INFLUENCE OF DEVELOPMENTAL STAGE, CULTIVAR, AND HEXAPEPTIDE AND CYCLODEXTRIN INHIBITORS ON POLYPHENOL OXIDASE ACTIVITY FROM TOMATO FRUITS

Two PPO preparations, one soluble and one particulate, were monitored at eight different developmental stages after anthesis of tomato fruits. Both PPO preparations showed significant decline after the 12th day postanthesis of fruit development. Although present in every cultivar tested, the two PPO preparations varied greatly in activity among the cultivars. The addition of 1‐mM synthetic hexapeptides caused inhibition over 10% on PPO activity measured in both preparations. The antibrowning effect of the β‐CD Cavasol W7M on PPO activity was also evaluated. Although the two families of compounds that we describe here, hexapeptides and CDs, react in very different ways either by physically blocking the active site of PPO or, alternatively, by chelating PPO substrates, they are common antibrowning compounds that can be commercially synthesized. These inhibitors are peptides and sugars, which are not likely to cause adverse health effects.     

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     

Prokaryotic expression and purification of Camellia sinensis polyphenol oxidase

BACKGROUND: Polyphenol oxidase (PPO) causes the postharvest loss of fruits and vegetables but is also a key factor in the quality development of tea. However, there are no reports on engineered active plant PPO purified from prokaryotic cells. RESULTS: In this study the ppo gene of about 1800 bp from Camellia sinensis cv. Yihongzao was successfully cloned and expressed in Escherichia coli. The PPOs purified from both the soluble fraction and the inclusion bodies showed activity. In addition, 1.0 × 10^?7 mol L^?1 Cu²⁺ and acidic conditions were found to be favourable for the engineered PPO catalysis of catechol oxidation. CONCLUSION: This paper represents the first report on C. sinensis ppo expression in E. coli and engineered active PPO purification. The results of the study provide a basis for the large‐scale preparation and application of PPO. Copyright © 2010 Society of Chemical Industry     

Purification of Polyphenoloxidase from the Purple‐fleshed Potato (Solanum tuberosum Jasim) and its Secondary Structure

ABSTRACT: Polyphenoloxidase (PPO) was purified from purple‐fleshed potatoes (Solanum tuberosum Jasim) using membrane concentration, ammonium sulfate fractionation, Resource Q ion exchange chromatography, and Sephacryl S‐200 HR gel permeation chromatography. PPO was purified 78‐fold from a crude extract. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis results showed that the purified enzyme has a major subunit molecular weight of 40 kDa. To elucidate the secondary structure of the purified PPO, circular dichroism (CD) was performed. The CD spectrum of the purified enzyme showed that PPO contains 35% α‐helix, 30% β‐turn, and 35% random coil structure.     

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%.     

Purification of polyphenol oxidase and the browning control of litchi fruit by glutathione and citric acid

Polyphenol oxidase (PPO, EC 1.10.3.2) was purified to homogeneity from litchi peel yielding a single protein with a molecular weight of about 75.6 kD by Sephadex G‐100 gel filtration, and a 108‐fold purification of PPO achieved. The enzyme was determined to be composed of two similar subunits. Glutathione, L ‐cysteine and citric acid suppressed PPO activity markedly, whereas ascorbic acid and n‐propyl gallate showed a little inhibition. Moreover, the effect was enhanced by the addition of citric acid. On the basis of the inhibition of PPO activity in vitro, the use of 10 mmol l ⁻¹ glutathione and 100 mmol⁻¹ l citric acid was found to give good control of the browning of litchi fruit, and an 80–85% inhibition of PPO activity was observed. It is suggested that application of glutathione in combination with citric acid may slow down the browning of litchi fruit. © 1999 Society of Chemical Industry     

PURIFICATION OF PEACH POLYPHENOL OXIDASE IN THE PRESENCE OF ADDED PROTEASE INHIBITORS1

Crude preparations of peach fruit (Prunus persica Batsch cv. Redskin) polyphenol oxidase (PPO) showed many apparent isoenzyme forms. Some of these forms were probably the result of proteolytic action of peach proteases while other forms were the result of association of PPO with carbohydrate materials. In the presence of protease inhibitors, Trasylol and phenylmethylsul-fonyl fluoride, three apparent isoenzyme forms of PPO were purified to homogeneity. The purification scheme included hydrophobic chromatography on phenyl sepharose CL-4B, hydroxylapatite chromatography, DEAE cellulose chromatography, and gel filtration on Ultrogel AcA 34. Minor contaminants remaining after these steps were separated from PPO by gel electrophoresis. The major PPO isoenzyme form (A) was purified 44 fold with an overall yield of 5.6% and contained no detectable carbohydrates. Isoenzyme forms A' and A' were purified 104 and 67 fold respectively, but still were associated with carbohydrate material. Cesium chloride centrifugation partially removed the carbohydrates associated with PPO A' and A'. Purified peach PPO A showed greater activity toward D-catechin (539%) and pyrogallol(l82%) than to catechol (100%). An apparent K₃ of 4, 0.3, and 2 mM was obtained with D-catechin, pyrogallol and catechol, respectively. The enzyme was severely inhibited by 10 μM 2,3-naphthalenediol (91%) and by 10 pM diethyl dithiocarbamate (100%).     

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.     

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.     

Cloning and some properties of Japanese pear (Pyrus pyrifolia) polyphenol oxidase,and changes in browning potential during fruit maturation

A PCR‐amplified genomic DNA fragment encoding Japanese pear (Pyrus pyrifolia) polyphenol oxidase (PPO) was cloned and sequenced. The DNA appears to encode a 66 kDa precursor protein consisting of a 56 kDa mature protein and a 9.5 kDa N‐terminal transit peptide. The amino acid sequence showed high homology with apple PPO. The PPO mainly existed as a soluble fraction in cells and was limitedly proteolysed, while the mature form (56 kDa) was detected in plastids. Immature fruits showing high browning potential had high PPO activity and a high level of phenolics, while mature fruits showing little browning had high PPO activity but a low level of phenolics. Copyright © 2003 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF BACILLUS SUBTILIS JM-3 PROTEASE FROM ANCHOVY SAUCE

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K_m and V_maxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine protease.     

Inhibitory effect of sericin on polyphenol oxidase and its application as edible coating

Sericin, a water‐soluble globular protein derived from silk industry wastewater, was investigated for food industrial applications. The results proved that sericin retarded polyphenol oxidase (PPO) activity. However, the degree of inhibition varied depending on the enzyme origins, the types of substrate, sericin content and sericin molecular size. Using catechol as a substrate, under the conditions studied, sericin lowered purified mushroom PPO and apple extract PPO activity by 40% and mango extract PPO by 75%. Kinetic studies on purified mushroom PPO indicated that the type of inhibition of sericin was dependent on the substrates used. Inhibitory effects of sericin increased as the sericin content increased. The reduction in sericin molecular size by enzymatic hydrolysis produced sericin hydrolysate with ability to decrease PPO activity approximately three times greater than that of sericin. Fresh‐cut Red Delicious apples coated with sericin showed significant reduction in weight loss and improvement in the colour and texture.     

Can grafting in tomato plants strengthen resistance to thermal stress?

Grafted and non‐grafted tomato plants were grown at 25 and 35 °C for 30 days. In tomato leaves we analysed total phenols, o‐diphenols, enzymatic activities (PAL, PPO and GPX) and dry weight. Our results indicate that heat stress in both grafted and non‐grafted tomato plants occurred at 35 °C and resulted in (1) increased PAL activity, (2) increased total phenols and o‐diphenols, (3) decreased PPO and GPX activities and (4) decreased dry weight. These results show that high temperature induces the accumulation of phenolics in tomato plants by activating their biosynthesis as well as inhibiting their oxidation. However, the impact of high‐temperature stress was smaller in grafted than in non‐grafted tomato plants. This indicates that grafted plants have a higher degree of resistance against thermal stress if the rootstock is more resistant to thermal stress. Copyright © 2003 Society of Chemical Industry     

Purification and kinetic characterization of polyphenol oxidase from Barbados cherry (Malpighia glabra L.)

Polyphenol oxidase (PPO) of Barbados cherry was extracted and purified through ammonium sulfate precipitation, gel filtration, and affinity chromatography. The purification factor for PPO was 60% with 8.3% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of PPO was approximately the sum of 52 and 38 kDa estimated by SDS–PAGE. The purity was checked by native PAGE, showing a single prominent band. The optimum pH was 7.2. The enzyme had a temperature optimum at 40 °C and was relatively stable at 60 °C, with 55% loss of activity. Sodium diethyl dithiocarbamate (SDDC), l-cysteine and ascorbate significantly inhibited PPO activity. 4-Methyl catechol and catechol were found to be efficient diphenolic substrates for cherry PPO, considering the _Vmax/_Km$V_{\text{max}}/K_{\mathrm{m}}$ ratio. The data obtained in this study may help to understand cherry fruit browning.     

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

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

IDENTIFICATION OF PROCYANIDIN A2 AS POLYPHENOL OXIDASE SUBSTRATE IN PERICARP TISSUES OF LITCHI FRUIT

Postharvest browning of litchi fruit results in short shelf life and reduced commercial value. Experiments were conducted to separate, purify and identify polyphenol oxidase (PPO ) substrates that cause litchi fruit to brown. PPO and its substrate were extracted from the pericarp tissues of litchi fruit. The litchi PPO substrate was purified using polyamide column, silica gel column and Sephadex LH‐20 column chromatography. The browning substrate was selected by a 0.5% FeCl₃ solution and then identified using a partially purified litchi PPO. Analyses of ultraviolet spectrometry, nuclear magnetic resonance and electrospray ionization mass spectrometry indicated that the PPO substrate was procyanidin A2. The substrate can be oxidized to ο‐quinones by litchi PPO and then form brown‐colored by‐products, resulting in pericarp browning of harvested litchi fruit.     

Effect of endo‐xylanase‐containing enzyme preparations and laccase on the solubility of rye bran arabinoxylan

Three commercial enzyme preparations with endo‐xylanase activity, namely Bio‐Feed Wheat, Bio‐Feed Plus and Grindamyl H 640, and laccase have been tested for their effects on the solubilisation of arabinoxylan (AX) in rye bran or autoclaved rye bran. Autoclaving efficiently increased the availability of AX for enzymatic degradation. Both Bio‐Feed Wheat (a monocomponent enzyme) and Bio‐Feed Plus (a multicomponent preparation with different enzymatic activities) efficiently degraded the autoclaved rye bran AX into lower‐molecular‐weight fragments. As much as 70% of the xylose residues and 58% of the arabinose residues in the autoclaved bran were soluble after treatment with Bio‐Feed Plus; the weight—average molecular weight of the detectable portion of these soluble polymers was 104 000 Da. Grindamyl H 640 solubilised only a small fraction of the AX in autoclaved rye bran; the molecular weight of these soluble fragments was higher than that of those released by the Bio‐Feed xylanases. Addition of laccase during treatment with Bio‐Feed Wheat or Grindamyl H 640 decreased the yield of water‐soluble AX. © 2003 Society of Chemical Industry     

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.     

Effect of thermal processing on oxide reductase inactivation and on colour fixing in erva‐mate (Ilex paraguariensis St. Hill) leaves

Leaves of erva‐mate (Ilex paraguariensis St. Hill) were heat treated with different combinations of time and temperature. Polyphenol oxidase (PPO) and peroxidase (POD) residual activities and colouring (L, a, b and ΔE) were studied after treatment for 3 weeks. The best results for enzyme inactivation after heat treatment were obtained with the leaves treated at 450 °C for 15 s and at 500 °C for 10 s, with reductions of 90.6% and 83.4% for PPO and 94.5% and 92.7% for POD. These groups also showed low levels of enzyme activity during the first few days of storage, and a few days later no enzyme activity was detected. The data on the colours, specially –a values (green colour), were in accordance with results for enzyme activity and helped in the research on the changes that occur in the leaves during storage. Data from this work can be suggested for the improvement of the current method of erva‐mate processing, which is still performed empirically.     

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.