205 8839
1 4 103 216
100 90 12 3
These values were obtained in a typical purification.
Figure 1. Elution profile by hydrophobic interaction. The column used was a phenyl-Sepharose 4-fast flow. Polyphenol oxidase activity was determined in collected fractions. Absorbance at 280 nm (mAU) (solid lines), specific activity (■), and conductivity (mS/cm) (dashed lines) are shown. Fractions with the highest specific activity are framed with dashed lines. York, NY, USA). All other reagents were from Sigma-Aldrich (St. Louis, MO, USA). Extraction Procedure. PPO crude extract was obtained from 350 g of Manila mango pulp mixed with 700 mL of 0.2 M sodium phosphate buffer, pH 7.0, 1% w/v poly(vinylpolypyrolidone) (PVPP), 1% v/v Triton X-100, and protease inhibitors (0.33 mg/100 mL aprotinin and 0.1 mM phenylmethanesulfonyl fluoride (PMSF)). PPO crude extract was left to rest overnight and centrifuged at 10650g for 30 min. The supernatant obtained was filtered and aprotinin (0.33 mg/100 mL) was added. All extraction procedures were carried out at 4 °C. PPO Activity Assay. PPO activity assay was performed in (1 mL) 0.2 M sodium phosphate buffer, pH 7.0, containing 50 mM catechol or pyrogallol as substrate; 33 μL of the enzyme solution was added to initiate the reaction. PPO assay was performed at 25 °C using an Agilent 8453 UV−vis (New York, NY, USA) diode array spectrophotometer. ΔAbs420 nm was recorded every 5 s during 5 min. One unit of PPO activity was defined as the change in absorbance of 0.001 min−1 mL−1 of enzyme.15 Purification Procedure. PPO was purified as described by10 briefly, filtered crude extract was subjected to (NH4)2SO4 precipitation (30−85% saturation) at 4 °C, then centrifuged at 10650g, 30 min. The precipitate was resuspended in a small volume of phosphate buffer (50 mM sodium phosphate, pH 7.0), 1.2 M ammonium sulfate, and 0.6 M potassium chloride and dialyzed overnight against three changes of the same buffer. The dialyzed solution was centrifuged at 34500g for 30 min and the supernatant collected. Aprotinin (0.33 mg/100 mL) was added to the supernatant. Protein concentration was determined by UV absorbance at 280 nm. Chromatography was carried out at room temperature using a Fast Protein Liquid Chromatography (FPLC) system from GE. The supernatant was loaded onto an XK 16/20 column packed with phenyl-Sepharose 4-fast flow previously equilibrated with buffer used in dialysis (equilibrium buffer). Then, the column was washed with
equilibrium buffer (70 mL) to remove unbound proteins. The equilibrium buffer was displaced stepwise from 100 to 0% with distilled water, and the bound proteins were eluted. Flow rate was 1.0 mL/min, and fractions (2.5 mL) were collected. PPO activity and protein concentration were determined, obtaining the elution profile. Fractions showing high specific activity were pooled and then dialyzed overnight against three changes of buffer A (20 mM Tris-HCl, pH 7.0) at 4 °C. Aprotinin (0.33 mg/100 mL) was added to the dialyzed enzyme. After that, the suspension was loaded onto anion exchange column Mono Q HR 10/10, previously equilibrated by washing with low ionic buffer A (40 mL) followed by high ionic buffer B (80 mL 20 mM TrisHCl pH 7.0, containing 1.0 M KCl) and finally with low ionic buffer A (80 mL). The flow rate was 2 mL/min,and fractions (2 mL) were collected. Protein was eluted by linearly increasing the ionic strength; four linear gradients were used in the elution process: (a) from 0 to 16% in 34 mL, (b) from 16 to 19% in 10 mL, (c) from 19 at 25% in 40 mL, and (d) from 25 to 100% in 40 mL. Then the column was washed with 100% elution buffer by using a volume of 24 mL. PPO activity and protein concentration were determined in each fraction, and the elution profile was obtained. PPO Molecular Weight (MW) Determination. Column chromatography was used for MW determination of the native enzyme and carried out at room temperature using an FPLC system. Briefly, a Superdex 200 10/300 GL column was washed with distilled water (50 mL) and then with elution buffer (50 mL of 50 mM phosphate, pH 7.0, and 0.15 M KCl) at a flow rate of 0.5 mL/min. After that, 350 μL of purified PPO was loaded onto the column and eluted with elution buffer (30 mL); fractions (1 mL) were collected, and for each fraction activity was determined. PPO molecular weight was determined by extrapolation of the PPO elution time in a calibration curve generated using a set of proteins with different molecular weights. B
dx.doi.org/10.1021/jf5029784 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
Figure 2. Elution profile by ion exchange chromatography. The column used was a Mono Q HR. Polyphenol oxidase activity was determined in collected fractions. Absorbance at 280 nm (mAU) (solid lines), specific activity (■), and conductivity (mS/cm) (dashed lines) are shown. Fractions with the highest specific activity are framed with dashed lines.
Figure 3. (A) SDS-PAGE of PPO purified from Manila mango: lane 1, molecular weight markers; lane 2, purified enzyme. (B) Elution profile by gel filtration from mango PPO. Gel Electrophoresis (SDS-PAGE). Enzyme purity was determined at different purification steps using SDS-PAGE.16 The concentration of the separating gel was 12%, and that of the stacking gel was 4%. Protein standard molecular weight markers were used. pH Dependence of PPO Activity. The effect of pH on PPO activity was measured in 1 mL of 50 mM catechol in 0.1 M citric acid− 0.2 M sodium phosphate buffer and in 2.5−8.0 M borate buffer, pH range 8.5−12.0 at 25 °C. Reaction was started by the addition of 33 μL of the isolated PPO. PPO specific activity (U/mg protein) was determined as described above. Optimum Temperature. PPO activity was assayed in 1 mL, by incubating 33 μL of the enzyme in 0.1 M citric acid−0.2 M sodium phosphate buffer, pH 6.0, containing 50 mM catechol or pyrogallol for 30 min at different temperatures (20−80 °C). PPO activity was determined as described above. Determination of PPO Kinetic Parameters. PPO velocity (v) at different substrate concentrations was measured in 0.2 M sodium phosphate buffer, pH 7.0, and various concentrations of substrates catechol, pyrogallol, and 4-methylcatecol (1−150 mM). The
Michaelis−Menten constant (Km) and maximum catalytic velocity (Vmax) for each substrate were determined by Lineweaver−Burk and nonlinear regression. PPO Substrate Specificity. PPO substrate specificity was determined using eight different substrates. PPO activity was monitored as the change in substrate absorbance (ΔAbs) at a given wavelength (λ) as follows: catechol, pyrogallol, 4-methylcatecol, epicatechin, caffeic acid, ferulic acid, and phenol at λ = 420 nm; L3,4-dihydroxyphenylalanine (DL-dopa) at λ = 480 nm. PPO activity assay solution was prepared in 0.2 M sodium phosphate buffer, pH 7.0, and variable concentrations of the given substrate (1, 2.5, and 10 mM).9,10 PPO specific activity (U/mg protein) was determined as described above. Effect of Inhibitors on PPO Activity. PPO activity was measured with 50 mM catechol (substrate) in 0.2 M sodium phosphate buffer, pH 7.0, in the presence of different concentrations of inhibitor (0.1, 1, and 10 mM): tropolone, kojic acid, ascorbic acid, sodium chloride, sodium metabisulfite, citric acid, EDTA disodium salt, succinic acid, C
dx.doi.org/10.1021/jf5029784 | J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
Article
vegetables and fruits, that is, in apple,9 mamey,10 d’Anjou pear,21 and peach,22 but has not been used in mango PPO. Figure 1 shows the elution profile of PPO. Unbound proteins were eluted at initial fractions, between 25 and 75 mL as monitored by absorbance at 280 nm. Then, the bound proteins began to be eluted at ≈195 mS/cm (fractions above 150 mL) (Figure 1). PPO activity was determined in collected fractions, and the highest activity was observed in fractions eluting at ≈160 mS/cm (182−232 mL). At this purification step, PPO showed a 103-fold increase in purity (Table 1). Fractions showing the highest specific activity were pooled and dialyzed against 20 mM Tris-HCl buffer, pH 7.0. Then, the dialyzed enzyme was loaded onto a Mono Q HR 10/10 anion exchange column and eluted; the above procedure has been useful in the purification of PPO from different sources including mamey,10 Goldnugget loquat,23 apple,9 banana pulp,8 Badami mango peel,13 and Raspuri mango sap (latex) (DEAE-Sephacel).24 On the other hand, in Kensington mango PPO sap and skin have been partially purified by Sephadex G-25.19 The elution profile (absorbance at 280 nm) showed a protein peak that elutes before the salt concentration increases (Figure 2). However, highly absorbing (280 nm) peaks were observed during elution with salt gradient; PPO activity was determined in collected fractions, and the highest PPO activities were located in fractions eluting between 32 and 42 mS/cm (108− 150 mL) (Figure 2). At this purification step, PPO was purified 216-fold (Table 1). Electrophoresis and PPO MW Determination. PPO purity was determined by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and protein stained with Coomassie brilliant blue. The Manila mango PPO was essentially pure as observed in SDS-PAGE and showed a MW of ≈31.5 kDa; however, a MW of 65 kDa was determined by gel filtration (GF) (Figure 3). Therefore, the Manila mango PPO seems to be a dimer in its native state. After GF elution, PPO was collected and enzyme activity assayed and, importantly, the eluted enzyme showed a specific activity similar to that previously loaded. PPO MW determination by combining both SDS-PAGE and GF has been used before in other PPOs from different plant sources with confident results: (a) MW for PPO from mamey was ≈18 kDa by SDS-PAGE and 16.1 kDa by GF, a monomer;10 (b) MW for PPO from loquat fruit was 58 kDa by SDS-PAGE and 55 kDa by GF, a monomer;25 (c) MW for PPO from pineapple was ≈25 kDa by SDS-PAGE and ≈104 kDa by GF, a tetramer;26 and (d) MW for PPO from Raspuri mango sap was ≈105 kDa by SDS-PAGE and ≈100 kDa by GF, a monomer.24 In this regard, most PPOs from plants are monomers, but it has been found that some fungi PPOs are mainly oligomers (tetramers with small subunits).27 Effect of pH on PPO Activity. The activity of purified PPO was determined at different pH values using catechol and pyrogallol as substrates. PPO activity was observed in the pH range between 3.0 and 7.0 with both substrates. The maximum PPO activity was observed at pH 6.0 with 100% activity. Therefore, it may be considered that the optimum pH for PPO activity is 6.0 (Figure 4). At pH >7.5, PPO activity decreased drastically. Interestingly, PPO from Manila mango showed the highest activity at pH 6.0 with both substrates. As shown in Table 2, usually PPOs from fruits are more active at neutral or slightly acid pH.26 For example, in PPO in flesh from varieties Tainong and Haden mango, the optimum pH values were shown at 7.0 and 6.0,11,12 respectively; on the other hand, for
benzoic acid, and potassium sorbate. The effect of inhibitors on PPO specific activity (U/mg protein) was determined as described above. Effect of Metal Ions on PPO Activity. PPO activity was measured with 50 mM catechol in 0.2 M sodium phosphate buffer, pH 7.0, and the presence of different concentrations (0.1, 1, and 10 mM) of the following salts: KCl, CaCl2, Na2SO4, MgSO4, MgCl2, ZnSO4, and MnSO4. For CuSO4 the concentrations used were 0.5, 2.5, 5, and 10 mM. PPO specific activity (U/mg protein) in the presence of ions was determined as described above.
■
RESULTS AND DISCUSSION PPO Extraction from Manila Mango. In plants, PPO is a plastid enzyme; in contrast, their phenolic substrates are located
Figure 4. PPO activity from Manila mango dependence on pH. Catechol (□) and pyrogallol (●) were used as substrate in 0.1 M acid citric−0.2 M disodium phosphate buffer (pH 2.5−8.0) and 0.2 M borate (pH 8.5−12). Activity was expressed as relative activity (%) compared with high activity determined. Lines are drawn to aid visualization. Values are means from three replicates. Standard deviations were
