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Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

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Abstract

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K+, other metal ions such as Zn2+, Cu2+, Fe3+, Ca2+, Fe2+ and Na+ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent Km and Vmax values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.

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... However, great diversity was found in the effect of metal ions on PPO extracted from different plants. For example, the acceleration effect of Cu 2? on PPO was also found in chufa (Eleocharis tuberosa) corms [25]. Acceleration of PPO activity by CuCl 2 was supposed as the result of PPO being a copper-containing enzyme [26]. ...
... [12], Welder (4.5), Noble muscadine (4.5-5.5) [25], Victoria (5.0) [8], and Ravat 51 and Niagara (5.5) [9]. ...
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... Moreover, Sun et al. have observed that adding Fe 2+ and Na + into the substrate-enzyme system increased the PPO activity. The plant PPO is suggested to have one binuclear Cu site per protein molecule for the reaction centre and various metal ions may affect the substrate by combining this reaction centre [36]. Similar dependencies were observed in our results (1 mM and 2 mM solutions of FeCl 2 and NaCl). ...
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... [2] It has a long history of cultivation in China and is widely planted. [3] The Chinese water chestnut peels are by-products that constitute approximately 20% (w/w) of the whole fruit, which were often discarded, resulting in wasted resources and environmental pollution. [4] Water chestnut peels are rich in brown pigments, which are an excellent water-soluble natural food coloring, and its main components are flavonoids (such as flavonoids, flavonols, flavonoids). ...
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... Hal tersebut mengakibatkan semakin efektif pula air dan panas dalam menginaktivasi enzim polifenolase, sehingga semakin lama blanching dapat meningkatkan nilai kecerahan dari fries uwi putih. Menurut Sun et al. (2010), polifenol oksidase mengkatalis oksidasi polifenol menjadi α-kuinon dengan adanya oksigen dan α-kuinon berpolimerisasi yang menyebabkan timbulnya pencoklatan atau timbulnya pigmen yang tidak diinginkan. ...
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Putri Syepty Sri Janatul Munawaroh, Widya Dwi Rukmi Putri, Lia Hapsari. 2018. Characteristics of Water Yam (Dioscorea alata) Fries with Study of Calcium Chloride Concentration and Blanching Time. Jurnal Teknologi Pertanian 19(1): 33-42. Indonesia has many kind of tubers that did not use optimally such as water yam (Dioscorea alata). Water yam can potentially developed into a product such as french fries. However, the problem of water yam has a less crispiness texture and has a colour distortion caused by browning process. The aims of this research was to determine the effect of calcium chloride concentrations (CaCl2) and blanching time on the chemical, physical, and organoleptic characteristics of water yam (Dioscorea alata) fries. The research used a Randomized Block Design (RDB) with 2 factors that were concentration of calcium chloride (CaCl2) (0, 1, and 2%) and blanching time (3, 6, and 9 minutes). Data were analyzed using ANOVA, followed by LSD with significant level 5%. The determination of the best treatment using multiple attribute Zeleny. The best treatment was concentration of calcium chloride (CaCl2) 2% and the blanching time of 6 minutes with water content 40.76%; starch 26.14%; fat 19.62%; protein 2.78%; ash 1.09%; texture 5.97 N; lightness (L *) 63.7; redness (a *) 1.34; yellowness (b *) 15.41; the value of the organoleptic taste 2.77; aroma 3.5; color 3.43; texture 2.93; and appearance of 3.17.
... Although optimum PPD temperatures vary among plants, the extraction methods and the types of substrates used, the activity of PPD is mostly obtained between 30-50 °C. It had been previously shown that different plant types exhibited different optimum temperatures, such as 35 °C for Chinese parsley (Lin et al., 2016), 30-40 °C for Hemşin apple (Aydin et al., 2015), 40 °C for artichoke (Dogan et al., 2005), 45 °C for chufa corns (Sun et al., 2010), 35 °C for mamey (Palma-Drozco et al., 2011), 8 °C for jackfruit (Tao et al., 2013) using catechol as the substrate. It is clear that the optimum temperature of purslane PPD is quite high compared to those reported for most PPD's from other sources. ...
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... Similar reports for the optimal temperature and optimal pH have been presented. The previous studies also raported that optimum temperature values were 40 ºC for artichoke (24), Barbados cherry (25) , Cape gooseberry (26) and 45 ºC for chufa corns (27), using catechol as a substrate; 30 ºC for aubergine (28) , 56 ºC for Amaya apple (29) using 4-methylcatechol as a substrate. ...
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... This herbal plant belongs to the sedge family, which is often found in wet farmlands or pool districts. It has been suggested that this fruit possesses some health benefits such as antimicrobial effects on bacteria, antioxidant activity, inhibition of inflammation and treatment for pharyngitis and laryngitis [8][9][10]. generally, the Chinese water chestnuts are washed, peeled, sliced and packaged, before being commercially sold for use in restaurants, hotels and homes [11]. ...
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Lychee polyphenoloxidase (PPO) was extracted and partially purified using ammonium sulphate precipitation and dialysis. The comparative analysis of PPO property was performed using its endogenous substrate (-)-epicatechin and exogenous substrate catechol. The pH optima for activity and activation temperature profiles of lychee PPO were very different when the enzyme reacted with endogenous and exogenous substrates. The addition of ethylenediaminetetraacetic acid disodium salt into the endogenous or exogenous substrate-enzyme system exhibited the same lowest inhibition of the PPO activity. However, l-cysteine was most effective in inhibiting enzymatic activity in the endogenous substrate-enzyme system while ascorbic acid was the best inhibitor in the exogenous substrate-enzyme system. Fe(2+) greatly accelerated the enzymatic reaction between endogenous substrate and PPO, but Cu(2+) exerted the same effect on the reaction between exogenous substrate and PPO. Based on the kinetic analysis, lychee PPO could strongly bind endogenous substrate but it possessed a higher catalytic efficiency to exogenous substrate. Copyright © 2007 Elsevier Ltd. All rights reserved.
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Polyphenoloxidase (PPO) presented different specific activities at different locations in the imperial tiger prawn (Penaeus japonicus), with the highest values in the carapace. The procedure achieved a degree of purification, close to 70 times, increasing relative activity by means of ammonium sulphate (0-40%) saturation. Isoelectric focusing showed two bands around pI 5.0. The optimum temperature for PPO reaction with DOPA was between 40 and 60°C, however thermal stability was greatest at temperatures below 35°C. The enzyme was most active at pH 5 and 8, but most stable at basic pH. Pressurization of the enzymatic extract was assayed within a range of 0.1-400 MPa, for 10 min at <10°C. Pressure-induced inactivation was evident, particularly at 300-400 MPa. Total inhibition of the extract was achieved only with ascorbic acid and citric acid at pH 3.0. 80 μg/ml sulphite, 150 μg/ml of kojic acid, 1 g/l of 4-hexylresorcinol or 0.1 g/l of sodium benzoate was required for 80% inhibition. © 2001 Elsevier Science Ltd. All rights reserved.
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This paper presents a kinetic study of the effect of sodium chloride on the catecholase activity of latent grape polyphenol oxidase. The modifier showed a strongly pH dependent inhibitory effect at pH values <5 but acted as an activator at higher values. Other salts such as potassium nitrate and sulfate also activated the enzyme, indicating an unspecific activation effect by ionic strength. Furthermore, at pH values <5, at which the enzyme followed Michaelis−Menten behavior, the presence of sodium chloride gave rise to a lag period in the product accumulation curves, generating positive kinetic cooperativity in the steady-state kinetics. In contrast, at higher pH values, at which the enzyme showed a lag phase and exhibited negative cooperativity, the presence of a salt decreased cooperativity. The inhibition data are consistent with a mechanism by which chloride ions bind to both protonated forms of the enzyme, free enzyme and the enzyme−substrate complex, the latter species undergoing a conformational change. This mechanism was simulated by computer, and good agreement was obtained with the experimental results. Keywords: Polyphenol oxidase; chloride; inhibition; activation; slow transition
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Chinese water chestnut (CWC) is a vegetable in which the edible storage parenchyma exhibits thermal stability to texture. This property has been attributed to the presence of large quantities of simple phenolics which exhibit pH-dependent autofluorescence. The identities of the major phenolic components were assessed by reverse-phase HPLC after sequentially extracting them with increasing strengths of alkali from purified CWC cell wall material. The levels of cold-alkali-liberated phenolics were very high (12·2 mg g−1 cell wall material). Ferulic acid dominated the monomeric phenol fraction (7·2 mg g−1), although small quantities of aldehydes were also released. A number of different diferulic acids were also identified, of which the 8-O-4′-linked form was the major component (2·8 mg g−1), with 8,5′-diferulic acid and the familiar 5,5′-form also present in smaller quantities (910 and 988 μg g−1, respectively). Hence, approximately 40% of the ferulic acid in the wall was present as dimers which are potentially available to form extensive, heat-resistant cross-links between polysaccharides within the wall, and between cells.
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Postharvest browning of litchi fruit results in a short life and a reduced commercial value. The experiments were conducted to separate, purify and identify the polyphenol oxidase (PPO) substrates that cause litchi fruit to brown. PPO and its substrates were, respectively, extracted from fruit pericarp tissues. The substrates for litchi PPO were separated and purified using polyamide column chromatography, silica gel column chromatography and preparative thin layer chromatography. The substrate was further identified by 0.5% FeCl3 solution and enzymatic reaction with litchi PPO. On the basis of UV, 1H NMR, 13C NMR, and ESI-MS data, the direct substrate for the PPO from litchi fruit pericarp tissues was identified to be (−)-epicatechin.
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Ascorbic acid (AA), dehydroascorbic acid (dehydroAA), isoascorbic acid (isoAA), ascorbic acid-2-phosphate (AA-2- PO4), and ascorbic acid-2-sulfate (AA-2-SO4) were tested as inhibitors of mushroom polyphenoloxidase (PPO). Kinetic analysis indicated that AA and isoAA were more effective than dehydroAA. The half times (t1/2) that decreased 50% of PPO activity for AA, isoAA and dehydroAA were 2.5, 3.1, and 1.9 hr, respectively, and the concentrations that inhibited half of PPO activity were as follows: ascorbic acid, 0.04 mM, isoAA, 0.25 mM; and dehydroAA, 7.5 mM. Electron spin resonance studies demonstrated that the Cu2+ of PPO was reduced to Cu+ by AA. AA-2-PO4 and AA-2-SO4 were not inhibitors for PPO. However, the digestion of AA-2-PO4 with acid phosphatase yielded AA to inhibit PPO activity. AA-2-SO4 was found to be a poor substrate for sulfatase.
Article
Table beet polyphenol oxidase (PPO) causes undesirable color in underblanched products. The effects of pH and temperature on PPO activity were studied. Varietal differences and the effects of blanching on PPO activity of table beets were measured. The enzyme was most active at pH 7.0 and temperature 25°C. The rate of heat inactivation increases rapidly with increasing temperature and follows pseudo-first order kinetics. The enzyme activity varied among different cultivars of table beets. Water blanching deactivated the enzyme but the time required for a complete inactivation varied according to the size of table beets.
Article
Application of chitosan coating to browning control and quality maintenance of fresh-cut Chinese water chestnut (CWC) was investigated. Fresh-cut CWC were treated with aqueous solution of 0.5, 1 or 2 g chitosan/100 mL, placed into trays over-wrapped with plastic films, and then stored at 4°C. Changes in color, total phenolic content and phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) activities were measured. The effects of chitosan coating on eating quality and overgrowth of spoilage organisms were also evaluated. Application of chitosan coating delayed discoloration associated with reduced activities of PAL, PPO and POD as well as lower total phenolic content, and slowed down the loss in eating quality associated with higher contents of total soluble solids, titrratable acidity and ascorbic acid of fresh-cut CWC. Disease development of the fresh-cut CWC-treated chitosan coating was also inhibited compared to the control. Increasing the concentration of chitosan coating markedly enhanced the beneficial effects. The results showed that application of chitosan coating effectively extended shelf life and maintained quality of fresh-cut CWC.
Article
The potential usage of salicylic acid (SA) as a powerful anti-browning agent in fresh-cut Chinese water chestnut (CWC) was investigated. The fresh-cut CWC were dipped for 1 min in solutions of 0, 1, 2 or 4 mM SA, then placed in trays over-wrapped with plastic films, and finally stored at 4 °C. Changes in color, eating quality, and disease incidence were evaluated, while activities of phenol-associated enzymes, polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), and concentrations of total soluble solid, titratable acidity and ascorbic acid were measured. SA treatment delayed discoloration, maintained eating quality with higher content of the quality attributers, and reduced activities of or delayed the increases in activities of PPO, POD and PAL in fresh-cut CWC. However, SA had no significant inhibition of the activities of PPO and POD in an in vitro test, indicating that the beneficial effect of SA was indirect. Further research is needed to elucidate the inhibition of the surface browning of the fresh-cut CWC by SA.
Article
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 Vmax and Km 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.
Article
Partial characterization of polyphenol oxidase activity in artichoke heads is described. Stable and highly active PPO extracts were obtained using 1.0% (w/v) polyethylene glycol (PEG), 1.5% (w/v) Triton X-100 and 0.1% NaCl in 0.2 M potassium phosphate buffer, pH 6.0. Three isoenzymes of the artichoke PPO were detected by polyacrylamide gel electrophoresis. The pH optimum for artichoke PPO was found to be a very broad (5.0–7.0) and the enzyme activity was stable in the range 6.0–7.0 at 25 °C for 60 min. The optimum temperature was 25 °C. The enzyme was heat-stable between 20 and 30 °C and completely inactivated at 80 °C after 5 min. The activation energy (Ea) with catechol was 15.8 kJ/mol at pH 6.0. PPO showed activity to catechol, pyrogallol and 4-methylcatechol, dl-dopa, l-dopa and gallic acid. (Km and Vmax values were 10.2 mM and 19,662 U/ml min for catechol, 12.4 mM and 12,500 U/ml min for 4-methylcatechol, 14.3 mM and 8065 U/ml min for pyrogallol, 37.7 mM and 5865 U/ml min for l-dopa 36.3 mM and 6060 U/ml min for dl-dopa, 43.6 mM and 4620 U/ml min for gallic acid, respectively). l-tyrosine was also tested but was not oxidized by artichoke PPO. The I50 values of inhibitors studied on PPO were determined by means of activity percentage (I50) diagrams. The values were 6.17 × 10−5 M, 6.32 × 10−5 M, 9.11 × 10−5 M, 1.76 × 10−5 M, 1.47 × 10−5 M, 8.33 × 10−5 M, 4.12 × 10−5 M, 1.94 × 10−4 M and 1.83 × 10−5 M for glutathione, thiourea, sodium azide, sodium metabisulfite, dithiothreitol, β-mercaptoethanol, sodium diethyl dithiocarbamate, oxalic acid and ascorbic acid, respectively. Therefore, the most effective inhibitor was dithiothreitol, followed in decreasing order by sodium metabisulphide and ascorbic acid. Metal ions (Zn++, Ba++, Cu++) were poor inhibitors of the enzyme at 10 mM.
Article
Taro (C. esculenta) is a staple food in many tropical regions. A comparative study of crude polyphenoloxidases from taro (tPPO) and potatoes (pPPO) was carried out to provide information useful for guiding food processing operations. Crude PPO was prepared by cold acetone precipitation using ascorbic acid as antioxidant. The PPO content of taro acetone powder was 770±17 units (mg protein)−1 as compared with 3848±180 units (mg protein)−1 in potato acetone powder. The pH-activity optimum was pH 4.6 for tPPO and pH 6.8 for pPPO. Both enzymes retained >80% activity after incubation at pH 4.5–8 but there was rapid activity loss at pH < 4. The temperature-activity optimum (Topt) was 30°C for tPPO and 25°C for pPPO with 75 and 27% of their respective maximum activity retained at 60°C. Both tPPO and pPPO were irreversibly inactivated by 10 min heating at 70°C. The activation enthalpy (ΔH#) and activation entropy (ΔS#) for tPPO heat-inactivation were 87.4 (±0.1) kJ mol−1 and −56.2 (±4) J mol−1 K−1, respectively. For pPPO, ΔH# was 59.1 (±0.1) kJ mol−1 whilst ΔS# was −141 (±4) J mol−1 K−1. The apparent substrate specificity was established from values Vmax/Km as: 4-methylcatechol>chlorogenic acid>dl-dopa>catechol>pyrogallol> dopamine>>caffeic acid for tPPO. There was no detectable activity towards caffeic acid. The substrate specificity for pPPO was: 4-methylcatechol>caffeic acid>pyrogallol>catechol>chlorogenic acid >dl-dopa>dopamine. According to the order of inhibitor effectiveness (sodium metabisulphite>ascorbic acid>NaCl≈ (EDTA), there was a significant lag-phase before increases occurred in the absorbance at 420 nm. Preincubation of PPO with inhibitors increased the extent of inhibition, indicating a direct effect on the structure of the enzyme.
Article
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 KM (5.5 mM) and the biggest Vmax/KM (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 Ki values for glutathione inhibitor, using catechol, 4-methylcatechol and pyrogallol substrates, were calculated, and it was found that the type of inhibition was competitive.
Article
Prevention of browning in pineapple puree by thermal inactivation of enzyme, Polyphenoloxisase (PPO), was examined between 40 and 90 °C and in relation to exposure time. The amount of inactivation was measured as a function of time and temperature under isothermal conditions. Reaction rate constant and activation energy (Ea) as well as Decimal reduction time (D) and z-value of thermal inactivation, were determined. The rate of inactivation varied with temperatures and follows a logarithmic law. Kinetic studies showed that the thermal inactivation (40–90 °C) of the PPO followed first-order kinetics.
Article
Half of the world's fruit and vegetable crops is lost due to postharvest deteriorative reactions. Polyphenol oxidase (PPO), found in most fruit and vegetables, is responsible for enzymatic browning of fresh horticultural products, following bruising, cutting or other damage to the cell. Chemical methods for controlling enzymatic browning include the use of sodium bisulfite, ascorbic acid and/or packaging under controlled atmospheres. Current approaches to understanding and controlling enzymatic browning are presented in this review article, with special focus on the use of antisense RNA as a control method.
Article
Polyphenol oxidase (PPO) was isolated from longan (Dimocarpus longan Lour.) fruit peel, with a 46-fold purification of PPO by ammonium sulfate, Sephadex G-200 and Phenyl Sepharose being achieved. Pyrogallol, 4-methylcatechol, and catechol were good substrates for the enzyme, and activity with chlorogenic acid, p-cresol, resorcinol, or tyrosine was not observed. The optimal pH for PPO activity was 6.5 with 4-methylcatechol. The enzyme had a remarkably temperature optimum (35°C) and was relatively stable, requiring a little more than 20 min at 50°C for 50% loss of activity. Reduced glutathione, l-cysteine, thiourea, FeSO4 and SnCl2 markedly inhibited PPO activity, whereas MnSO4 and CaCl2 enhanced PPO activity. Data obtained in this study might help to better understand longan fruit peel browning.
Article
Litchi (Litchi chinensis Sonn.) fruit peel polyphenol oxidase (PPO) was partially purified 21 fold by ammonium sulfate fractionation and gel filtration. Pyrogallol, catechol, and 4-methylcatechol were good substrates for the enzyme; with no activity observed with chlorogenic acid, p-cresol, resorcinol, or tyrosine. The optimal pH for PPO activity was 7.0 with 4-methylcatechol, with the enzyme being most stable at pH 7.4. The enzyme was relatively temperature stable with maximum activity at 70 °C and requiring a little less than 10 min at 90 °C for 50% loss of activity. The Km and Vmax for the enzyme, with 4-methylcatechol, were 10 mM and 1.47 × 104 units/min per mg protein, respectively. The enzyme was not activated by SDS. Reduced glutathione, l-cysteine, tropolone, thiourea, FeSO4, and SnCl2 markedly inhibited PPO activity, whereas MnSO4 and CaCl2 enhanced PPO activity. Data obtained in this study might help to better understand and control commercially, litchi fruit peel browning.
Article
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Article
A spectrophotometric assay method for the analysis of polyphenol oxidase (PPO), in apple and tobacco leaves, has been optimized to increase efficiency in the screening of large numbers of transgenic plants. Crude protein extracts from leaf punches were prepared in a FastPrep homogenizer. The addition of Triton X-100 during extraction resulted in 44 and 74% increases in the PPO activity recovered, from apple and tobacco, respectively. The enzyme kinetics differed markedly between apple and tobacco. Apple leaf PPO was isolated in a latent state and was activated by the addition of SDS. In contrast, tobacco PPO activity was inhibited by SDS, particularly at acidic pH. Apple PPO showed a pronounced pH optimum around pH 6, whereas the pH profile for tobacco PPO was much flatter, with a broad optimum around pH 4. The calculated Km' value for apple PPO, using 4-methylcatechol as substrate, was 8.1, and for tobacco the Km was 4.3. The PPO reaction was strongly inhibited by tropolone, a Cu competitor, and restored by the addition of Cu2+. Several factors affecting variability in leaf PPO activity levels in plants are discussed.
Acetonitrile was HPLC grade and obtained from Merck Other reagents were analytical grade Optimum conditions for bonding of plant phenols to insoluble polyvinylpyrrolidone
  • Chemicals Standardsst
  • Louis
  • Usa Mo
  • R A Andersen
  • J A Sowers
Chemicals and reagents Standards (À)-gallocatechin gallate, (À)-epicatechin gallate and (+)-catechin gallate were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile was HPLC grade and obtained from Merck (Darmstadt, Germany). Other reagents were analytical grade. References Andersen, R. A., & Sowers, J. A. (1968). Optimum conditions for bonding of plant phenols to insoluble polyvinylpyrrolidone. Phytochemistry, 7, 293–301.
The biochemical properties of polyphenol oxidase from banana leaf
  • H.-L Chu
  • D.-B Yeh
  • J.-F Shaw
Chu, H.-L., Yeh, D.-B., & Shaw, J.-F. (1993). The biochemical properties of polyphenol oxidase from banana leaf. Botanical Bulletin of Academia Sinica, 34, 169–174.
The biochemical properties of polyphenol oxidase from banana leaf
  • Chu