Polyphenol Oxidase Substrates Research Articles

Polyphenol Oxidase and o‐Diphenols Inhibit Postharvest Proteolysis in Red Clover and Alfalfa

Many forages experience significant proteolytic losses when preserved by ensiling. Such losses in alfalfa (Medicago sativa L.) are especially high, with degradation of 44 to 87% of the forage protein to nonprotein N (NPN). In contrast, red clover (Trifolium pratense L.) has up to 90% less proteolysis during ensiling. Here we demonstrate that the combination of polyphenol oxidase (PPO) and o‐diphenol PPO substrates, both abundantly present in red clover, is responsible for postharvest proteolytic inhibition in this forage crop. Proteolysis in red clover leaf extracts increased nearly fivefold when endogenous o‐diphenols were removed by gel filtration but returned to starting levels by adding back an exogenous o‐diphenol. Proteolysis in leaf extracts of red clover plants silenced for PPO expression was dramatically increased compared to control plants. Leaf extracts of transgenic alfalfa expressing a red clover PPO gene showed a nearly fivefold o‐diphenol–dependent decrease in proteolysis compared to those of control alfalfa. We also demonstrate that PPO levels 10‐ to 20‐fold lower than those typically found in red clover are sufficient for proteolytic inhibition, that as little as 0.25 μmol o‐diphenol mg−1 protein has a substantial impact on proteolysis, that a wide variety of o‐diphenols are functional substrates in proteolytic inhibition, and that proteolysis is reduced for PPO‐expressing alfalfa in small‐scale ensiling experiments. Together, these results indicate that PPO and o‐diphenols can be an effective treatment to prevent protein loss in ensiled forage crops.

Polyphenol Oxidase in Wheat Grain: Whole Kernel and Bran Assays for Total and Soluble Activity

ABSTRACTColor is a key quality trait of wheat products, and polyphenol oxidase (PPO) is implicated as playing a significant role in darkening and discoloration. In this study, total and soluble PPO activities were characterized in whole kernel assays and bran extracts. In whole kernel assays similar to AACC Approved Method 22–85, four wheat cultivars were ranked the same for both total and soluble (leached) PPO activity with L‐DOPA (diphenol) as the substrate. Total kernel PPO activity was much greater than soluble PPO activity in three hexaploid wheat cultivars, indicating that insoluble PPO was the major contributor to kernel PPO measurements. Tyrosine (monophenol) was an excellent PPO substrate in kernel assays as expected but had no activity as a substrate for soluble PPO. However, soluble PPO activity with tyrosine was activated by the addition of the diphenols chlorogenic acid and caffeic acid. When PPO was assayed in homogenized bran, 89–95% of total PPO activity remained insoluble, associated with the bran particles. The kernel assay detected <2% of PPO measured in an equivalent amount of homogenized bran. However, total PPO activity was 2‐fold higher in Klasic than in ID377s, both when measured in the kernel assay and in homogenized bran, indicating that the kernel assay was an accurate predictor of relative total extracted PPO activity in these two cultivars. Adding detergents (0.1% SDS plus 0.2% NP‐40) to the bran extraction buffer increased both soluble and insoluble PPO activity. Results indicate that relative PPO activities among wheat cultivars are similar in whole kernel and kernel leachate assays, and that the predominant insoluble fraction of PPO, which is relatively uncharacterized, may be largely responsible for wheat product discoloration.

Spraying of 4-hexylresorcinol based formulations to prevent enzymatic browning in Norway lobsters ( Nephrops norvegicus) during chilled storage

A comparison was made of the effects on melanosis development in Norway lobsters ( Nephrops norvegicus) of treatment by dusting with a commercial sulphite-based product and of spraying with a formulation containing 4-hexylresorcinol (0.1% and 0.05%), in combination with organic acids and chelating agents. The following tests were performed during chilled storage: polyphenol oxidase (PPO) activity, melanosis score, colour parameters, tyrosine and tyramine content, as the main substrate of PPO. Differences among treatments were evaluated by means of statistical analyses (ANOVA, principal components and discriminant analyses). All formulations diminished PPO activity during storage successfully. The melanosis score was higher in sulphite-treated Norway lobsters, and a formulation with 0.05% 4-hexylresorcinol was enough to prevent the appearance of melanosis for 12 days. The tyrosine content decreased during storage, but the tyramine content was insignificant. Formulations with 4-hexylresorcinol improved the appearance of Norway lobsters, in comparison with the commercial sulphite-based product.

Specific roles of enzymatic browning and lignification in apple disease resistance

Abstract The roles of enzymatic browning and lignification as resistance mechanisms against Penicillium expansum were investigated in ‘Golden Delicious’ apples picked at different maturity stages. To determine enzymatic browning, the browning potential of the pulp was determined on different fruit, with or without ascorbate, and with or without 4-methylpyrocatechol, substrate of polyphenol oxidase (PPO), and guaiacol, to obtain maximal peroxidase (POX) activity. Moreover, the activity of PPO and POX enzymes was determined at each harvest date and lignin content was measured at harvest and after storage in order to correlate it with disease resistance. Initially, when pulp tissue was treated with ascorbate, decay incidence was negatively correlated with browning. In this mechanism, the enzymatic activities of PPO and POX did not limit the reaction. In contrast, the availability of their substrates was limiting. When fruit were pre-incubated with 4-methylpyrocatechol, an increase in susceptibility to the pathogen was observed in immature apples (harvest 1). On the other hand, pre-incubation with guaiacol led to increased resistance in the immature fruit. Lignin content was highly negatively correlated with decay incidence. These results provide evidence that enzymatic browning is not a determining factor of apple resistance. In contrast, POX activity appeared to be an important factor especially through its action on lignification, which appeared to be involved in the resistance of apple fruit to P. expansum .

Purification and characterization of polyphenol oxidase from Ferula sp.

Polyphenol oxidase (PPO) of several Ferula sp. was extracted and purified through (NH 4) 2SO 4 precipitation, dialysis, and gel filtration chromatography. Leaf and stem extracts were used for the determination of enzyme properties. Optimum conditions, for pH, temperature, and ionic strength were determined. The best substrates of PPO were catechol for leaf and (−) epicatechin for stem samples. Optimum pH and temperature were determined. K M and V max values were 2.34 × 10 −3 M and 8541 EU/ml for catechol, and 2.89 × 10 −3 M and 5308 EU/ml for (−) epicatechin. The most effective inhibitor was sodium diethyl dithiocarbamate for leaf samples and sodium metabisulphite for stem samples. Both inhibitors indicated competitive reactions. PPO showed irreversible denaturation after 40 min at 60 °C.

Changes in concentrations of phenolic compounds and polyphenol oxidase activity in banana peel during storage

Phenolic compounds were analyzed in the outer, middle and inner layers of the peel and in the pulp of bananas during ripening from the ripening-initiation. The major phenolic compound found was dopamine followed by 3, 4-dihydroxyphenylalanine (L-DOPA), tyramine and tyrosine in all portions of the bananas. These compounds located mainly in the outer layer of peel followed by the middle and inner layers of the peel and the pulp. The concentration of dopamine decreased during ripening in all portions of the fruit, particularly in the outer layer of the peel in which senescent spotting appeared at a late stage of storage. On the other hand, those of the other compounds, namely L-DOPA, tyrosine and tyramine increased during ripening. Polyphenol oxidase (PPO) activity was detected in all portions of the fruit ; however, it increased only in the outer layer of the peel. Short-term (24h) nitrogen-gas treatment of the ripening-initiated banana delayed the induction of senescent spotting and the decrease in dopamine content, while it increased the PPO activity of the nitrogen-gas-treated bananas to the same extent as that of air-treated bananas. These results indicate that dopamine is used as substrate of PPO for the formation of brown pigments of senescent spotting.

Method for the determination of molar absorptivities of thiol adducts formed from diphenolic substrates of polyphenol oxidase

Metabolic thiols such as cysteine and glutathione are involved in the biosynthetic pathway of phaeomelanins. They attack the o-quinones generated by polyphenol oxidase in its action on mono and o-diphenols and thus generate adducts. Determination of the molar absorptivities of these adducts is useful for spectrophotometric studies of phaeomelanin biosynthesis, antibrowning reagents in plants, and polyphenol oxidase assay methods. For their calculation, a method based on the depletion of o-diphenol by the action of polyphenol oxidase in the presence of thiol has been proposed. However, the method is slow and presents certain problems, for which reason we propose a new and faster method based on the action of polyphenol oxidase on o-diphenols which are in excess with respect to oxygen. Under these assay conditions there is rapid enzymatic formation of o-quinones, which react stoichiometrically with a thiol giving rise to the corresponding thiol–diphenol adduct. The method has been successfully applied to adducts of cysteine and glutathione with several o-diphenolic substrates of polyphenol oxidase involved in phaeomelanin biosynthesis in skin, neurones, and plants.

Colour of blackspot bruises in potato tubers during growth and storage compared to their discolouration potential

Abstract Two potato (Solanum tuberosum) cultivars, Dali and Oleva, with, respectively, low and high susceptibility to blackspot bruise, were grown in 1998 and 1999. Tubers from plants of Dali and Oleva were harvested three and four times during the final growth period, respectively. The tubers were analysed for blackspot susceptibility and colour of blackspots four times during storage and the tuber cortices were analysed for discolouration potential, activity of polyphenol oxidase (PPO) and concentration of PPO substrates (free tyrosine, chlorogenic acid, caffeic acid). During tuber growth, an increasing abundance of red- and yellow-coloured intermediates and final black-coloured compounds in the assay of potential discolouration were observed. The overall correlation coefficients (r) between red intermediates and free tyrosine were 0.74 and 0.64 for Dali and Oleva, respectively, whereas they were 0.49 and 0.68, respectively, between yellow intermediates and free tyrosine. The overall correlation coefficients between black end-products and free tyrosine were 0.69 and 0.67 for Dali and Oleva, respectively. No overall correlation was found between the discolouration potential and other phenols. Changes in blackspot susceptibility and in colour of blackspots during growth and storage of the tubers could not be ascribed to changes in the discolouration potential (colour development of blended tuber tissue), PPO activity or concentration of phenols in the tuber cortices. Contrary to the discolouration potential, the blackspots were significantly darker at the beginning of the storage period compared to later in storage. Furthermore, during tuber growth the stable colour of bruises did not reflect the concomitant increase in discolouration potential. The results provide supporting evidence that intracellular compartmentation is the determining factor for blackspot bruise susceptibility in potato tubers.

Properties of chlorogenic acid quinone: relationship between browning and the formation of hydrogen peroxide from a quinone solution.

Chlorogenic acid is the major polyphenol in foods derived from plants and is a good substrate for polyphenol oxidase. Chlorogenic acid quinone (CQA-Q), which is an oxidative product of chlorogenic acid by polyphenol oxidase, is an important intermediate compound in enzymatic browning. CQA-Q was prepared, and its properties and the relationship with browning were examined. The quinone solution was yellow or orange, and its molecular absorption coefficient was estimated to be 1.7 x 10(3) for 325 nm and 9.7 x 10(2) for 400 nm in an acidic aqueous solution. Chlorogenic acid and H2O2 were spontaneously generated in the CQA-Q solution as the yellowish color of the solution gradually faded. A pale colored polymer was the major product in the reaction solution. Amino acids such as lysine and arginine added to CQA-Q solution did not repress the fading of the yellowish color of the solution. We concluded from these results that CQA-Q itself and a mixture of CQA-Q and amino acids did not form intensive brown pigments in the acidic aqueous solution. H2O2 spontaneously formed in the CQA-Q solution, and other polyphenols might have played an important role in the formation of the brown color by enzymatic browning.

Role of anthocyanin degradation in litchi pericarp browning

Abstract Pericarp browning is the main problem of post-harvest litchi fruit, resulting in an accelerated shelf life and reduced commercial value of the fruit. Underhill and Critchley (1994) . Anthocyanin decolorisation and its role in lychee pericarp browning. Australian Journal of Experimental Agriculture , 34 , 115-122 found that there was not an obvious change in the content of anthocyanins when the fruit browned. This work was conducted with a view to explaining this unexpected observation. Litchi pericarp browning index increased while the content of anthocyanins decreased with storage time when 0.1 M HCl was used as the extract solution instead of acidic methanol. The visible spectum of the anthocyanin extract, at a range of 400–600 nm and pH values of 1.0, 3.0 and 5.0, were recorded, with an absorbance peak of about 510 nm. The colour of the extract depended on the pH values and the half-degradation constants for anthocyanins at pHs 1.0, 3.0 and 5.0 were, respectively, 29, 15.3 and 10.5 days, as calculated from the kinetics of the degradation. Compared with the anthocyanin extract, anthocyanidin is more vulnerable, with a half-degradation of about 5.3 min at pH 5.0. Furthermore, the product from the anthocyanidin degradation had a similar structure to catechol (a good substrate for polyphenol oxidase), which, in turn, could accelerate enzymatic browning reaction by the enzyme polyphenol oxidase. In addition, an anthocyanase, catalyzing anthocyanin hydrolysis and producing anthocyanidin was extracted from litchi fruit pericarp. High activity of the enzyme was observed in the pericarp. Thus, it is suggested that anthocyanase might contribute to the browning of litchi pericarp involved in the anthocyanase-anthocyanin-PPO reaction.

PHYSICO-CHEMICAL PROPERTIES of POLYPHENOL OXIDASE FROM d'ANJOU and BARTLETT PEARS (PYRUS COMMUNIS L.)

Polyphenol oxidase (PPO) and phenolics were studied in two pear cultivars, d'Anjou and Bartlett. PPO activity, total phenolics and chlorogenic acid concentration differed significantly with respect to cultivar. PPO activity, total phenolics and chlorogenic acid all decreased in overripe fruits. The pH optimum (d'Anjou, 4.7 and Bartlett, 5.5), and optimum temperature (d'Anjou 40C and Bartlett, 20C) for maximum PPO activity were determined. Among substrates, 4-methylcatechol followed by catechol and dopamine, was the most readily oxidized substrate of PPO from both pear cultivars. The enzyme from both cultivars did not show any activity with any of the monohydroxy substrates. K m of 13.33 and 10.92 mM were determined with 4-methylcatechol for d'Anjou and Bartlett pear PPO, respectively. Benzoic and cinnamic acid series compounds were poor inhibitors of PPO. However, L-cysteine, sodium metabisulfite, ascorbic acid and thiourea proved to be effective inhibitors of this enzyme. Heating at 65C for 30 min resulted in a loss of 60-72% of PPO activity.

Comparison of Enzymatic Browning of Japanese Pear and Apple.

When Japanese pear and apple are cut, the section of Japanese pear does not turn brown as readily as apple, although both fruits belong to the same family. Here, the enzymatic browning of the two fruits is compared. The activity of polyphenol oxidase (PPO) of Japanese pear was higher than that of apple. The PPO of Japanese pear existed uniformly throughout the fruit, while the PPO of apple was localized around the core. The content of chlorogenic acid, (+)-catechin, and (-)-epicatechin in Japanese pear was much lower than in apple. When these polyphenols were added to Japanese pear juice, the juice turned as strong a brown color as apple juice. Therefore, the reason why Japanese pear does not turn brown as easily as apple is that the amount of substrate of PPO in the former is much less.

Myricetin, an antioxidant flavonol, is a substrate of polyphenol oxidase

The present study demonstrates the antiradical efficiency of myricetin, a flavonol widely distributed in fruits and vegetables, by testing its ability to react with two different free radicals, ABTS+· and DPPH·. The polyphenolic nature of myricetin led us to consider the possibility of its oxidation by polyphenol oxidase (PPO). The results reported show that myricetin can be oxidised by PPO extracted and partially purified from broad bean seeds. The reaction was followed by recording spectral changes with time, maximal spectral changes being observed at 372 nm. The presence of two isosbestic points (at 274 and 314 nm) suggested that only one absorbing product was formed. The spectral changes were not observed in the absence of PPO. The oxidation rate varied with the pH, reaching its highest value at pH 5.5. The myricetin oxidation rate increased in the presence of SDS, an activing agent of polyphenol oxidase. Maximal activity was obtained at 1.3 mM SDS. The kinetic parameters were also determined: V m = 1.35 µM min−1, K m = 0.3,mM, V m/K m = 4.5 × 10−3 min−1. Flavonol oxidation was inhibited by a selective PPO inhibitor such as cinnamic acid (KI = 1 mM). The results reported show that myricetin oxidation was strictly dependent on the presence of polyphenol oxidase. © 1999 Society of Chemical Industry

Myricetin, an antioxidant flavonol, is a substrate of polyphenol oxidase

Myricetin, an antioxidant flavonol, is a substrate of polyphenol oxidase

Polyphenol Oxidase from Malatya Apricot (Prunus armeniaca L.)

Polyphenol oxidase (PPO) of Malatya apricot was isolated by (NH4)2SO4 precipitation and dialysis. PPO showed activity to catechol, l-dopa, and gallic acid. Catechol was the most suitable substrate for Malatya apricot PPO (lowest Km value). The optimum pH for the PPO was 8.5. Heating for 40 min at 40 °C did not cause a significant loss of enzymic activity. The times required for 50% inactivation of activitiy at 60 and 80 °C were found to be 47 and 16 min, respectively. The I50 values of inhibitors studied on PPO were determined by means of activity percentage [I] diagrams. The values were 2.7 × 10-5, 5.03 × 10-5, 3.62 × 10-5, 3.68 × 10-5, and 8.30 × 10-4 M for sodium metabisulfite, ascorbic acid, 2-mercaptoethanol, thiourea, and salicylic acid, respectively. Ascorbic acid, 2-mercaptoethanol, sodium metabisulfite, and thiourea inhibited the reaction strongly. Keywords: Malatya apricot; Prunus armeniaca L.; polyphenol oxidase; inhibitors

Biochemical Potential of Potato Tubers To Synthesize Blackspot Pigments in Relation to Their Actual Blackspot Susceptibility

In a study of six potato varieties (Solanum tuberosum L.), which were grown under two different fertilization regimes, the biochemical potential of tuber tissue to synthesize blackspot pigments was distinguished from the actual blackspot susceptibility exhibited after impact application. Blackspot susceptibility and biochemical potential for pigment synthesis were not correlated, which supports the hypothesis that the extent to which the blackspot potential is actually being used is mediated by the sensitivity to cell decompartmentation. Quantification of polyphenol oxidase (PPO), soluble protein, and endogenous PPO substrates demonstrated that the content of free tyrosine is the predominant determinant for the biochemical potential for blackspot synthesis. PPO was not a limiting factor for pigment synthesis, even if PPO content was relatively low. It was therefore concluded that manipulation of PPO activity may offer a solution to the problem of blackspot formation only if it leads to a virtually complet...

Phenolic Composition, Browning Susceptibility, and Carotenoid Content of Several Apricot Cultivars at Maturity

Phenolic composition and susceptibility to browning were determined for nine apricot (Prunus armeniaca L.) cultivars. Chlorogenic and neochlorogenic acids, (+)-catechin and (-)-epicatechin, and rutin (or quercetin-3-rutinoside) were the major phenolic compounds in apricots. In addition to these compounds, other quercetin-3-glycosides and procyanidins have been detected. Chlorogenic acid content decreased rapidly during enzymatic browning, but the susceptibility to browning seemed to be more strongly correlated with the initial amount of flavan-3-ols (defined as catechin monomers and procyanidins). As chlorogenic acid is certainly the best substrate for polyphenol oxidase, the development of brown pigments depended mainly on the flavan-3-ol content.

Prediction of Polyphenol Oxidase Activity in Model Solutions Containing Various Combinations of Chlorogenic Acid, (−)-Epicatechin, O2, CO2, Temperature, and pH by Multiway Data Analysis

Controlled or modified packaging (CA/MA) of lettuce is simulated in model solutions containing lettuce polyphenol oxidase (PPO), chlorogenic acid (CG), and/or (−)-epicatechin (EPI) under various combinations of CO2, O2, N2, temperature, and pH conditions. Enzymatic browning is measured by PPO activity, color formation, substrate consumption, and development of products. Results were analyzed by multiway data analysis. CO2 minimized the chemical polymerization and reduced PPO activity slightly. CG was a better substrate for PPO than EPI. CG was, in particular, involved in the primary enzymatic catalyzed oxidation, whereas EPI was important for the subsequent chemical polymerization in mixtures of CG and EPI. A very descriptive five-parameter multiplicative model was obtained from the PPO data by the multivariate method PARAFAC. This model describes the importance of substrate, atmospheric composition, pH, and temperature for PPO activity. Low pH (3.0) reduced PPO activity strongly (89%), whereas low temper...

Comparison of β-Cyclodextrin Effect on Polyphenol Oxidation Catalyzed by Purified Polyphenol Oxidase from Different Sources

The effects of β-cyclodextrin (β-CD) on polyphenol oxidation catalyzed by apple polyphenol oxidase (PPO), endive PPO, or mushroom tyrosinase have been compared. β-CD forms a complex with phenolic substrates of PPO by inclusion. Assuming a 1:1 β-CD/phenol stoichiometry, and assuming that PPO is inactive on the complex β-CD/phenol, KD values were similar when determined kinetically by inhibition of apple PPO or endive PPO. However, the experimental velocities found during inhibition of mushroom tyrosinase by β-CD were higher than the values predicted by this model. In this latter case, it was assumed that mushroom tyrosinase is able to act on the complex β-CD/phenol. A new model based on this assumption allows experimental and calculated velocities to be fit in presence of β-CD. Keywords: Enzymatic browning; polyphenol oxidase; apple; endive; mushroom; inhibition; cyclodextrin

Monophenolase Activity of Polyphenol Oxidase from Haas Avocado

Avocado polyphenol oxidase (PPO) has been isolated and partially purified by using Triton X-114, reported here for the first time. The enzyme showed both monophenolase and diphenolase activities. The monophenolase activity of avocado PPO was characterized using 4-hydroxyanisole (4HA) as substrate with 3-methyl-2-benzothiazolinone hydrazone as coupled nucleophile. This continuous spectrophotometric method was reliable, with high sensitivity and precision. 4HA was a very good substrate for avocado PPO, showing a higher kcat than that for l-dopa at the optimum pH. Monophenolase activity of PPO showed a lag period (τ) prior to the attainment of the steady state rate (Vss). The optimum pH was 5, the same for the monophenolase and diphenolase activities when the p-hydroxyphenylpropionic acid/3,4-dihydroxyphenylpropionic acid pair was assayed, as well as when 4HA was assayed. A reaction mechanism for explaining the kinetic behavior of the monophenolase activity of avocado PPO has been proposed and characterized. Keywords: Avocado; enzyme kinetics; 4-hydroxyanisole; MBTH; monophenolase; polyphenol oxidase