Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

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.     

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.     

Characterization of Sultaniye grape (Vitis vinifera L. cv. Sultana) polyphenol oxidase

Polyphenol oxidase (PPO) was extracted from Sultaniye grapes grown in Turkey, and its characteristics in terms of pH and temperature optima, thermal inactivation, kinetic parameters and potency of some PPO inhibitors were studied. Optimum pH and temperature for grape PPO were found to be 3.4 and 30 °C, using catechol as substrate. K_m and V_max values were found to be 44.5 ± 5.47 mm and 0.695 ± 0.0353 OD₄₁₀ min^?1, respectively. Four inhibitors were tested in this study and the most potent inhibitor was sodium metabisulphite, followed by ascorbic acid. From the thermal inactivation studies in the range of 65–80 °C, the half‐life values of the enzyme ranged between 2.6 and 49.5 min. Activation energy (E_a) and Z values were calculated to be 208.5 kJ mol^?1 (r² = 0.9544) and 10.95 °C (r² = 0.9517), respectively.     

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.     

Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape

Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera ‘Thompson Seedless’), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol l-Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent Michaelis–Menten constant (K _m) and maximum velocity of the reaction (V _max) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD_400 nm/min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD_478 nm/min for D, L-DOPA, respectively. The obtained similar specificity values of V _max/K _m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was l-cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg²⁺ and Cu²⁺ increased, while Zn²⁺ and K⁺ reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25 °C with 10 mM catechol as substrate. The enzyme was heat stable between 10 and 25 °C, but showed significant activity loss at temperatures higher than 40 °C and completely inactivation at 70 °C for 10 min. Thermal inactivation of PPO showed a first-order kinetic with an activation energy (E _a) of 146.1 ± 10.8 kJ/mol at pH 6.0.     

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.     

Characterization of Polyphenoloxidase from Stanley Plums

Five plum cultivars were investigated for polyphenoloxidase. Stanley cultivar, which showed highest PPO activity, was selected for characterization of this enzyme. The activity of crude enzyme was 3.5 times greater in the flesh than in the skin of the fruit. The enzyme showed a K_m of 20 raM of catechol and V_max of 5.41 × 10^?1 O.D./ min at pH 6.0. Its pH and temperature optima were 6.0 and 20°C respectively. The enzyme lost activity below pH 4.5, but was stable even at 70°C. Among substrates, 4-methylcatechol was oxidized more rapidy, although catechol, dopamine, pyrogallol and caffeic acid were also good substrates. The enzyme was strongly inhibited by sodium metabisulfite (Na₂S₂O₅), L-cysteine and ascorbic acid.     

Determination of some biochemical properties of polyphenol oxidase from Emir grape (Vitis vinifera L. cv. Emir)

Polyphenol oxidase (PPO) was extracted from Emir grapes grown in Turkey and its characteristics in terms of pH and temperature optima, thermal inactivation, kinetic parameters and potency of some PPO inhibitors were studied. The optimum pH and temperature for grape PPO were found to be 4.2 and 25 °C respectively using catechol as substrate. K_m and V_max values were found to be 25.1 ± 2.72 mmol L⁻¹ and 0.925 ± 0.04 OD₄₁₀ min⁻¹ respectively. Of the inhibitors tested, the most potent was sodium metabisulfite, followed by ascorbic acid. The thermal inactivation curve was biphasic. Activation energy (E_a) and Z values were calculated as 251.4 kJ mol⁻¹ (r² = 0.996) and 8.92 °C (r² = 0.993) respectively. Copyright © 2006 Society of Chemical Industry     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

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.     

Purification and some properties of polyphenol oxidase of longan fruit

Polyphenol oxidase (PPO) was isolated from longan (Dimocarpus longan Lour.) fruit peel, with a 46-fold purification of PPO by ammonium sulfate, Sephadex G-200 and Phenyl Sepharose being achieved. Pyrogallol, 4-methylcatechol, and catechol were good substrates for the enzyme, and activity with chlorogenic acid, p-cresol, resorcinol, or tyrosine was not observed. The optimal pH for PPO activity was 6.5 with 4-methylcatechol. The enzyme had a remarkably temperature optimum (35°C) and was relatively stable, requiring a little more than 20 min at 50°C for 50% loss of activity. Reduced glutathione, l-cysteine, thiourea, FeSO₄ and SnCl₂ markedly inhibited PPO activity, whereas MnSO₄ and CaCl₂ enhanced PPO activity. Data obtained in this study might help to better understand longan fruit peel browning.     

Biochemical characterization and process stability of polyphenoloxidase extracted from Victoria grape (Vitis vinifera ssp. Sativa)

Polyphenol oxidase (PPO) was isolated from Victoria grapes (Vitis vinifera ssp. Sativa) grown in South Africa and its biochemical characteristics were studied. Optimum pH and temperature for grape PPO activity were pH 5.0 and T = 25 °C with 10 mM catechol in McIlvaine buffer as substrate. PPO showed activity using the following substances: catechol, 4 methyl catechol, d, l-DOPA, (+) catechin and chlorogenic acid. K_m and V_max values were 52.6 ± 0.00436 mM and 653 ± 24.0 OD_400 nm/min in the case of 10 mM catechol as a substrate. Eight inhibitors were tested in this study and the most effective inhibitors were found to be ascorbic acid, l-cysteine and sodium metabisulfite. Kinetic studies showed that the thermal inactivation of Victoria grape PPO followed first-order kinetics, with an activation energy, E_a = 225 ± 13.5 of kJ/mol. Both in semipurified extract and in grape juice, PPO showed a pronounced high pressure stability.     

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.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

Selected Properties of Polyphenol Oxidase Obtained from Ispir Sugar Bean

Polyphenol oxidase enzyme was isolated from Ispir sugar bean by ammonium sulphate precipitation and its biochemical properties were investigated. For this purpose, K_M and V_max values for optimum conditions of pH, temperature, and ionic strength were determined for catechol, catechin, and chlorogenic acid as substrates. Enzyme activities were measured spectrophotometrically at 420 nm using the same substrates at optimum conditions. K_M values were found to be 2.4875, 1.3154, and 2.2487 M for catechol, catechin, and chlorogenic acid, respectively. V_max values were 3.1480, 0.6130, and 0.5039 EU/ml.min for the same substrates, respectively. These results indicated that catechol was used as a subsrate for inhibition studies. For catechol substrate, dithiothreitol, glutathione, thiourea, and L-cysteine chlorid were inhibitors. For these inhibitors, K_i constants were calculated from Lineweaver-Burk plots and inhibiton types were estimated. Moreover, I₅₀ values were also determined. The most effective inhibitor was found to be glutathione.     

Partial purification and characterisation of polyphenol oxidase and peroxidase from marula fruit (Sclerocarya birrea subsp. Caffra)

Marula fruit, native to sub-Saharan Africa, is of growing commercially importance. Polyphenol oxidase (PPO) and peroxidase from the fruit were partially purified by a combination of temperature induced phase separation in Triton X-114, DEAE-ion exchange and Sephadex G100 gel filtration. PPO activity was purified 58-fold with 75% recovery while the purification factor for peroxidase was 19% with 25% recovery. The enzymes were characterised for enzyme concentration–reaction rate relationship, thermal stability, pH activity and stability, molecular weight, isoelectric point (pI) and kinetic parameters. PPO and peroxidase shared the same molecular weight (71 kDa) and pI (5.43). Thermal deactivation curves were bi-phasic for both activities. Peroxidase displayed maximal activity at pH 4.0 with ABTS (2,2′-azino-(bis-3-ethylbenzthiazoline-6-sulfonic acid)) and a K_M of 1.77 mM for hydrogen peroxide. The pH optimum for PPO was 7.0 with catechol. Marula PPO had K_M values of 1.41, 1.43, 3.73 and 4.99 mM for catechin, 4-methylcatechol, 3,4-dihydroxyphenylpropanoic acid (DHPPA) and catechol, respectively.     

Characterization of polyphenol oxidase from Uapaca kirkiana fruit

Polyphenol oxidase (PPO), the enzyme responsible for the postharvest spoilage of fruits, was extracted and purified from Uapaca kirkiana peel and pulp by ammonium sulfate precipitation and dialysis. Further purification of peel PPO was carried out by gel filtration chromatography. Optimum pH values were 7 and 8 for peel and pulp PPO, respectively. The optimum temperatures for peel and pulp PPO were 45 and 35 °C, respectively. Inhibition studies of the PPO enzyme were performed using citric acid, sodium azide, sodium metabisulfite and thiourea. The most effective inhibitors were sodium azide and citric acid for both peel and pulp PPO. V_max and K_m values were 13.63 units min^?1 and 4.923 mmol L^?1, respectively, for peel PPO and 14.03 units min^?1 and 5.43 mmol L^?1, respectively, for pulp PPO. Three isoenzymes of Uapaca kirkiana PPO were detected by polyacrylamide gel electrophoresis. One of the isoenzymes could be identified as having a molecular weight of 26 625 Da. Copyright © 2005 Society of Chemical Industry     

Polyphenoloxidase from DeChaunac grapes

Polyphenoloxidase (PPO) from red grape cultivar, DeChaunac, grown in New York State was isolated and purified 17-fold by using Phenyl Sepharose CL-4B column. Disc gel electrophoresis revealed near homogeneity of three isoenzyme bands. The molecular weight of this enzyme ranged from 73 000 to 85 000. The temperature and pH optima of the purified enzyme were 20 °C and 6.0, respectively. Kinetic studies showed that the thermal inactivation of the PPO followed first-order kinetics, with the activation energy, Ea = 52.39 Kcal mol^?1. The substrate specificity showed a high degree of PPO activity toward o-diphenolic compounds with the highest affinity toward caffeic acid among substrates studied. The apparent K_m values for caffeic acid and 4-methylcatechol as substrates for the Dechaunac PPO were 16 mmol and 25 mmol, respectively. The most potent inhibitors of the PPO were D.L-dithiothreitol and sodium metasulphite at the concentration level of 0.5 mmol.     

Effect of various inhibitors on enzymatic browning,antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa)

In this study, polyphenol oxidase (PPO) was isolated from fresh lettuce. Its optimum temperature and pH were found to be 40 °C and 7.0, respectively. Lettuce PPO was shown to use catechin, catechol, chlorogenic acid, caffeic acid and gallic acid as substrates. Among the substrates used, the greatest substrate specificity was observed with chlorogenic acid. Lettuce PPO was sensitive to some inhibitors. Ascorbic acid, cysteine, oxalic acid and citric acid were tested as potential inhibitors of lettuce PPO. Cysteine was the most effective inhibitor. Total phenol and total antioxidant activity contents were also determined in the presence of these inhibitors at room and refrigerator temperatures. Ascorbic acid and cysteine increased the total antioxidant activity of lettuce while citric and oxalic acids had no effect on the total antioxidant activity. Lettuce phenolics were protected from oxidation by ascorbic acid and cysteine.