Characterization and Inhibitors of Polyphenol Oxidase from Chinese Toon

Chinese toon is the unique and traditional woody vegetable in China. Enzymatic browning catalyzed by polyphenol oxidase (PPO) is prone to happen during the harvest, storage, and processing of Chinese toon so that the sensory quality and the nutritional content of Chinese toon products are seriously influenced. In order to prolong shelf life and storage period, the characterization of Chinese toon PPO (CtPPO) has been analyzed in this study. The PPO was extracted and fractionated by 25–70% (NH₄)₂SO₄, and the biochemical characteristics were analyzed. Based on the V_max /K_m ratio, pyrogallol was the most suitable substrate, followed by catechol and gallic acid. CtPPO exhibited no affinity with methyl gallate. The molecular mass of CtPPO was approximately 84.55 kDa estimated by SDS-PAGE. The native PAGE showed four prominent bands. Optimal pH and temperature were 6.2, 40°C and 8.5, 80°C for catechol and pyrogallol, respectively. CtPPO showed high and stable activity at pH ranging from 5.0 to 7.2 for catechol and pH 7.4 to 9.5 for pyrogallol. Activation energy (Ea) values were 263.79 KJ/mol for catechol and 103.91 KJ/mol for pyrogallol. In addition, CtPPO showed stronger heat resistance above 70°C for pyrogallol than catechol in the half-life values, D-values and other thermodynamic parameters. Ascorbic acid was the most effective inhibitor, followed by L-cysteine and citric acid. Purple onion peel extract and pomegranate rind extract were effective natural inhibitors, but the former was more valid.     

APPLICATION OF A NEW CONTINUOUS FLOW SPECTROPHOTOMETRIC METHOD FOR THE CHARACTERIZATION OF POLYPHENOL OXIDASE NATURALLY IMMOBILIZED ON COCONUT FIBER

The association of continuous flow injection and spectrophotometry affords a simple, novel and rapid way of monitoring continuously the activity of naturally immobilized enzymes in their natural environment, thus eliminating cumbersome purification. The method was applied to determine the activity of polyphenol oxidase (PPO) enzymes naturally immobilized on coconut (Cocus nucifera, L.) fiber tissues. Maximum enzyme activity occurred at a temperature of 25C and at pH 6.0 using catechol as substrate. Thermal stability was assayed in a temperature range of 20 to 75C. The PPO exhibited excellent thermal stability, with only 50% loss in its activity at 75C after 4.3 min exposure. For catechol apparent Michaelis‐Menten constant (apparent Km), apparent Vmax and the apparent activation energy were 9.1 × 10^?3 mol L^?1, 0.20 abs min^?1 and 10.5 kcal mol^?1, respectively. The immobilized PPO showed high activity for o‐diphenols. The reactivity order was caffeic acid > pyrogallol > catechol. Complete inhibition of the enzyme was observed with 1 × 10^?3 mol L^?1 concentration of cyanide, thiourea, L‐cysteine, ascorbic acid, sodium sulfite, nitrates of cadmium, zinc and mercury, individually. Benzoic acid, 3‐hydroxy‐benzoic acid, 4‐acetamidephenol, sodium azide, resorcinol, L‐cystine and EDTA at equal concentrations inhibited PPO partially.     

PARTIAL PROPERTIES OF POLYPHENOL OXIDASE IN MANGO (MANGIFERA INDICA L. CV. "TAINONG") PULP

The properties of polyphenol oxidase (PPO, EC 1.14.18.1) from an extract of mango pulp were studied. PPO, with catechol as substrate, had an optimum pH at 7.0 and optimum temperature at 30C. PPO showed activity with dihydroxyphenols and trihydroxyphenols, but not with monohydroxyphenols. Kinetic parameters maximum velocity and Michaelis constant for PPO were 256.28 U/min and 6.30 mM with catechol, and 199.61 U/min and 47.81 mM with pyrogallol. The activity of PPO was well retained after heating the extract for 15 min at 50C, and 98% of the activity was lost after the extract was heated for 5 min at 80C. PPO was effectively inhibited by ascorbic acid as well as by β‐mercaptoethanol and L‐cysteine, and was enhanced by sodium dodecyl sulfate.     

Purification and Characterization of Polyphenol Oxidase from Akko XIII Loquat (Eriobotrya japonica cv Akko XIII)

Akko XIII is an important loquat variety grown in Turkey. As with many fruits and vegetables, enzymatic browning catalyzed by polyphenol oxidase (PPO) also occurs in loquats. PPO from Akko XIII loquat was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis and ion exchange chromatography. The enzyme showed several peaks with PPO activity on DEAE-Toyopearl 650 M column, of which only two (isoenzyme A and isoenzyme B) were characterized. Assay of activity of the isoenzymes between pH 3.04 and 7.80 using catechol as substrate showed two activity peaks, one at acidic pH and the other at neutral pH. pH optima of isoenzyme A and B were found to be at 7.4 and 4.98, respectively. The Km values of isoenzyme A and B using catechol as substrate were found to be 152.3 mM and 5.4 mM, respectively. They both displayed maximal activity at 30^oC. The two isoenzymes displayed different heat resistance and sensitivity towards various inhibitors.     

Partial characterization of lettuce (Lactuca sativa L.) polyphenol oxidase

Polyphenol oxidase (PPO) from garden lettuce (Lactuca sativa L.) was partially purified by ammonium sulphate ((NH₄)₂SO₄) precipitation and dialysis, and then some of its kinetic properties such as optimum pH and temperature, substrate specificity, thermal inactivation and inhibition were investigated. The total phenolic and protein contents of Lactuca sativa L. extracts were determined according to the Folin-Ciocalteu and Bradford methods, and found to be 304 mg/100 g on a fresh weight basis and 494 μg/mL, respectively. PPO activity was determined using 4-methylcatechol, catechol and pyrogallol as substrates. Kinetic parameters, K _m and V _max, were calculated from Lineweaver–Burk plots. According to V _max/K _m ratio, pyrogallol was the most suitable substrate, followed by catechol and 4-methylcatechol. The optimum temperature and pH values were 30, 40 and 30 °C; and 6.5, 8.0 and 7.5 for 4-methylcatechol, catechol and pyrogallol substrates, respectively. The thermal inactivation of PPO was investigated at 35, 55 and 75 °C. The enzyme activity decreased with increasing temperature. The effect of different inhibitors on partially purified Lactuca sativa L. PPO was spectrophotometrically investigated. For this purpose, tropolone, glutathione, ascorbic acid and 4-aminobenzoic acid were used to inhibit the activity of Lactuca sativa L. PPO at different concentrations. From the experimental results, it was found that glutathione was found to be the most potent inhibitor for Lactuca sativa L. PPO.     

Inhibition kinetics of polyphenol oxidase by glutamic acid

The inhibition of polyphenol oxidase (PPO) by glutamic acid was investigated. Application of different concentrations of glutamic acid to mushroom solution and Ocimum basilicum L. extract showed that glutamic acid appeared to be an effective browning inhibitor. Glutamic acid showed uncompetitive inhibition for mushroom and Ocimum basilicum L. polyphenol oxidases using 4-methylcatechol as a substrate, for mushroom PPO using catechol as a substrate and for Ocimum basilicum L. polyphenol oxidase using pyrogallol as a substrate; mixed-type inhibition for mushroom polyphenol oxidase using pyrogallol as a substrate; and non-competitive inhibition for Ocimum basilicum L. polyphenol oxidase using catechol as a substrate. Furthermore, sodium azide was used as an inhibitor for comparison with the inhibition potency of glutamic acid. It was found that glutamic acid was a more power inhibitor than sodium azide. The type of inhibition observed depended on the substrate, inhibitor and enzyme source used.     

Polyphenoloxidase from Amasya Apple

Polyphenoloxidase (PPO) of Amasya apple was partially purified by (NH₄)₂SO₄ precipitation and dialysis. The sample was used for characterization of the PPO. Optimum pH were 7.0, 9.0, 8.6 and 6.6 on substrates catechol, 4-methyl catechol, pyrogallol and L-dopa respectively. Catechol was the most suitable for Amasya apple PPO. The optimum temperature for maximum PPO activity was 18°C with catechol. Of seven inhibitors tested, the strongest was L-cysteine. Effectiveness of inhibitors increased in the order: thiourea, glutathione, β-mercaptoethanol, sodium cyanide, ascorbic acid, sodium metabisulfide, and L-cysteine. The K_M was 34 mM of catechol. The activation energy with catechol was 107 cal/mol. In electrophoretic separation, three isoenzymes were detected with both catechol and L-dopa substrates.     

Inhibition of polyphenol oxidase obtained from various sources by 2,3‐diaminopropionic acid

This paper reports for the first time the inhibition of the catecholase activities of mushroom, artichoke (Cynara scolymus L) and Ocimum basilicum L polyphenol oxidase by 2,3‐diaminopropionic acid. Polyphenol oxidases from artichoke and O basilicum L were purified by ammonium sulfate precipitation, dialysis and a Sepharose 4B‐L ‐tyrosine‐p‐aminobenzoic acid‐affinity column. In inhibition studies, 2,3‐diaminopropionic acid showed uncompetitive inhibition for mushroom PPO using catechol and pyrogallol as substrates, competitive inhibition for O basilicum L PPO using catechol as a substrate, and uncompetitive inhibition for artichoke PPO using catechol as a substrate. Furthermore, sodium azide, which is an inhibitor of PPO, was used as an inhibitor for comparison with the inhibition potency of 2,3‐diaminopropionic acid. The highest 2,3‐diaminopropionic acid inhibition observed with O basilicum L (K_i = 0.89 mM ), followed by artichoke (K_i = 1.42 mM ) and mushroom (K_i = 2.47 mM ), respectively. Copyright © 2005 Society of Chemical Industry     

Strawberry Polyphenoloxidase: Its Role in Anthocyanin Degradation

Two purified fractions of strawberry polyphenoloxidase (PPO) were used in model systems containing D-catechin alone or in combination with either pelargonin or cyanin. Km values using D-catechin were 0.5 mM and 0.41 mM and Vmax 82,700 and 18,800 nmoles O²/min/ mg protein, respectively, for each isozyme. Reaction of PPO with D-catechin led to the formation of yellow-brown pigments with maximum absorbance at 390 nm. Little PPO activity could be detected using cyanin as a substrate and no activity with pelargonin. PPO and D-catechin together caused the loss of 50 to 60% of the anthocyanin pigments after 24 hr at room temperature with the formation of a precipitate.     

Characterization of polyphenoloxidase from 2 peach (Prunus persica L.) varieties grown in Argentina

Polyphenoloxidase was extracted from September peach (_SEPPO) and Summerset peach (_SUPPO) and its physicochemical characteristics were analyzed. The optimum pH was 6.5 for _SEPPO and 5.5 for _SUPPO. The optimum temperature was 35°C for _SEPPO and 39.4°C for _SUPPO. Activation energy (Ea) from thermal activation was 41.5 kJ/mol for _SEPPO and 37.5 kJ/mol for _SUPPO. Heating at 60°C by 5 min, _SUPPO was denatured whereas _SEPPO retained 2.6% of activity. Activation enthalpy (ΔH^#) and activation entropy (ΔS^#) for _SEPPO heat-inactivation were 69.9 J/mol and −83.5 kJ/mol·K for _SUPPO, ΔH^# was 91.8 J/mol while ΔS^# was −21.0 kJ/mol·K. Substrate specificity (V_max/K_M) was 4-methylcatechol>catechol>pyrogallol for _SEPPO and 4-mehtylcatechol>pyrogallol>catechol for _SUPPO. For both enzymes, the order of inhibition effectiveness using reductor agents was metabisulphite>ascorbic acid. Benzaldehyde, 4-hydroxybenzaldehyde, and dl-dopa were competitive inhibitors, and their K_I values were 38.86, 8.43, and 2.08 mM, respectively.     

Polyphenol Oxidase from Apple (Malus domestica Borkh. cv Bramley's Seedling): Purification Strategies and Characterization

ABSTRACT Polyphenol oxidase (PPO) was isolated from Bramley's Seedling apples with 75.7‐fold purification and 26.5% recovery by ammonium sulfate precipitation, phenyl sepharose chromatography, ion exchange chromatography, and hydroxyapatite chromatography. Molecular weight was estimated to be about 45 kDa by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS PAGE). Optimum PPO activity was at pH 6.5 and greater than 50% activity was retained during storage for 72 h at pH 5.5 to 6.5. Optimum temperature for activity was 30 °C and the enzyme had residual activity of greater than 50% during storage for 72 h at 20 °C to 30 °C and for 24 h at 40 °C to 50 °C. Of the substrates tested, activity was greatest with 4‐methylcatechol followed by catechol, pyrogallol, and (?)epicatechin. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid.     

Inhibition kinetic and mechanism of polyphenol oxidase from various sources by diethyldithiocarbamic acid

Inhibition kinetics and mechanism of polyphenol oxidases (PPO) partially purified from various sources such as Thymbra spicata L. var. spicata and Ocimum basilicum L., and of mushroom PPO bought from Sigma by diethyldithiocarbamic acid have been described using catechol, 4-methylcatechol and pyrogallol as substrates. The inhibition type was competitive for O. basilicum L. PPO using catechol and 4-methylcatechol as substrates, for mushroom PPO using catechol, 4-methylcatechol and pyrogallol as substrates, and for T. spicata L. var. spicata PPO using 4-methylcatechol as a substrate; uncompetitive inhibition for T. spicata L. var. spicata PPO using pyrogallol as a substrate; and non-competitive inhibition for O. basilicum L. and T. spicata L. var. spicata PPO using pyrogallol and catechol as substrates, respectively. The inhibition effect of diethyldithiocarbamic acid on enzymatic browning varied greatly from one phenol to another and from one enzyme to another. Hence, no general rule can easily be established with regard to the type of inhibition observed.     

Characterization and inhibition of <Emphasis Type="Italic">Rosmarinus officinalis</Emphasis> L. polyphenoloxidase

Polyphenoloxidase (PPO) from Rosmarinus officinalis L. was fractionated by ammonium sulfate ((NH₄)₂SO₄) precipitation and dialysis, and then some of its kinetic properties such as optimum pH and temperature, substrate specificity, thermal inactivation, and inhibition were investigated using 4-methylcatechol, catechol, and pyrogallol as substrates. The protein content of Rosmarinus officinalis L. extracts was determined according to Bradford’s method. Kinetic parameters, K _m and V _max, were calculated from Lineweaver–Burk plots. According to V _max/K _m ratio, 4-methylcatechol was the most suitable substrate. The optimum temperature and pH values were 20, 30 and 30 °C, and 7, 8 and 8 for 4-methylcatechol, catechol, and pyrogallol substrates, respectively. The thermal inactivation of PPO was investigated at 35, 55, and 75 °C. The enzyme activity decreased with increasing temperature. The effect of different inhibitors on partly purified Rosmarinus officinalis L. PPO was spectrophotometrically investigated. For this purpose, ascorbic acid and l-cysteine were used to inhibit the activity of Rosmarinus officinalis L. PPO at different concentrations. From the experimental results, it was found that l-cysteine is a more effective inhibitor than ascorbic acid due to lower K _i values.     

Polyphenol oxidase from yali pear (Pyrus bretschneideri)

Polyphenol oxidase (PPO) was isolated from Yali pear (Pyrus bretschneideri R). At the end of purification by ion exchange chromatography on DEAE-cellulose, 10.8-fold purification was achieved. The enzyme showed activity to catechol, pyrogallol, chlorogenic acid and DL-DOPA; of these four, chlorogenic acid was the best substrate. The optimum pH for the PPO was 7.0. PPO activity was not destroyed by heating to 30° for 30 min. The effects of various compounds as inhibitors of the reaction catalysed by the enzyme were tested.     

Peroxidase and polyphenol oxidase in long-term frozen stored papaya slices. Differences among hermaphrodite and female papaya fruits

The effects of freezing and frozen storage on hermaphrodite and female papayas (cv Sunrise, solo group) polyphenol oxidase (EC 1.10.3.1; PPO) and peroxidase (EC 1.11.1.7; POD) were evaluated. The freezing process produces a significant increase in both enzymatic activities (22–11%, PPO and 13%, POD) depending on the kind of papaya fruit. During frozen storage, the soluble PPO activity shows a continuous increase up to 9 months of storage only in hermaphrodite tissues. This same sample also showed a significant activation of soluble POD at 3 months of storage. Female frozen samples maintained a continuous decrease in soluble POD activity during storage, while PPO activity showed some increase up to 12 months. Isoenzyme pattern of PPO in freshly frozen papaya tissues showed an intensification of the most cationic forms, D and E (R_f=0·55 and R_f=0·64, respectively) and a disappearance of band B (R_f=0·28). This band B was not regenerated during frozen storage. However, female tissues only showed a continuous inactivation of bands D and E through storage. POD isoenzyme pattern showed different changes depending on the kind of papaya fruit. In both frozen papayas a new isoenzyme (R_f=0·39) form appeared at three months of storage. From this date, hermaphrodite frozen samples lost this isoenzyme together with original the most anionic POD form (R_f=0·58), increasing the intensity of the only remaining form (R_f=0·24). © 1998 SCI.     

PARTIAL PURIFICATION OF POLYPHENOL OXIDASE FROM PLUMS (Prunus domestica L., cv. STANLEY)

Polyphenol oxidase (PPO) was extracted and purified from Stanley plums (Prunus domestica L.) Crude PPO showed pH optima of 5.8 to 6.4 with different substrates. Heating for 5 min at 75C completely inactivated this enzyme. Plum PPO was stable at -20C for 16 weeks. K_mof this enzyme ranged from 17.5 mM with 4-methylcatechol to 31.2 mM with chlorogenic acid. The enzyme was purified 36-fold through (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose and Sephadex G-100. PAGE of crude and purified plum PPO showed 7 and 3 bands, respectively, when stained for activity with catechol. The molecular weight of 3 subunits of purified PPO was estimated in the range of 45–66 kD.     

Use of contact prints for recording polyphenoloxidase isoenzymes separated by electrophoresis

Crude extracts of polyphenoloxidase (PPO) (E.C. 1.14.18.1) from a variety of food and plant sources were subjected to polyacrylamide gel electrophoresis. Following electrophoresis, the gels were stained for enzyme activity using catechol and L-dopa as substrates. Contact prints were made of the enzyme stained gels 30 min after enzyme staining and on the following day. The negative images (white bands on a dark background) clearly showed the number, type, mobility and variation in enzyme staining of isoenzyme forms of PPO separated by electrophoresis. Contact prints were used to identify PPO isoenzyme forms in the leaves, stipules, roots, and stems of broad beans. This method was also used to demonstrate that a single isoenzyme form of PPO in broad bean leaves decreased with leaf age. Photographs, using professional rapid process X-ray copy film, were also taken of enzyme stained gels for comparison to contact prints. Using contact prints, permanent records of PPO isoenzyme forms can be obtained rapidly, are inexpensive, and reproduce actual gel patterns.     

Determination of kinetic properties of polyphenol oxidase from Thymus (Thymus longicaulis subsp. chaubardii var. chaubardii)

A partial characterization of polyphenol oxidase (PPO) activity of Thymus longicaulis subsp. chaubardii var. chaubardii is described. Polyphenol oxidase of Thymus was isolated by (NH₄)₂SO₄ precipitation and dialysis. The effects of substrate specificity, pH, temperature, heat-inactivation and glutathione inhibitor on polyphenol oxidase activity obtained from T. longicaulis subsp. chaubardii var. chaubardii were investigated. Polyphenol oxidase showed activity toward catechol, 4-methylcatechol and pyrogallol. Pyrogallol was the most suitable substrate, due to the lowest K_M (5.5 mM) and the biggest V_max/K_M (1260/min) values. It was found that the optimum pH values did not change with temperature, and were 6.5 for catechol and pyrogallol and 5.5 for 4-methycatechol at all temperatures. Optimum temperatures were 25 °C for catechol and 4-methylcatechol, and 35 °C for pyrogallol. Again, it was found that optimum temperature did not change with pH. Activation energy values were calculated from the Arrhenius equation and found to be in the range −1.72 and −7.48 kcal/mol for catechol, −3.56 and −9.17 kcal/mol for 4-methylcatechol, and −1.60 and −3.98 kcal/mol for pyrogallol as substrates, respectively. From heat-inactivation studies, the required times for 50% inactivation, using catechol, 4-methylcatechol and pyrogallol substrates, were 68.9, 66.4 and 96.3 min at 45 °C, 19.9, 17.9 and 34.3 min at 65 °C, and 4.1, 2.1 and 11.9 min at 85 °C, respectively. I₅₀ and K_i values for glutathione inhibitor, using catechol, 4-methylcatechol and pyrogallol substrates, were calculated, and it was found that the type of inhibition was competitive.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM FRESH-CUT CHINESE WATER CHESTNUT

The characteristics of polyphenol oxidase (PPO) from Chinese water chestnut (CWC) and its potential inhibitors for browning reactions were investigated. PPO was isolated from fresh‐cut CWC and was purified on a Sephadex G‐100 column, with a yield of total activity close to 10%. The molecular weight, Michaelis constant (K_m), substrate specificity, optimal pH and temperature of CWC PPO were examined. Kinetic studies indicated that the K_m and V_max values of CWC PPO for catechol were 10.32 mmol/L and 6.452 × 10⁴ U/min, respectively. The optimal pH and temperature for CWC PPO was 6.5 and 40C, respectively. Among the browning inhibitors tested, 4‐hexylresorcinol, at a concentration of 0.3 mmol/L, showed the strongest inhibition (70%) against the PPO activity of CWC, followed by 3.0 mmol/L N‐acetyl‐L‐cysteine with an inhibition of 53%.     

Partial characterization of polyphenoloxidase from a hybridized wheat (Triticum aestivum L.)

Polyphenol oxidase was extracted and partially purified from wheat leaves by a procedure that included ammonium sulfate fractionation followed by dialysis and gel filtration chromatography. These procedures led to 35.21-fold purification with 17.65% recovery. Optimum pH, temperature, and ionic strength were determined with six substrates. Some kinetic properties of the enzyme such as V _max, K _M, and k _cat were calculated for the substrates. The k _cat/K _M values of the PPO for catechol, catechin, pyrogallol, l-dopa, dopamine, and 4-methyl catechol were 31408, 31167, 28404, 15378, 4865, and 4967 mM/min, respectively. The best substrate of wheat PPO was found to be catechol. The native molecular weight of the PPO was estimated to be 243 kDa based on its mobility in gel filtration column. The inhibitory effects of glutathione, sodium azide, ascorbic acid, oxalic acid, l-cysteine, and thiourea on the reaction catalyzed by the enzyme were tested, and I ₅₀ values were estimated to be 8.0 mM, 10.12 mM, 11.18 mM, 77.33 mM, 183 mM, and 413 mM, and K _i constants were also calculated as 0.416 ± 0.244 mM, 0.317 ± 0.208 mM, 0.820 ± 0.111 mM, 13.80 ± 1.179 mM, 14.10 ± 5.069 mM, and 130 ± 62.45 mM, respectively, by means of Lineweaver–Burk graphs. The most effective inhibitor was glutathione. Glutathione, sodium azide, oxalic acid, and thiourea were competitive inhibitors, whereas ascorbic acid and l-cysteine were also noncompetitive inhibitors.