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 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 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.     

Characterisation of ‘Starking’ apple polyphenoloxidase

Experiments were performed to optimise the extraction conditions of ‘Starking’ apple fruit polyphenoloxidase (PPO), to evaluate the affinity and specificity towards several substrates and to study the stability of enzyme extracts from apple samples stored under different conditions. Sodium phosphate buffer (0·2 M , pH 6·5) plus 0·25% Triton X₁₀₀ and 1% or 2% PVPP was found to be the most efficient extraction medium. Chlorogenic acid, dopamine and 4-methylcatechol showed similar specificity towards PPO, and chlorogenic acid was found to be the best substrate for the enzyme. Enzyme extracts from frozen cut apple stored at −4°C, and extracts from lyophilised apple samples stored at 4°C were more stable than extracts obtained from fresh-cut or acetone powder samples. © 1998 SCI.     

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.     

Polyphenol oxidase properties,anti-urease,and anti-acetylcholinesterase activity of Diospyros lotus L. (Plum Persimmon)

Diospyros lotus fruit polyphenol oxidase was purified using affinity chromatography, resulting in a 15-fold enrichment in specific activity. The purified enzyme, having 16.5 kDa molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibited the highest activity toward 4-methylcatechol. Maximum diphenolase activity was reached at pH 7.0 and 60°C in the presence of 4-methylcatechol. K_m and V_max values were calculated as 3.8 mM and 1250 U/mg protein, respectively. Ascorbic acid was a promising inhibitor with an IC₅₀ value of 0.121 µM. The activity of the purified enzyme was stimulated by Fe²⁺, Sr²⁺, Zn²⁺, and K⁺ and deeply inhibited by Hg²⁺, at 1 mM final concentration. Aqueous extract of Diospyros lotus L. fruit showed strong substantial urease and acetylcholinesterase inhibition, with IC₅₀ values of 1.55 ± 0.05 and 16.75 ± 0.11 mg/mL, respectively.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

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.     

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.     

Strawberry Polyphenoloxidase: Extraction and Partial Characterization

Mixing strawberries ground under liquid nitrogen with polyvinylpyrrolidone alone or combined with Amberlite XAD-4 in pH 4.5 buffer yielded stable polyphenoloxidase (PPO) extracts. Temperature stability of the enzyme varied with substrate and followed first order kinetics of inactivation. pH optima were 5.5 with catchol and 4.5 with 4-methylcatechol. Maximum activity was with D-catechin, followed by 4-methylcatechol and pyrogallol with no cresolase activity. Inhibition occurred with diethyldithiocarbamate, potassium metabisulfite, KCN and dithiothreitol. Benzenesulfinic acid, cysteine and ascorbic acid blocked the browning reaction without enzyme inhibition. Extraction with Dowex AG 2-X8 resulted in browning. No loss in activity after 95 days occurred with quick-frozen fruit stored under nitrogen at -40°C.     

Quality and biochemical changes of longan (Dimocarpus longan Lour cv. Daw) fruit under different controlled atmosphere conditions

Sulphur dioxide has been used to control pericarp browning in longan fruit. However, due to health and regulatory concerns, alternative treatments should be tested. The objective of this study was to find the tolerance levels of longan fruit to low O₂ (2%, 5%, 10% and 15%) and elevated CO₂ (5%, 10%, 15% and 20%) at 2 °C. According to the tolerance study, controlled atmospheres (CA) of 5% O₂ + 5% CO₂, 5% O₂ + 10% CO₂ and 5% O₂ + 15% CO₂ were compared with normal air (control) at 2 °C. Pericarp browning and decay incidence of longan were significantly (P ≤ 0.05) higher in control than all the CA treatments. CA storage reduced polyphenol oxidase (PPO) activity, maintained L* value and slowed down a decrease in total phenolic contents (TPC). Pericarp browning was highly correlated with PPO, L* and TPC.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

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.     

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     

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.     

Effect of hexanal vapour on longan fruit decay,quality and phenolic metabolism during cold storage

The effects of postharvest treatment with hexanal vapour on longan fruit decay, quality, hexanal residue, phenolic compound content, and polyphenoloxidase (PPO) and peroxidase (POD) activities were studied during storage at 5 °C for 30 days. Hexanal exposure for 2 h at 900 μL L^?1 before cold storage reduced the percentage of fruit with decay and was deemed the optimum treatment. Hexanal exposure resulted in a pericarp that was more reddish brown and less intense in colour. Hexanal residue in the pericarp and aril of fumigated fruit was several fold higher than that of nonfumigated fruit, although levels were low at the end of cold storage. Electrolyte leakage of pericarp increased during 5 °C storage and was further increased by hexanal exposure. Hexanal reduced pericarp phenolic content, and increased PPO and POD activities. Overall, use of hexanal vapour reduced postharvest disease of longan fruit but increased the likelihood of pericarp browning.     

Inactivation Kinetics of the Most Baro-Resistant Enzyme in High Pressure Processed Litchi-Based Mixed Fruit Beverage

This work focused on a litchi-based mixed fruit beverage, comprising of coconut water and lemon juice, mixed in an optimized proportion. Based on preliminary studies, three resistant spoilage enzymes were identified in the beverage, viz. polyphenol oxidase (PPO), peroxidase (POD), and pectin methyl esterase (PME). The response surface methodology (RSM) based on central composite face-centered design (FCCD) screened out PPO as the most resistant enzyme within the high pressure processing (HPP) domain of 200–600 MPa/30–70 °C/0–20 min. A detailed kinetic study was conducted on PPO inactivation within the same HPP domain along with a set of thermal treatments (0.1 MPa/30–70 °C). A synergistic effect of pressure and temperature on PPO inactivation was observed, throughout the HPP domain. However, PPO was almost completely inactivated at 500 MPa/70 °C/20 min. The inactivation order (n) values for PPO were 1.10 and 1.25 for thermal and HPP treatments, respectively. For every 10 °C rise in temperature, the inactivation rate constant (k, U^n-1 min^?1) increased approximately by 1.5 times, within 50–70 °C (at 0.1 MPa), while a 10-fold increase was obtained in the case of HPP treatments. The activation energy (E _a ) and the activation volume (V _a), depicting the temperature and pressure dependence of k, was found to decrease slightly, with an increase in pressure and temperature, respectively. The PPO inactivation rate constant was modeled as a function of both temperature and pressure conditions by combining both Arrhenius and Eyring equations.     

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.     

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.     

Purification and characterisation of a polyphenol oxidase from Boletus erythropus and investigation of its catalytic efficiency in selected organic solvents

Polyphenol oxidase (PPO) was purified from Boletus erythropus using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. Optimum pH and temperature were found to be 8.0 and 20 °C, respectively, using 4-methylcatechol as a substrate. The enzyme was extremely stable between pH 3.0 and 9.0 after 24 h incubation at 4 °C. B. erythropus PPO was also quite stable between 10 and 30 °C after 4 h incubation. The K_m and V_max values were calculated as 2.8 mM and 1430 U/mg protein by Lineweaver–Burk curve, respectively. The enzyme activity was inhibited by sodium metabisulfite, ascorbic acid, sodium azide and benzoic acid. It was seen that the mushroom PPO was an effective biocatalyst in selected organic solvents, such as dichloromethane, dichloroethane and toluene, when catechin was used as a substrate. All data support that B. erythropus has a highly active PPO, possessing similar biochemical and kinetic characteristics to other plant PPOs.