Pear Polyphenol Oxidase Research Articles

Pear polyphenol oxidase enhances theaflavins in green tea soup through the enzymatic oxidation reaction

AbstractPolyphenol oxidase (PPO) is a necessary medium for converting catechins into theaflavins in tea. This study aimed to provide an efficient and cost‐effective method for the purpose of developing a tea beverage rich in four main theaflavins (TFs) by optimizing the key factors affecting the enzymatic oxidation reaction to produce TFs. Autumn green tea (AT) soup was mixed with pear PPO crude enzyme solution and then reacted for different time at varying reaction temperatures, pH, ratios of tea soup to crude enzyme solution, and ratios of tea to water. The content of TFs in tea beverage was monitored by UPLC‐Q‐Orbitrap‐MS/MS to select the optimal process parameters to produce the maximum TFs. The TFs content after the reaction increased 968.49 times compared with that before the reaction. The results showed that the use of the enzymatic oxidation reaction of pear PPO crude enzyme solution to improve the TFs in AT soup was efficient. This method helped regulate the strong bitter and astringent taste sensation in AT, and it is unnecessary to purify the PPO enzyme source. This method is expected to be a breakthrough to use AT to produce high TFs tea beverages in a cost‐effective way with high value.

Differential inhibitory effects of organic acids on pear polyphenol oxidase in model systems and pear puree

The inhibitory effects of ascorbic, citric, ferulic and salicylic acid on pear polyphenol oxidase (PPO) were investigated in model and food system. All four organic acids showed strong inhibition effect in model system. However, compared to model system, inhibitory effect of PPO in pear puree was quite different. Ascorbic acid showed a much weaker effect in pear puree compared to the model system. Similarly, ferulic and salicylic acid had limited inhibitory effect in pear puree. Interestingly, the inhibition of PPO activity was not correlated to browning control in pear puree. Ascorbic acid in low concentration was not effective in inhibiting PPO activity in pear puree but exhibited an excellent anti-browning effect. The study indicates that the level of PPO activity is not always a good indicator of susceptibility to browning in foods. Overall, ascorbic acid showed the strongest inhibition of browning and increased the total phenolic content of pear puree.

Influence of Nucleophilic Amino Acids on Enzymatic Browning Systems.

In the present study the enzymatic oxidation of gallic acid and catechin catalyzed by nashi pear polyphenol oxidase (PPO) in the presence of the amino acids lysine, arginine, or cysteine was investigated for polyphenol-amino acid adducts. HPLC analyses revealed the formation of two novel dihydrobenzothiazine carboxylic acid derivatives (8-(3',4'-dihydro-2 H-chromene-3',5',7'-triol)-3,4-dihydro-5-hydroxy-2 H-benzothiazine-3-carboxylic acid and 7-(3',4'-dihydro-2 H-chromene-3',5',7'-triol)-3,4-dihydro-5-hydroxy-2 H-benzothiazine-3-carboxylic acid) from 2'-cysteinyl catechin and 5'-cysteinyl catechin in cysteine incubations, respectively. In contrast, arginine and lysine did not lead to any amino acid adducts. Target compounds were separated by high-performance countercurrent chromatography and preparative HPLC and unequivocally characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Mechanistic incubations starting from the catechin-cysteine adducts showed that both catechin and PPO are crucial components in the formation of the dihydrobenzothiazines. The cysteine incubations showed a red-brown coloration, which coincided with formation and degradation of the dihydrobenzothiazines finally leading to the formation of high-polymeric melanins. Therefore, these compounds might be the key intermediates to understand development of color during cysteine-driven enzymatic browning reactions.

The Inactivation Kinetics of Soluble and Membrane-Bound Polyphenol Oxidase in Pear during Thermal and High-Pressure Processing

Polyphenol oxidase (PPO) from pear was characterized with catechol as substrate. The Michaelis constant of soluble and membrane-bound PPO were 15.6 and 23.8 mM, respectively, and their optimum pH for activity were 6.0 and 6.5, respectively. The inactivation kinetics of soluble and membrane-bound PPO during thermal (45–75 °C) and high-pressure thermal processing (600 MPa, 40–80 °C) were studied. The inactivation kinetics of pear PPO were described by a first-order model at all processing conditions. Compared to soluble PPO, membrane-bound PPO was more sensitive to thermal and high-pressure inactivation. Both soluble and membrane-bound PPO displayed higher sensitivity towards thermal inactivation at pH 3.5 (pH of pear puree made from pears dipped in citric acid prior to blending) compared to pH 4.4 (pH of non-acidified pear puree). High pressure and temperature had synergistic inactivation effects on pear PPO at pH 4.4 while slight antagonistic effects were observed at pH 3.5.

Effective synthesis of theaflavin-3,3′-digallate with epigallocatechin-3-O-gallate and epicatechin gallate as substrates by using immobilized pear polyphenol oxidase

In the present study, pear polyphenol oxidase (PPO) was purified, immobilized and applied for the synthesis of theaflavin-3,3′-digallate (TF3). Firstly, PPO of pear (Pyrus bretschneideri Rehd cv. Huangguan) was purified 24.36-fold in specific activity with a recovery of 6.77% by sequential use of precipitation with 70% saturated ammonium sulfate and chromatography of DEAE-Sepharose Fast Flow column. The purified pear PPO, found to be a dimer of identical subunits of molecular mass 35kDa, was then successfully immobilized onto Fe3O4/chitosan nanoparticles via glutaraldehyde coupling reaction. Finally, TF3 was effectively synthesized by applying the immobilized PPO as biocatalyst with epicatechin gallate (ECG) and epigallocatechin-3-O-gallate (EGCG) as substrates, and the maximum yield of TF3 was 42.23% based on the amount of ECG added. In addition, the immobilized enzyme still remained 85% of its initial ability after successive usage of 8 cycles and could be stored for 30days with less loss of activity. All the results suggest that the developed procedure is quite useful and has great potential for synthesis of TF3 and other theaflavins.

Alternative pathway implicated as an influencing factor in the synthesis of theaflavin

The principal pigments present in black tea, theaflavins (TF), have been indicated to be of potential clinical significance in various fields of research which has been hampered by the very low levels of TFs from black tea extractions, being the original method employed to acquire TFs. Forelle pear (44 µM TF/g dry weight/h) and Yacon leaf (65 µM TF/g dry weight/h) homogenates were tested for their TF synthesis capacity and found to have a larger TF synthesis capacity than a green tea leaf homogenate (26 µM TF/g dry weight/h) based upon the flavognost method. In an incubation system of green tea leaf extract utilizing endogenous enzymes present in Forelle pear and Yacon homogenates to synthesize TF, the formation of an unknown peak [m/z 563.1349; (23.95)5; C26H28O14] was detected by mass spectrometry with a molecular mass similar to TF. This is in contrast to TF being solely synthesized in an in vitro model incubation system using isolated catechins and purified Forelle pear polyphenol oxidase. The preferential formation of the unknown compound could explain the low levels of TFs in black tea.

Construction and transformation for the antisense expression vector of the polyphenol oxidase gene in Yali pear

To inhibit the browning process in fruits of Yali pear, in this paper, antisense gene techniques were used to reduce the expression of PPO gene. A cDNA fragment of 450 bp, which is located at the 3′ terminal of the polyphenol oxidase (PPO) gene, was amplified from Yali pear using the RT-PCR method, then the antisense expression vector was constructed by inserting the fragment of the Yali pear PPO gene between the CaMV promoter and NOS terminator of the expression vector pBI121 in a reverse orientation. After that, with the agrobacterium-mediated method, the PPO antisense gene was transformed into Yali pear shoots. Northern blot analysis and enzyme activity assay showed that the PPO activities in the transgenic Yali pear shoots were significantly decreased, compared with the non-transformed Yali pear shoots. This lays a good foundation for breeding new varieties of pears with browning resistance in the future.

CHARACTERIZATION OF POLYPHENOLOXIDASE FROM WILD PEAR (PYRUS ELAEGRIFOLIA)

ABSTRACT Wild pear polyphenoloxidase (PePPO) was extracted and purified using a Sepharose 4B-l-tyrosine-p-amino benzoic acid affinity column. Optimum conditions for pH, temperature and heat inactivation were determined. At the optimum pH and temperature, KM and Vmax values for PePPO with catechol and pyrogallol were determined. The Vmax/KM showed that PePPO has the greatest activity toward catechol. Optimum pH for PePPO was pH 6.0 using catechol as substrate. Optimum temperatures of PePPO for pyragallol and catechol were 65 and 35C, respectively. Enzyme activity decreased because of heat denaturation with increasing temperature. Inhibition of PePPO was investigated using p-aminobenzoic acid, ethyleneglycol, l-cysteine, l-tyrosine, sodium azide, p-aminobenzenesulfonamide, β-mercaptoethanol and dithiothreitol and catechol as substrate. Competitive-type inhibition was obtained with ethyleneglycol, l-cysteine, l-tyrosine, p-aminobenzenesulfonamide and dithiothreitol. Uncompetitive inhibition was obtained with β-mercaptoethanol, sodium azide and p-aminobenzoic acid. These results show that the most effective inhibitor for PePPO was dithiothreitol and that the type of inhibition depended on the origin of PPO. PRACTICAL APPLICATIONS In this present work, the properties of polyphenoloxidase in Pyrus elaegrifolia, including optimum temperature, optimum pH, substrate specificity and response to inhibitors, were studied.

Ascorbic Acid and 4‐Hexylresorcinol Effects on Pear PPO and PPO Catalyzed Browning Reaction

The effects of ascorbic acid (AA) and 4-hexylresorcinol (4-HR) on pear polyphenoloxidase (PPO) activity and stability have been investigated in vitro. AA does not interact directly with PPO but prevents browning by reducing oxidized substrates. The 4-HR exerts a dual role on PPO. If no substrates are present, it interacts preferably with the deoxy form of PPO inactivating it. If substrates and 4-HR are both present they compete for the catalytic site. The 4-HR behaves then as a canonical enzyme inhibitor, binding to the met form of PPO. Simultaneous addition of 4-HR and AA has synergistic inhibition or inactivatory effects depending on the presence or the absence of PPO substrates.

Prevention of enzymatic browning of pear by onion extract

Onion extract was prepared by extracting onion with water, and the effects of the extract on pear polyphenol oxidase and browning of pear were investigated. The polyphenol oxidase of pear was inhibited by onion extract, and the inhibitory effect of onion extract toward pear polyphenol oxidase was increased with the heated extract. The inhibitory effect of the extract was increased with increasing heating temperature and time. The browning of pear juice was retarded by addition of both fresh and heated onion extracts. The onion extract inhibited the pear polyphenol oxidase non-competitively. Therefore, the inhibitory effect of onion extract against pear browning seems to be due to the inhibitory effect of onion extract against pear polyphenol oxidase.

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

AbstractA 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

Effects of Pulsed Electric Field Processing on Apple and Pear Polyphenoloxidases

The feasibility of inhibiting polyphenoloxidase from apple and pear by pulsed electric field processing was evaluated. These treatments significantly lowered polyphenoloxidase activity of enzyme extracts from apple ( Golden deliciousvar.) and pear ( Blanquillavar.). Exponential decay pulses were generated by a laboratory scale electric pulse generator and applied in bipolar mode. Pulse duration was 0.02 ms and electric field intensities were up to 24.6 kV/cm. The temperature of samples never exceeded 15 ºC during pulsed electric field processing treatments. Polyphenoloxidase activities were reduced up to 3.15% and 38.0% initial value in apple extract at 24.6 kV/cm and pear extract at 22.3 kV/cm both for 6 ms total treatment time, respectively. Apple and pear polyphenoloxidase exposed to pulsed electric field processing diminishes their activities following first order kinetics. Rate constants ranged from 132 to 440 ms 1 for apple polyphenoloxidase, whereas for pear 1 and changed exponentially with the applied electric field intensity. Residual polyphenoloxidase activity was correlated to energy density by an exponential decay model.

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.

Monophenolase activity of polyphenol oxidase from blanquilla pear

Pear polyphenol oxidase (PPO) has been isolated by using two sequential phase partitionings with Triton X-114 (TX-114). The enzyme showed monophenolase activity when assayed on p-hydroxyphenyl propionic acid (PHPPA) with 3-methyl-2-benzothiazolinone hydrazone (MBTH) in a continuous spectrophotometric method, with high sensitivity and precision. The initial monophenolase activity showed a lag period (τ) prior to the attainment of the steady state rate ( V ss ). Both kinetic parameters, V ss and τ, depended on the enzyme and monophenol concentrations, as well as on the presence of catalytic amounts of o-diphenol. The enzyme showed an optimum pH of 4.3 and the value of K m toward PHPPA was 0.5 mM.

Continuous Spectrophotometric Method for Determining Monophenolase and Diphenolase Activities of Pear Polyphenoloxidase

ABSTRACTA continuous spectrophotometric method was based on the coupling reaction between 3‐methyl‐2‐benzothiazolinone hydrazone (MBTH) and the quinone product of the oxidation of p‐hydroxyphenyl propionic acid (PHPPA) or 3,4‐dihydroxyphenyl propionic acid (DHPPA) in the presence of polyphenol oxidase. The monophenolase activity of pear PPO was characterized for the first time. Solubility and stability of the adduct formed at the optimum pH (4.3) enabled the system to reach steady‐state, making it possible to determine monophenolase activity with short lag periods. This, together with the value of ε for the MBTH‐quinone adduct, makes this method more sensitive than other continuous methods for assaying monophenolase and diphenolase activities.

An experiment in membrane enzyme purification with triton X-114: Avocado pear polyphenoloxidase

An experiment in membrane enzyme purification with triton X-114: Avocado pear polyphenoloxidase

Purification of d'Anjou Pear (Pyrus communis L.) Polyphenol Oxidase

Polyphenol oxidase (PPO) was extensively purified to homogeneity from d'Anjou pear (Pyrus communis L.) by extraction in the presence of the phenolic binder AG 2-X8 andTriton X-100. Chlorophyll pigment was removed by chromatography resulting in a clear, colorless enzyme extract. Purification of pear PPO was achieved after chromatography on Phenyl Sepharose CL-4B, DEAE-cellulose, and hydroxylapatite columns. Only after the columns were run at room temperature rather than at 4 degrees C were sharp peaks and good resolution obtained. Reproducibility of the entire scheme was excellent with chromatography on the hydrophobic resin as a key to successful purification. Three separate fractions of pear PPO were homogeneous when stained for protein with the silver stain after polyacrylamide slab gel electrophoresis and corresponded to relative mobilities of 0.41, 0.43, and 0.73. The effect of dimethylsulfoxide on enzyme activity was investigated and found to increase significantly the activity of purified pear PPO over the control.

POLYPHENOL OXIDASE OF d'ANJOU PEARS (Pyrus communis L.)

ABSTRACTStability, substrate and inhibitor specificity and electrophoretic properties of a crude polyphenol oxidase (PPO) preparation extracted from d'Anjou pears (Pyrus communis L.) were investigated. The pH optimum of PPO occurs at 7.0. Heat inactivation of PPO followed first order kinetics and approximately 50% of PPO activity was inactivated after heating for 11.7, 6.25, 2.24 and 1.1 min at temperatures of 70°, 75°, 80° and 85°C, respectively. Crude PPO extract was active towards o‐dihydroxyphenols, but inactive towards monophenols. Disc electrophoresis revealed eight active isozymes toward catechol, 4‐methyl catechol, chlorogenic acid, caffeic acid, dopamine, d‐catechin and DL‐dopa. Similar electrophoretic patterns were observed with all substrates. No differences in the electrophoretic patterns were observed between fresh PPO preparations and preparations which had been frozen or dialzyed. Pear PPO was relatively sensitive to most inhibitors tested with the exception of sodium chloride. Of the inhibitors studied L‐cysteine and diethyldithiocarbamate were the most effective against pear PPO.

Inhibition of Friction Discoloration on ‘d’Anjou’ Pears by 2-Mercaptobenzothiazole1

Abstract Treatment with 2-mercaptobenzothiazole (MBT) inhibited the friction discoloration of fruit of ‘d’Anjou’ pear (Pyrus communis L.). The inhibition was apparently due to inactivation of the pear polyphenoloxidase.

Polyphenoloxidase in Bartlett Pearsa

SUMMARYThe characteristics of polyphenoloxidase in Bartlett pears were investigated. In a citrate‐phosphate buffer containing 0.03M catechol as the substrate, activity of the pear polyphenoloxidase was greatest in the pH range 5.8‐6.4, being optimum at pH 6.2. The Michaelis constant of the enzyme was 0.048M at pH 6.2 in a citrate‐phosphate buffer. It was active only on phenolic compounds having an ortho‐diphenolic configuration. Neither the meta‐ nor para‐dihydroxy phenolic compounds nor phenol was attacked. The energy of activation for pear polyphenoloxidase on catechol was 4.9 kcal per mole. Oxygen was necessary for browning of catechol to take place in the presence of pear polyphenoloxidase, and the activity was greatly decreased when the concentration of oxygen in the reaction mixture was lowered. Diethyldithiocarbamate, a copper‐chelating agent, and phloroglucinol, a competitive inhibitor, reduced browning markedly, but ascorbic acid was most effective of all. It was noted that ascorbic acid acts as an antioxidant rather than as a true enzyme inhibitor. Iodoacet‐amide, a sulfhydryl inhibitor, had no effect on rate of browning. Methods for preventing brown discoloration in canned pears are discussed.