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.     

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.     

Partial characterization of peroxidase from the leaves of thymbra plant (Thymbra spicata L. var. spicata)

Peroxidase (EC 1.11.1.7; donor: hydrogen-peroxide oxidoreductase) catalyses the oxidation of various electron donor substrates (e.g. phenols, aromatic amines). In this study, the peroxidase was extracted from Thymbra spicata L. var. spicata and, then partially purified with (NH₄)₂SO₄ precipitation and dialysis. The substrate specificity of peroxidase was investigated using 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS), o-dianisidine, o-phenylenediamine, catechol and guaiacol as substrates. Furthermore, the effects of buffer concentration, pH, temperature and thermal inactivation on enzyme activity were also studied. The results obtained have shown that (i) the best substrate is o-dianisidine, followed by ABTS, catechol, guaiacol and o-phenylenediamine, respectively; (ii) the best buffer concentration is 40 mM for o-dianisidine and catechol, 10 mM for ABTS and guaiacol, and 100 mM for o-phenylenediamine; (iii) optimum pH is 2.5 for ABTS and o-phenylenediamine, 6.0 for o-dianisidine, and 7.0 for catechol and guaiacol; (iv) optimum temperature is 20 °C for catechol, 40 °C for ABTS, guaiacol and o-dianisidine, and 50 °C for o-phenylenediamine; and (v) the enzyme activity in the thermal inactivation experiments was enhanced with increase in temperature with o-dianisidine as a substrate while its activity decreased with o-phenylenediamine.     

Purification and Characterization of Polyphenol Oxidase From Agaricus bisporus

A polyphenol oxidase was isolated from Agaricus bisporus using ammonium sulfate precipitation, combined with DEAE–Sepharose Fast Flow chromatography and Phenyl Sepharose CL-4B chromatography. The molecular weight of the purified polyphenol oxidase was determined to be about 43 kDa with N-terminal sequence Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation. The optimum temperature and pH for the polyphenol oxidase activity was 20°C and a pH of 6.5–7.0. Moreover, it was stable at 30 and 40°C over a 60 min pre-incubation time period. The K_m and V_max values of this polyphenol oxidase for catechol were 0.67 mM and 3333 U/ml min^?1, respectively. In addition, following the cross linking with polyphenol oxidase, the emulsifying activity index, foam capacity, and foam stability of the whey proteins were evaluated to be modified. This work was useful to better understand polyphenol oxidase from A. bisporus, and may lead to its practical use in the future.     

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 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.     

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.     

Lysyl oxidase from jumbo squid (Dosidicus gigas) muscle: purification and partial characterization

Lysyl oxidase (LOX; E.C.1.4.3.13) was purified from jumbo squid muscle (Dosidicus gigas) with 1900‐fold and yield 1.9%, and characterized for the first time. The purification procedure consisted of fractionation with urea and a combination of size‐exclusion and anion‐exchange chromatography. The enzyme had a molecular weight of 32 kDa, as estimated by SDS‐PAGE. Using a specific LOX substrate (1,5‐diaminopentane), its optimum activity was determined at pH 8.2 and 65 °C. Activation energy (E_a) of the enzyme was 69.94 kJ K^?1 mol^?1. The enzyme was strongly inhibited by β‐aminopropionitrile fumarate (BAPN), a specific LOX inhibitor. Moreover, purified LOX was able to work at different temperatures (20–90 °C) at pH 8.2. Although further research is needed, the results from this work suggest that based on LOX activity, this enzyme may be of practical use in preventing textural changes in jumbo squid during storage or processing.     

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     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

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 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.     

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.     

Characterization of a β-Glucosidase from an Edible Mushroom,Lycoperdon Pyriforme

A β-glucosidase from Lycoperdon pyriforme, a wild edible mushroom, was characterized biochemically. The enzyme showed a maximum activity at pH 4.0 and 50°C when p-nitrophenyl-β-D-glucoside was used as a substrate. K_m and V_max values were calculated as 0.81 mM and 1.62 U/mg protein, respectively. The enzyme activity was conserved about 85% over a broad range of pH (3.0–9.0) at 4°C after 24 h incubation. The activity was fully retained after 60 min incubation at 20–40°C. Na⁺, Li⁺, Mg²⁺, Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺, and Cu²⁺ did not affect the enzyme activity and 0.25% sodium dodecylsulfate inhibited the enzyme activity approximately 76%. Ethylenediamine tetra-acetic acid, phenylmethanesulfonylfluoride, and dithiothreitol showed no or a little negative effect on the enzyme activity. The resistance of the enzyme to some metal ions, chemicals, and ethanol along with the pH stability, can make it attractive for future applications in industry.     

Immobilization and characterization of naringinase for the hydrolysis of naringin

The degree of hydrolysis of naringin was investigated at various temperatures (40, 50, 60 °C), enzyme concentrations (0.01–0.30 mg ml⁻¹), and pH values (2.5–5.5) for naringinase enzyme. Naringinase was immobilized on celite by simple adsorption. Naringin content was determined by HPLC method. The degree of hydrolysis of naringin showed a linear increase up to an enzyme concentration of 0.2 mg ml⁻¹ that corresponds to 82% hydrolysis. The optimum values of pH for the hydrolysis of naringin were 4.0 for free and 3.5 for immobilized enzymes. Maximum enzyme activities were found to be 70 and 60 °C for free and immobilized enzymes, respectively. The values of K _m,app and V _max,app calculated were 1.22 mM and 0.45 μmol min⁻¹ mg enzyme⁻¹ for free and 2.16 mM and 0.3 μmol min⁻¹ mg enzyme⁻¹ for immobilized enzyme, respectively. The mathematical modelling was applied to the experimental data for hydrolysis of naringin as a function of time at 30, 40 and 50 °C. The increase in temperature from 30 to 50 °C increased the rate constant 3.09 times for free enzyme. However, the rate constants found for immobilized enzyme applications did not increase in a similar trend as a function of temperature. The retained activity of celite-adsorbed naringinase was found to be 83% at their optimum conditions. The retained activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60 °C).     

Kinetic behaviour and thermal inactivation of pectinlyase used in food processing

Kinetic properties and thermal inactivation of pectinlyase (PL) were assayed in commercial pectinase preparations (Rapidase C80, Pectinase CCM, Pectinex 3XL and Grindamyl 3PA) by using apple pectin as substrate. The PL activity of Rapidase C80 showed substrate inhibition, while the other enzyme preparations followed typical Michaelis–Menten kinetics. The optimum pH and temperature values for PL activity lay within the range of 5.5–6.5 and 35–40 °C, respectively. PL was heat‐inactivated with simple first order kinetics. With respect to this, thermodynamic activation parameters (ΔH^#, ΔS^# and ΔG^#) using a non‐linear Arrhenius plot were calculated. The Pectinase CCM and Pectinex 3XL PL showed a half‐life (t_1/2) at 50 °C of 2.0 min and 11.8 min, 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.     

Polyphenol Oxidase System in Red Delicious Apples

The polyphenol oxidase system (E.C. 1.14.18.1) in‘Red Delicious’apples (Malus domestica) was investigated. Polyacrylamide gel electrophoresis (PAGE) revealed two active isoenzymes as detected with pyrocatechol substrate. The crude enzyme extract (acetone powder) was found to have one optimum pH of 6.2 and one optimum temperature for the browning reaction of 30°C with the experimental conditions described. The Michaelis constant (Km) as an average of the two active enzyme forms was 2.2 × 10^?lM pyrocatechol and the average maximum velocity (Vmax) was 4.8 × 10^?l AA390 per minute.     

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.     

Acid phosphatase in the tubers of Dioscorea species and its purification from the white yam (D. rotundata poir)

Acid phosphatase activity was determined in 15 cultivars from four species of yam. A 12-fold purification of the enzyme from Dioscorea rotundata (cv. chikakwondo) gave a homogeneous preparation as demonstrated by polyacrylamide gel electrophoresis. This enzyme preparation has an apparent molecular weight of 115 000 ±2000 and an optimum activity at a pH of 5·20 and a temperature of 50°C. The K_m of the enzyme is 3·81 mM with disodium p-nitrophenylphosphate (p-NNP) as a substrate. The energy of activation, heat of activation, energy of inactivation and heat of inactivation are 7·0, 6·4, 4·41 and 4·34 kcal M^?1, respectively. Although it has very little activity with most organic phosphoric acid esters, it is significantly inhibited by Ca²⁺, Hg²⁺ and EDTA and activated by Mg²⁺. The enzyme has a half-life of 50,17 or 13 days, respectively, when stored at 6-8°C, 0°C or room temperature (29±2°C).