Abstract
The polyphenol oxidase (PPO) enzyme was purified and characterized from Hemşin Apple (Malus communis L.), which was organically grown in Hemşin, in the Rize province of Turkey. Enzyme (PPO) activation was determined with catechol substrate. Apples were homogenized with homogenate buffer (pH 8.5). This process was followed by precipitation with (20–80%) saturated solid (NH4)2SO4 and dialysis. Finally, purification with DE52-Cellulose ion-exchange and Sephadex G-25 columns was performed. Experiments were performed at an optimum pH (5.5) and optimum temperature (30–40°C). The kinetic and thermal parameters Km (3.40 mM), Vmax (333.3 EU/mL.min), Ea (3.57 kcal), ∆H (2.968 kcal/mol), Q10 (1.33), kcat (24.57 min−1) and V0 (7.2x103 mM−1.min−1) were assessed. Additionally, the effects of Mg2+, Pb 2+, Fe2+, Fe3+, Cd2+, Cu2+, Zn2+, Co2+, Al3+, Mn2+ and Na+ on enzyme activity was recorded, and the IC50 values, Kİ values and inhibition types were determined.
INTRODUCTION
Polyphenol oxidase (PPO; EC 1.14.18.1) is a copper-containing protein that is widely distributed on a phylogenic scale. This protein is responsible for the enzymatic browning reaction that occurs when tissues are damaged during the handling, storage and processing of fresh fruits and vegetables, as well as some animal products.[Citation1,Citation2] Browning reactions are made up of the following five basic types; ascorbic acid oxidation,[Citation3] caramelization,[Citation4] the enzymatic reactions of phenols,[Citation5] the chemical reaction of amino groups (Maillard reaction)[Citation6] and the oxidation of lipids to create browned polymers.[Citation7] The oxidation of phenolic compounds to unstable o-quinones is known as “enzymatic browning”.[Citation8,Citation9] The PPO enzyme catalyzes the following two reactions: the hydroxylation of monophenols to o-diphenols (cresolase activity) and the oxidation of o-diphenols to quinones (catecholase activity).[Citation10,Citation11] This browning reaction occurs in fruits and vegetables that are damaged during harvesting, handling, storage and processing operations.[Citation12,Citation13]
Heat treatment and sulfites inhibit enzyme browning; ascorbic acid, cysteine, and glutathione have also been reported to prevent browning in many articles.[Citation14,Citation15] Some reducing agents are oxidized, such as cysteine, β-mercaptoethanol and ascorbic acid. Other reducing agents are phenol scavengers, such as PVPP (polyvinyl polypyrrolidone) and PEG (polyethylene glycol); these compounds are used in PPO homogenate buffer to prevent to o-quinone formation.[Citation8] PPO enzyme extraction requires the presence of detergents, e.g., Triton X-100 or Triton X-114.[Citation16,Citation17] This is because the enzyme is localized to thylakoid membranes within plant cells.[Citation18] PPO enzymes from fruits and vegetables vary in their properties. The properties of PPO enzymes from a single species can vary depending on the growth conditions and stage of maturity.[Citation19] PPO enzymes have been previously purified from apple sources[Citation20] such as grand Alexander apples,[Citation21,Citation22] red delicious apple peels,[Citation23] delicious apples,[Citation24] apples,[Citation25] and Amasya apples.[Citation26]
In this study, PPO from Hemşin apple (Malus communis L.) was purified and partially characterized. The Hemşin apple is known as the “Demir apple” by people in the region. An additional goal of this research was to determine kinetic values and thermal parameters for this enzyme. The effects of some metal ions including Mg2+, Pb 2+, Fe2+, Fe3+, Cd2+, Cu2+, Zn2+, Co2+, Al3+, Mn2+ and Na+ on Hemşin apple (Malus communis L.) PPO were also evaluated.
MATERIALS AND METHODS
Plant Materials and Chemicals
The Hemşin apples (Malus communis L.) used in this study were obtained from the Hemşin district of the Rize province in Turkey. They were washed and were maintained at +4°C until use. All of the reagents used in these experiments were obtained from Sigma or Merck.
Purification of PPO from Hemşin Apple (Malus communis L.)
Fifty grams of fresh apple (Malus communis L.) tissue were collected and stored at –83°C. The sample was weighed and filmed in a blender for 2 min. Tissue was homogenized in 30 mL phosphate buffer (0.1 M, pH 8.5) containing 1% Triton-X 100, 2% PEG (polyethylene glycol), and 10 mM ascorbic acid. The sample was stirred for 15 min and homogenized at a stable pH for 3 h at +4°C. The homogenate was centrifuged at 20.000 × g for 30 min in a refrigerated centrifuge. The pulp was removed from the homogenate by filtration. Ammonium sulfate at 4°C was slowly added to the sample in an ice bath until the supernatant reached 20–80% saturation.[Citation27] Next, the sample was precipitated by centrifugation at 20.000 × g for 30 min at 4°C. The precipitate was dissolved in a small volume of 0.1 M Tris/HCl buffer (pH 8.5) and dialyzed in the same buffer overnight.
DE52-Cellulose Ion-Exchange Chromatography
The DE52-Cellulose (Diethyl aminoethyl cellulose) column material was dissolved in 150 mL 0.2 M Tris-HCl buffer (pH 7.3). Once the gel had collapsed to the bottom of the container, the liquid was remaining at the top was discarded. The gel was then washed with 150 mL Tris-HCl buffer. This process was repeated three times. Afterward, the gel was loaded onto the column (5 cm2 × 30 cm) and balanced with 0.05 M Tris-HCl buffer (pH 7.5) using a gradient mixer apparatus (Pharmacia Fine Chemicals). Dialyzed PPO extract was loaded onto the gel, and the flow rate of the column was adjusted to 20 mL/h. Fractions (5 mL) were collected using 150 mL 0.05 M Tris-HCl (pH 7.5) buffer with a NaCl gradient between 0.1–1 M. For the determination of enzyme activity, the absorbance of samples was recorded at 420 nm. Proteins were spectrophotometrically observed at 280 nm.
Concentration and Purification of PPO by Sephadex G-25
Three milliliters of enzyme solution, which contained the high activity fractions collected from DE52 cellulose chromatography, was blended with 1 g Sephadex G-25 and centrifuged at 1000 × g (MSE Mistral 2000, U.K.) for 3 min.[Citation28] The enzyme activity of the upper layer of the solution was detected using 0.1 M catechol as the substrate; the absorbance at 420 nm was measured using a spectrophotometer. Throughout all of the purification steps, the protein concentration was determined using the Bradford method with Coomassie Brilliant Blue and bovine serum albumin as a standard.[Citation28–Citation30]
PPO Enzyme Activity Assay and Enzyme Unit Definition
Enzyme activity was measured using the method described by Gülçin[Citation31] with some modification. For this purpose, 100 μL enzyme extract was added to a cuvette containing 200 μL pure water, 400 μL phosphate buffer (0.1 M), and 300 μL catechol (0.1 M) at room temperature. The absorbance at 420 nm was measured using a spectrophotometer and compared against a blank sample that did not contain enzyme extract. One unit of PPO enzyme activity was defined as an increase in absorbance of 0.001 per min.
Determination of Optimum pH
A buffer system between pH 3.0–9.5 was used to determine the pH at which enzyme activity was the greatest. For the pH studies, acetate buffer (for a pH range of 3.0–4.0), phosphate buffer (for a pH range of 4.0–7.0), and Tris-HCl buffer (for a pH range of 7.0–9.5) were used.[Citation32] Enzyme activity was measured spectrophotometrically in this buffering range according to the procedure described for the PPO activity assay.
Determination of pH Stability
To determine the pH stability of the apple (Malus communis L.) PPO enzyme, 0.5 mL of the enzyme solution was incubated in 1 mL buffer solution in a pH range of 3.0–9.5 for 30 h at +4°C. Aliquots were then taken at intervals and the PPO activity was determined with catechol substrate. The enzyme was then added to this mixture, and the activity was measured. Increases in absorbance were recorded over a 3 min period. The stable pH values obtained from this assay were used for all of the other experiments.
Determination of Ionic Strength
The effect of ionic strength on the apple (Malus communis L.) PPO enzyme was studied using 1–100 mM concentrations of phosphate buffer using catechol as a substrate at an optimum pH.
Optimum Temperature and Thermodynamic Parameters
A digital water bath was used to determine the optimum temperature for enzyme activity between 0 and 80°C. The optimum enzyme temperature, activation energy (Ea), enthalpy (∆H) and Q10 values were found. The logarithm of activity at each temperature (log k) was calculated; the temperature values were converted to kelvin and expressed as 1/T × 1000–1 in a graph.[Citation33] The slope of the Arrhenius was obtained using this graph. The Ea value was obtained using this slope, as given below:
The enthalpy (∆H) was calculated according to the following formula:[Citation34–Citation36]
The Q10 value, which is the difference in activity between two temperatures that are 10°C degrees apart, was determined. This value was calculated by dividing the high activity by the low activity.[Citation37]
Enzyme Kinetics
KM and Vmax values were determined from a plot of 1/V against 1/(S) using Lineweaver-Burk graphs, as described previously.[Citation38–Citation40] These values were obtained using five different volumes of 0.1 M substrate under optımal temperature and pH. The total amount of enzyme (ET) used to determine the KM and Vmax values was quantitatively measured. The kcat value, which indicates enzyme turnover, was calculated using the following formula:
The specificity constant (Vo) of a reaction allows for the comparison of the catalytic activity of various enzymes or the conversion of various products by the same enzyme.
The Effects of Metal Ions
The effect of Mg2+, Pb2+, Fe2+, Fe3+, Cd2+, Cu2+, Zn2+, Co2+, Al3+, Mn2+, and Na+ on enzyme activity was determined using catechol as a substrate. Five different volumes of 0.05 M inhibitors were used to determine enzyme activity. An activity (%) graph was drawn for each inhibitor. The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of ions in inhibiting the activity of an enzyme.[Citation41–Citation43] IC50 values for each metal ion were determined and are shown in .
Enzyme activity in the presence and absence of an inhibitor was measured at five different substrate concentrations.[Citation44] Three different inhibitor concentrations were selected, and these values were close to the IC50. Lineweaver-Burke graphs were drawn using 1/V and 1/[S] values.[Citation45] After all of the inhibition types were determined, Ki values were determined with the help of these graphs.[Citation46,Citation47]
RESULTS AND DISCUSSION
Purification of PPO from Apple (Malus communis L.)
Fifty grams of apple (Malus communis L.) tissue was placed in a Dewar flask under liquid nitrogen for 10 min to decompose cell membranes. The cold apple was homogenized using a blender in 100 mL of 100 mM cold phosphate buffer (pH 8.5) containing PEG (polyethylene glycol), Triton X-100 and ascorbic acid. The homogenate was filtered and kept at 4°C before being centrifuged at 20.000 × g for 30 min at 4°C. The supernatant was used as a crude enzyme extract. The crude extract was further fractionated with ammonium sulfate. The crude extract was first brought to 20% saturation; the sample was centrifuged, and the supernatant collected. This supernatant was then brought to 80% saturation by the addition of ammonium sulfate. Samples were subjected to ammonium sulfate saturation, dialysis, DE52-Cellulose ion-exchange chromatography and concentration by Sephadex G-25, and the results were tabulated (). The specific activity, protein content, purification fold and yield (%) of the PFO are shown in . Dialyzed enzyme after 20–80% ammonium sulfate saturation was loaded onto a DE52-Cellulose ion-exchange column. After the enzyme was loaded, the column was thoroughly washed with the same buffer until the UV absorbance of the eluate returned to the base line. The bound enzyme was eluted with a linear gradient of NaCl (0.1–1.0 M) at a flow rate of 0.5 mL/min–1. The protein content of the whole fraction was measured at 280 nm, and fractions with high protein content () were collected. These fractions (peaks 10–20) corresponded to NaCl concentrations between 0.1–0.3 M. The protein content of the elution was determined using Coomassie Brilliant Blue and measured at 595 nm. Enzyme activity was determined using catechol and measured at 420 nm (). Active fractions, which had high enzyme activities, were collected and pooled. The pooled fractions were concentrated by ammonium sulfate precipitation. Finally, the precipitated protein was re-dissolved in a small volume of 100 mM phosphate buffer (pH 6.0) and dialyzed against the same buffer. Finally, 3 mL of purified enzyme was added to a Sephadex G-25 column to further purify and concentrate the protein.
TABLE 1 Purification of polyphenol oxidase (PPO) from Hemşin apple (Malus communis L.)
FIGURE 1 DE52-Cellulose (Diethylaminoethyl cellulose) column. (-●-) symbolize the protein content, which was spectrophotometrically measured by the absorbance at 280 nm. (-○-) symbolize the PPO activity, which was measured by the activity assay. (-∆-) symbolize the linear gradient buffer which containing (0,1–1 M) NaCl.
Characterization Studies
Optimum pH is a critical parameter that affects enzyme activity. The optimum pH was determined using three different buffers over a range of 3.0 to 9.5 and with catechol as a substrate (). PPO activity was determined in the pH range of 3.0–5.5 in 0.2 M phosphate/0.1 M citrate buffer. Activity was determined in the pH range of 5.5–7.0 in 0.2 M phosphate buffer and in the pH range of 7.5–9.5 in 0.2 M Tris/HCl buffer. Maximum activity with catechol was obtained at pH 5.5, as shown in . This value is the same as the optima reported for PPO from strawberry at pH 5.5[Citation48] and PPO from “Anna” apples at pH 5.4.[Citation49] At pH 6.0, enzyme activity declined approximately 30%. At pH 9.0, the enzyme was still active; however, approximately 60% of enzyme activity was lost. Several pH optima have been reported by other authors for PPO using various substrates. An optimal pH of 8.0 has been reported for PPO from kiwi,[Citation50] an optimal pH of 8.5 has been reported for PPO from Malatya apricot (Prunus armeniaca),[Citation51] and an optimal pH of 6.0 has been reported for PPO from avocado[Citation52] and Stanley plums.[Citation53]
TABLE 2 The optimum conditions for and the kinetic and thermodynamic characteristics of purified Hemşin apple (Malus communis L.) PPO activity. (kcat: turnover number, Vmax: maximum velocity, Km: Michaelis-Menten constant, Vo: catalytic efficacy, Ea: activation energy, ∆H: enthalpy)
FIGURE 2 Optimum temperature of PPO from Malus communis L. The Arrhenius plots for heat inactivation of the purified PPO.
pH stability of PPO was determined using 0.1 M buffer solutions. Acetate buffer was used for a pH range of 3.0–4.0, phosphate buffer was used for a pH range of 4.0–7.0, and Tris HCl buffer was used for a pH range of 7.0–9.0. One milliliter of crude enzyme solution was mixed with 3 mL buffer solution. After mixing, the samples were incubated at 4°C for 30 h. PPO activity was measured prior to the PPO activity assay every 6 h. As seen in , a stable pH was found at 8.5 for apple (Malus communis L.) PPO.
FIGURE 3 Determination of stable pH for PPO from Malus communis L.
The ionic strength of a solution is a measurement of the concentration of ions in that solution. The effect of ionic strength on the apple (Malus communis L.) PPO enzyme at a catechol concentration 0.1 M was determined.
The optimum temperature for the apple (Malus communis L.) PPO enzyme activity is shown . Activity was measured over a temperature range of 10–80°C using catechol as a substrate (0.1 M) in phosphate buffer (0.1 M; pH 5.5) at 420 nm as quickly as possible. The optimum temperature for apple (Malus communis) PPO was 30°C. shows that enzyme activity was lost at temperatures above 50°C. The optimum temperatures for PPO enzymes from different sources using catechol as a substrate have been previously reported; 12°C is the optimum temperature for PPO from ferula sp.,[Citation54] 15°C is optimum for PPO from Amasya apple,[Citation26] 40°C is optimum for PPO from Barbados cherry (Malpighia glabra),[Citation55] and 55°C is optimum for PPO from medlar fruits (Mespilus germanica).[Citation56] The thermodynamic parameters activation energy (Ea), enthalpy (∆H), and Q10 for the PPO enzyme were determined between 30 and 70°C. These results are shown in .
Kinetic Parameters
The dissociation constant of the enzyme-substrate complex and the maxima velocity (KM and Vmax) of the apple (Malus communis L.) PPO enzyme when catechol is used as a substrate are shown in . As seen , Km (3.40 mM) and Vmax (333.3 EU/mL.min) values were found for catechol. The kcat (24.57 min–1) value was calculated using the equation kcat = Vmax /ET. Vo, the catalytic power of the enzyme, was calculated as kcat/Km.
Effect of Metal Ions
The effect of metal ions on apple (Malus communis L.) PPO enzyme activity was analyzed. Activation, the type of inhibition and the inhibition constant (Ki) are shown in . Mg2+, Cd2+, Zn2+, and Al3+ inhibited enzyme activity. Fe3+ and Cu2+ moderately activated the enzyme. Mn2+, Pb2+, Fe2+, and Co2+ partially activated enzyme activity, and Na+ did not affect enzyme activity. Cd2+ was the most effective inhibitor of PPO activity when catechol was used as substrate. In descending order of effectiveness, Al+, Zn2+, and Mg2+ also inhibited PPO activity. Cd2+ and Zn+ acted as competitive inhibitors, and Mg2+ and Al3+ acted as uncompetitive inhibitors. The Ki values for Mg2+ (21.33 mM), Cd2+ (4.87 mM), Zn2+ (10.25 mM) and Al3+ (5.54 mM) were calculated using the 1/V and 1/S values from Lineweaver-Burk graphs. As seen in , the other metals activated Hemşin apple (Malus communis L.) PPO.
TABLE 3 The inhibition effects of metal ions on Hemşin apple (Malus communis L.) PPO activity
TABLE 4 The activation effects of metal ions on Hemşin apple (Malus communis L.) PPO activity
CONCLUSION
In the present study, the polyphenol oxidase enzyme was purified and characterized from Hemşin apple (Malus communis L.). The enzyme behaved optimally at pH 5.5 and at 30°C when catechol was used as a substrate. Some kinetic parameters, including Vmax (333.3 EU/mL.min), Km (3.40 mM), and kcat (24.57), were determined. Some thermal parameters, including Ea (3.57 kcal), ∆H (2.968 kcal/mol), Q10 (1.33), and V0 (7.2×103 mM–1.min–1), were calculated for apple (Malus communis L.) PPO. The effects of some metal ions were determined, and the IC50 value, Kİ value and inhibition type were found for the metal ions that possessed inhibition properties.
FUNDING
IG and SE would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this research, RGP-VPP-254.
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