Grape Polyphenol Oxidase Research Articles

Effects of Guankou grape polyphenol oxidase on enzymatic browning

Polyphenol oxidase (PPO) was found to be the main cause of Guankou wine browning by investigating the browning index (BI) and the phenolic substances during the winemaking process. PPO was purified via temperature-induced phase partitioning, ammonium sulfate precipitation, and chromatography. The estimated molecular weights obtained by SDS-PAGE were 38 and 45 kDa. PPO was found to have an optimum pH and temperature of 4.6 and 30°C, respectively. The minimum time required to achieve inactivation was 10 min at 75°C. PPO from Guankou grape showed the highest binding affinity with caffeic acid, and a higher affinity for catechin than that from other grape varieties. The addition of ascorbic acid effectively reduced the browning of Guankou wine. Practical applications Guankou grape is the main variety in Jianshi County, Hubei Province, China. The enzymatic browning process of Guankou grapes and PPO characterization after purification were investigated, and it was found that the unique PPO was responsible for the rapid browning of Guankou wine. A suitable inhibitor was also found to prevent that from happening. These results provide a more comprehensive understanding of the features of Guankou grape PPO, and may assist with the effective control of browning occurring during wine making.

An approach to recombinantly produce mature grape polyphenol oxidase

Polyphenol oxidases (PPOs) are important enzymes that are widely found in both prokaryotes and eukaryotes including grapes. Studies of grape PPO to date have mostly relied on enzymes extracted and purified from plants. In this work, we describe the production of the mature form of Shine Muscat grape PPO by using an Escherichia coli expression system. We have optimised the purification procedure to obtain pure and active recombinant enzymes and characterised the catalytic efficiency of the recombinant grape PPO by using ultraviolet/visible (UV/Vis) spectrophotometry. Our work provides a simple protocol of obtaining pure and active recombinant grape PPO that will enable further studies about the catalytic mechanism and inhibition of this enzyme.

Wine-making with Protection of Must against Oxidation in a Warm, Semi-arid Terroir

To protect varietal aromas from oxidation before alcoholic fermentation, two grape must samples were prepared from white grapes potentially low in copper, pre-cooled and supplemented with ascorbic acid and solid CO2 (trial ACO2) or SO2 (trial BSO2). The wines prepared from musts protected from oxidation had aroma descriptors that included “passion fruit” and “grapefruit skin”. The lower concentrations of flavanols in the ACO2 trial demonstrated that the use of solid CO2 as an oxidation preventative instead of SO2 reduced the extraction of these polyphenols from the grape solids. The higher concentration of hydroxycinnamoyl tartaric acids of the wine from the A CO2 trial with respect to BSO2 was ascribed to the lower grape polyphenoloxidase activity induced by the lower oxygen level in the A CO2 trial, or to the combination of caftaric acid quinone with the SO2 in B SO2. Although the grapes were very ripe (alcohol in wines ~ 14.5% vol), the wines made with musts prepared by the two techniques were characterised by aroma descriptors like “passion fruit” and “grapefruit skin”, and these aromas were not detected in the wines prepared from unprotected musts.

The Use of Polyphenol Oxidase Activity to Identify a Potential Raisin Variety

ABSTRACTThe biochemical characterization of polyphenol oxidase (PPO) in the white grape Bianchetta Trevigiana is addressed in this research to fulfil the requirement for raisin varieties with high concentrations of phenolic compounds and low PPO activity not only to avoid enzymatic browning but also for maintaining the nutritional value of raisins. Spectrophotometric assays and electrophoresis were used to study this ancient variety exhibiting low PPO activity (only cathecolase activity) which reached at the low pH value of grape berry and was about 15% of the maximum enzymatic activity measured. In addition, the visualization of the active isoforms of grape PPO after 2-DE separation of proteins was a key step for further studies using MALDI-TOF-MS and bioinformatic tools. Based on the thermal inactivation profiles, heating treatment at 70°C may be considered a way to inhibit Bianchetta Trevigiana PPO activity by preserving also its high phenolic content. Therefore PPO characterization may be proposed as a marker for identifying potential high value raisin varieties.

Biochemical Properties of Emir Grape Polyphenol Oxidase as Affected by Harvest Year

Effect of harvest year on biochemical properties of polyphenol oxidase (PPO) from Narince grapes was studied in 2006 and 2007.The optimum pH of the enzyme in both years were found to be 5.49 whereas optimum temperature for enzyme activity was 30° C in the year 2006 and 35° C in the year 2007. The apparent substrate specificities were established from Vmax/Km as: caffeic acid > 4- methylcatechol >catechol >pyrogallol for two enzymes in both years. There were marked differences between the two enzymes in terms of thermal stability. The effects of inhibitors varied in a dose dependent manner and according to the harvest year.

Effects of defoliation and water restriction on total phenols and antioxidant activities in grapes during ripening

<p style="text-align: justify;"><strong>Aim</strong>: To compare the effect of water restriction and defoliation on the phenol contents and oxidant and antioxidant activities of Tempranillo grapes grown in the region of Extremadura, Spain.</p><p style="text-align: justify;"><strong>Methods and results</strong>: The results showed that at harvest, the water restriction treatment altered total foliar area, pH, and total soluble solid (TSS) and phenol contents but not polyphenol oxidase (PPO) and superoxide dismutase (SOD) activities. By contrast, the defoliation treatment did not affect TSS and pH, however, titratable acidity and phenol compounds were enhanced and PPO and SOD activities were decreased. Moreover, defoliation resulted in advanced grape ripening and reduced total foliar area. </p><p style="text-align: justify;"><strong>Conclusion</strong>: At harvest, water restriction led to alterations in the levels of peroxidation and peroxidase (POX) activity, without any effect on the other parameters. Defoliation induced a lowering of PPO and SOD activities and an increase in phenol content.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: Given the environmental characteristics of the Extremadura region, defoliation may be suitable and efficient for regulating Tempranillo grape PPO activity, which is œnologically relevant since it could lead to lower levels of phenol oxidation and hence prevent wine discolouration.</p>

Kinetic Study of Hydroxytyrosol Oxidation and Its Related Compounds by Red Globe Grape Polyphenol Oxidase

Red Globe grape polyphenol oxidase, partially purified using phase partitioning with Triton-X114, was used to study the oxidation of hydroxytytosol (HT) and its related compounds tyrosol (TS), tyrosol acetate (TSA), and hydroxytyrosol acetate (HTA). The enzyme showed activity toward both monophenols (monophenolase activity) and o-diphenols (diphenolase activity) with a pH optimum (pH 6.5) that was independent of the phenol used. However, the optimal temperature for diphenolase activity was substrate-dependent, with a broad optimum of 25-65 °C for HT, compared with the maximum obtained for HTA (40 °C). Monophenolase activity showed the typical lag period, which was modulated by pH, substrate and enzyme concentrations, and the presence of catalytic amounts of o-diphenols. When the catalytic power (Vmax/K(M)) was determined for both activities, higher values were observed for o-diphenols than for monophenols: 9-fold higher for the HT/TS pair and 4-fold higher for HTA/TSA pair. Surprisingly, this ratio was equally higher for TSA (2.2-fold) compared with that of TS, whereas no such effect was observed for o-diphenols. This higher efficiency of TSA could be related to its greater hydrophobicity. Acetyl modification of these phenols not only changes the kinetic parameters of the enzyme but also affects their antioxidant activity (ORAC-FL assays), which is lower in HTA than in HT.

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 (Km) and maximum velocity of the reaction (Vmax) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD400 nm/min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD478 nm/min for D, L-DOPA, respectively. The obtained similar specificity values of Vmax/Km 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 (Ea) of 146.1 ± 10.8 kJ/mol at pH 6.0.

Molecular Modeling of Peroxidase and Polyphenol Oxidase: Substrate Specificity and Active Site Comparison

Peroxidases (POD) and polyphenol oxidase (PPO) are enzymes that are well known to be involved in the enzymatic browning reaction of fruits and vegetables with different catalytic mechanisms. Both enzymes have some common substrates, but each also has its specific substrates. In our computational study, the amino acid sequence of grape peroxidase (ABX) was used for the construction of models employing homology modeling method based on the X-ray structure of cytosolic ascorbate peroxidase from pea (PDB ID:1APX), whereas the model of grape polyphenol oxidase was obtained directly from the available X-ray structure (PDB ID:2P3X). Molecular docking of common substrates of these two enzymes was subsequently studied. It was found that epicatechin and catechin exhibited high affinity with both enzymes, even though POD and PPO have different binding pockets regarding the size and the key amino acids involved in binding. Predicted binding modes of substrates with both enzymes were also compared. The calculated docking interaction energy of trihydroxybenzoic acid related compounds shows high affinity, suggesting specificity and potential use as common inhibitor to grape ascorbate peroxidase and polyphenol oxidase.

Antibrowning potential of Brassicacaea processing water

The effects of Brassicacaea processing water on polyphenol oxidases from different plant sources were investigated. Results showed that processing water (PW) prepared by cooking young Brassicacaea leaves with water has the capacity to inhibit both commercial tyrosinase and plant polyphenol oxidases. PW was freeze-dried (LPW) in an attempt to increase its utility and improve its efficiency at inhibition of polyphenol oxidases, however this resulted in a loss of activity, probably due to the stresses of the freezing and/or drying processes. The addition of PW to ascorbic acid resulted in complete inhibition of grape polyphenol oxidase, suggesting that the combination of the two antibrowning agents may be a promising tool to control this enzymatic activity.

Kinetic characterisation and thermal inactivation study of polyphenol oxidase and peroxidase from table grape (Crimson Seedless)

Polyphenol oxidase (PPO) and peroxidase (POD) were extracted from a table grape (Crimson Seedless) using Triton X-114 and characterized using spectrophotometric methods. Both PPO and POD were activated by acid shock. However, in the presence of the anionic detergent sodium dodecil sulphate (SDS), PPO was activated whereas POD was inactivated. The enzymes were kinetically characterized and both followed Michaelis–Menten kinetics, although with different values of their kinetic parameters. The V m/ K m ratio showed that Crimson Seedless grape PPO presents a similar affinity for 4- tert-butyl-catechol (TBC) whether activated by acid shock (0.018 min −1) or SDS (0.023 min −1). With regards to POD, the K m and V m values for 2,2′-azinobis(3-ethylbenzothiazolinesulphonic acid) (ABTS) were 0.79 mM and 1.20 μM/min, respectively. In the case of H 2O 2, the K m and V m value were 0.4 mM and 0.93 μM/min, respectively. PPO and POD showed similar thermostability, losing >90% of relative activity after only 5 min of incubation at 78 °C and 75 °C, respectively. In addition, PPO´s activation energy was similar to that obtained for POD (295.5 kJ/mol and 271.9 kJ/mol, respectively).

IZMIR GRAPE POLYPHENOL OXIDASE (VITIS VINIFERA L.): PARTIAL PURIFICATION AND SOME KINETIC PROPERTIES

ABSTRACT Polyphenol oxidase (PPO) was extracted from the Izmir grape fruit (Vitis vinifera L.) and its characteristics were studied. The PPO enzyme was purified 2.2-fold by (NH4)2SO4 precipitation and 26.1-fold by gel filtration chromatography. This partially purified enzyme obtained from dialysis migrated as one band with catechol substrate, four bands with Coomassie brilliant R-250 (NATIVE-PAGE) native system and as six bands of 34, 30, 22, 18, 16 and 15 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The sample obtained from dialysis after ammonium sulfate precipitation was used for the characterization of partially purified enzyme. Substrate specificity experiments were carried out with catechol, 4-methyl catechol, l-tyrosine, pyrogallol, p-cresol, gallic acid and caffeic acid. Michael–Menten constant and maximum velocity values were 3.65 mM and 1,971.6 ΔA/dak, respectively, with catechol substrate. Optimum pH for the PPO enzyme were determined at 7.2, 7.0, 7.4, 7.2, 6,4, 6.7 and 5.7 using catechol, 4-methyl catechol, l-tyrosine, pyrogallol, p-cresol, gallic acid and caffeic acid substrates, respectively. Optimum temperature for maximum PPO activity was 25C for catechol. Heat inactivation studies showed a significiant decrease in enzymatic activity at temperatures above 45C. Activation energy was calculated to be 12.4 kcal/mol. The most potent inhibitors for the grape PPO were thiourea, sodium diethyldithiocarbamate and sodium azide. Inhibition constants were calculated for L-ascorbic acid, l-cysteine and β-mercaptoethanol at the optimum pH of PPO activity. PRACTICAL APPLICATIONS The polyphenol oxidase enzymes isolated from different species have been used to control the pollution in water, to remove and transform toxic compounds of industrial processes and to determine catechol and other biologically active catecholamines in biologic liquids as an enzymatic biosensor.

THERMAL AND HIGH PRESSURE INACTIVATION KINETICS OF VICTORIA GRAPE POLYPHENOL OXIDASE: FROM MODEL SYSTEMS TO GRAPE MUST

ABSTRACT Polyphenol oxidase (PPO) was extracted from white grapes (var. Victoria, South Africa) and partially purified by ammonium sulfate precipitation. Detailed kinetic studies on thermal and high‐pressure inactivation of grape PPO were preformed in McIlvaine buffer (pH = 4.0), in reconstituted grape must and in grape must. Thermal inactivation of PPO in McIlvaine buffer followed a first‐order model while in reconstituted grape must and grape must a biphasic inactivation behavior has been observed. Under all conditions investigated, a first‐order kinetic model could describe the pressure inactivation data. It was found that PPO is more thermostable and pressure‐stable in must than in reconstituted grape must and McIlvaine buffer at the same pH = 4.0.

Characterization of polyphenol oxidase from Napoleon grape

Polyphenol oxidase (PPO) from Napoleon grape was isolated using a two-phase partitioning approach with Triton X-114. The enzyme was purified in a latent form and could be optimally activated by the presence of 0.2% of sodium dodecyl sulphate (SDS) at pH 6.0. In the absence of SDS, the enzyme showed maximum activity at acid pH (3.0). The enzyme was kinetically characterized at pH 3.0 and pH 6.0 in the presence of 0.2% of SDS, using 4- tert-butylcatechol (TBC) as a substrate. The V m/ K M ratio showed that Napoleon grape PPO presents greater affinity for TBC at acid pH (0.1 min −1) that at pH 6.0 in the presence of SDS (0.02 min −1). The enzyme was highly heat stable, 80% of activity remaining at 70 °C. Selected inhibitors were also studied, tropolone being the most active with a K i value of 27 μM at acid pH and pH 6.0 in the presence of 0.2% SDS.

Polyphenol Oxidase from Dominga Table Grape

Dominga grape polyphenol oxidase (PPO) was extracted using phase partitioning with Triton X-114. The enzyme was obtained in latent state and could be optimally activated by the presence of 0.2% sodium dodecyl sulfate (SDS) at pH 6.0. In the absence of SDS, the enzyme showed maximum activity at acid pH. The kinetic parameters of the enzyme at pH 3.0 and 6.0 in the presence of SDS were calculated. The effect of several inhibitors was studied, tropolone being the most effective with a K(i) value of 18 muM. The effect of cyclodextrins was also studied, and the complexation constant K(c) between G(2)-beta-cyclodextrins and 4-tert-butylcatechol was calculated using the enzymatic method (K(c) = 13960 M(-)(1)). The evolution of the color parameters (L, a, b) of liquefied grape berries was inhibited by inhibitors of PPO activity, such as diethyldithiocarbamate, metabisulfite, and G(2)-beta-cyclodextrins, indicating that enzymatic browning by PPO is the main process involved in the browning of Dominga grape juice at room temperature.

Biochemical characterization and process stability of polyphenoloxidase extracted from Victoria grape ( Vitis vinifera ssp. Sativa)

Polyphenol oxidase (PPO) was isolated from Victoria grapes ( Vitis vinifera ssp. Sativa) grown in South Africa and its biochemical characteristics were studied. Optimum pH and temperature for grape PPO activity were pH 5.0 and T = 25 °C with 10 mM catechol in McIlvaine buffer as substrate. PPO showed activity using the following substances: catechol, 4 methyl catechol, d, l-DOPA, (+) catechin and chlorogenic acid. K m and V max values were 52.6 ± 0.00436 mM and 653 ± 24.0 OD 400 nm /min in the case of 10 mM catechol as a substrate. Eight inhibitors were tested in this study and the most effective inhibitors were found to be ascorbic acid, l-cysteine and sodium metabisulfite. Kinetic studies showed that the thermal inactivation of Victoria grape PPO followed first-order kinetics, with an activation energy, E a = 225 ± 13.5 of kJ/mol. Both in semipurified extract and in grape juice, PPO showed a pronounced high pressure stability.

Purification and Analysis of Wheat Grain Polyphenol Oxidase (PPO) Protein

ABSTRACTWheat (Triticum aestivum L.) breeding programs have used various whole kernel assays to estimate polyphenol oxidase (PPO) activity, thereby identifying germplasm that has a greater chance of producing consumer products with superior color. However, the enzymes involved in these assays are poorly understood and the purification and characterization of a wheat kernel PPO protein has not been reported previously. A PPO from wheat bran was purified using ammonium sulfate precipitation, ion and size‐exclusion chromatography, and continuous elution electrophoresis. The purified protein migrated at 67 kDa on SDS‐PAGE under denaturing and reducing conditions, exhibited PPO activity in the presence of SDS, and eluted at 45 kDa on SDS‐PAGE under nondenaturing and nonreducing conditions. N‐terminal sequence analysis of peptide fragments obtained from tryptic digests confirmed the purified wheat bran protein as a PPO. This wheat PPO protein showed the greatest sequence identity to grape (Vitis vinifera) and pineapple (Ananas comosus) PPO. The purified wheat PPO shares no more sequence identity with the deduced amino acid sequence of a previously isolated partial wheat PPO sequence than it does to PPO from other plant taxa widely divergent from wheat. Based on immunoblot analysis, purified PPO from wheat bran appears to be a processed, mature form lacking an estimated 14–16 kDa transit peptide required for plastid localization.

PH-Dependent Effect of Sodium Chloride on Latent Grape Polyphenol Oxidase

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 ch...

Structural determination of colourless and yellow dimers resulting from (+)-catechin coupling catalysed by grape polyphenoloxidase

(+)-Catechin oxidation was carried out in aqueous systems using grape polyphenoloxidase as catalyst. Eight fractions corresponding to the major products formed at pH 3 and 6 were purified using HPLC at the preparative and semi-preparative scale. Structural characterization using UV-visible detection and mass spectrometry indicated that they corresponded to (+)-catechin dimers including two yellow pigments. Mono- and bi-dimensional 1H and 13C NMR analyses provided structural hypotheses for five oxidation products whereas the other three fractions were mixtures of several isomers. Colourless products, with C-C or C-O interflavan linkages, were dehydrodicatechins of the B-type. One of the two pigments corresponds to dehydrodicatechin A, already identified in other oxidation models, and the other pigment is a new structure of the quinone-methide type.

Mechanisms of anthocyanin degradation in grape must‐like model solutions

AbstractOxidative degradation of o‐diphenolic (eg cyanidin‐3‐glucoside) and non‐o‐diphenolic (eg malvidin‐3‐glucoside) anthocyanins in the presence of caffeoyltartaric acid and grape polyphenoloxidase was studied in model solutions. Both anthocyanins reacted with the enzymically generated caffeoyltartaric acid o‐quinone. Kinetic studies indicated that cyanidin‐3‐glucoside was degraded mostly by coupled oxidation whereas malvidin‐3‐glucoside formed adducts with caffeoyltartaric acid quinone. In solutions containing equimolar amounts of the anthocyanins, both reactions took place but coupled oxidation of cyanidin‐3‐glucoside occurred at a faster rate, partly protecting malvidin‐3‐glucoside. The occurrence of coupled oxidation resulted in sparing of caffeoyltartaric acid. Several oxidation products, including red pigments, were detected by HPLC. The UV‐Vis spectra of the coloured condensation products suggested that they contained both caffeoyltartaric acid and anthocyanin moieties. All these pigments were gradually degraded to colourless compounds either by enzymatic oxidation or by further reaction with quinones.