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.     

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.     

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.     

Quantification and characterisation of polyphenol oxidase from vanilla bean

Polyphenol oxidase (PPO) of Vanilla planifolia Andrews beans was extracted and purified through ammonium sulphate precipitation, dialysis, and gel filtration chromatography. PPO activity was measured by improved UV technique using 4-methylcatechol and catechol as substrates increasing substantial sensitivity of previous procedure. The optimum pH and temperature for PPO activity were found to be 3.0 and 3.4 and 37 °C, respectively. K_m and V_max values were found to be 10.6 mM/L and 13.9 OD₃₀₀ min⁻¹ for 4-methylcatechol and 85 mM/L and 107.2 OD₃₀₀ min⁻¹ for catechol. In an inhibition test, the most potent inhibitor was found to be 4-hexylresorcinol followed by ascorbic acid. The thermal inactivation curve was biphasic. Activation energy (E_a) and z values were calculated as 92.10 kJ mol⁻¹ and 21 °C, respectively.     

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.     

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.     

Comparison of Polyphenol Oxidases Prepared From Different Parts of Artichoke (Cynara Scolymus L.)

Artichoke polyphenol oxidases (PPOs) were obtained by (NH₄)₂SO₄ precipitation using ascorbic acid, polyvinylpyrrolidone, and Triton X-100. The PPO content of artichoke head (AHPPO) was 8820 units (mg protein)^?1 as compared with 3370 units (mg protein)^?1 in artichoke leaves-and-stem (ALSPPO) by using catechol as a substrate. The substrates of both AHPPO and ALSPPO are o-diphenols, such as catechol, pyrogallol, and L-DOPA. Optimum pH and temperature of both PPOs were determined. AHPPO had higher thermal stability than ALSPPO. Also, T_m (the midpoint of thermal inactivation) and t_1/2 (half-life) values were determined. K_m and V_max of both PPOs were observed to be similar. Twelve inhibitors were tested and their I₅₀ values were determined. The most effective inhibitors were found to be potassium cyanide, ascorbic acid, L-cysteine, and thiourea. Sodiumdodecylsulfate, urea, and cupric sulfate caused an increase about 20–30% in the PPO activity.     

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 of polyphenol oxidase from broccoli (Brassica oleracea var. botrytis italica) florets

Polyphenol oxidase (PPO) from broccoli florets was extracted and purified through (NH₄)₂SO₄ precipitation, ion-exchange and gel filtration chromatography. The molecular weight was estimated to lie between 51.3 and 57 kDa by sodium dodecyl sulphate-polyacrylamide gel electophoresis (SDS-PAGE) and gel filtration. The effects of substrate specificity, pH, and sensitivity to various inhibitors: citric acid, ascorbic acid, sodium sulphate and EDTA (sodium salt of ethylenediaminetetraacetic acid) of partially purified PPO were investigated. Polyphenol oxidase showed the best activity toward catechol (K_M = 12.34 ± 0.057 mM, V_max = 2000 ± 8736 U/ml/min) and 4-methyl catechol (K_M = 21 ± 0.087 mM, V_max = 28.20 ± 0.525 U/ml/min). The optimum pH for broccoli PPO was 5.7 with catechol and 4-methylcatechol as substrates. The most effective inhibitor was sodium sulphate.     

Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.     

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.     

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     

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 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.     

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     

Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.     

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%.     

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.     

CHARACTERIZATION AND INACTIVATION BY THERMAL AND PRESSURE PROCESSING OF STRAWBERRY (FRAGARIA ANANASSA) POLYPHENOL OXIDASE: A KINETIC STUDY

Polyphenol oxidase (PPO) was isolated from strawberries (Fragaria ananassa) and some of its biochemical characteristics were studied. The optimum pH for strawberry PPO activity was pH = 5.0. K_mand V_maxvalues were 5.95 mm and 133.8 A_420nm/min using a 50 mm catechol substrate solution. Kinetic studies showed that the thermal inactivation of strawberry PPO followed biphasic kinetics, resulting in an activation energy of 314.1 kJ/mol for the labile fraction and 321.3 kJ/mol for the stable fraction. Pressure/temperature inactivation of the stable fraction of strawberry PPO can be adequately described by a first‐order model. Pressure and temperature were found to act synergistically, except in the high temperature–low pressure region where an antagonistic effect was observed. A second‐degree polynomial model was successfully applied to describe the temperature/pressure dependence of the inactivation rate constants of the stable strawberry PPO fraction.