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The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (K i : 0.07 ± 0.03 μM), mushroom (K i : 0.7 ± 0.3 μM), and eggplant (K i : 4.8 ± 1.2 μM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (K i : 1112 ± 324 μM), mushroom (K i : 567 ± 81 μM), and eggplant (K i : 2016.7 ± 805.6 μM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Acetes chinensis (belonging to the Decapoda Sergestidae genus) is widely distributed in East Asian waters and is extremely widespread and present in the shallow coastal areas of China. Polyphenol oxidase (PPO), which was extracted from Acetes chinensis , was purified in a four-step procedure involving phosphate-buffered saline treatment, ammonium sulphate precipitation, DEAE-Cellulose chromatography, and Phenyl-Sepharose HP chromatography, and then, its biochemical characterization was measured. The specific activity of the purified enzyme was increased to 643.4 U/mg, which is a 30.35 times increase in purification, and the recovery rate was 17.9%. L-dopa was used as the substrate, the enzymatic reactions catalyzed by PPO conformed to the Michaelis equation, the maximum reaction velocity was 769.23 U/mL, and the Michaelis constant K m was 0.846 mmol/L. The optimal pH of PPO from Acetes chinensis was 7.5, and the optimal temperature was 35 °C. The metal ions experiment showed that Mn 2+ and K + could enhance the activity of PPO; that Ba 2+ and Ca 2+ could inhibit the activity of PPO; and that Cu 2+ had a double effect on PPO, increasing the PPO activity at low concentrations and inhibiting the PPO activity at high concentrations. The inhibitor experiment showed that the inhibitory effects of EDTA and kojic acid were weak and that ascorbic acid and sodium pyrophosphate had good inhibitory effects. The purification and characterization of Acetes chinensis serve as guidelines for the prediction of enzyme behavior, leading to effective prevention of enzymatic browning during processing.

Fennel seeds were recognized as a promising polyphenol oxidase (PPO) source upon investigating some edible green plants (carob, jujube, coriander, fennel, and licorice). The fennel PPO enzyme was purified by three-phase partitioning and biochemically characterized in detail for the first time. The purification fold and activity recovery values were determined as 20-fold and 120%, respectively. Its molecular weight was 27.8 kDa. The temperature for the selected substrates (catechol, 4-tert-butylcatechol, 4-methylcatechol, and pyrogallol) was 30 °C, while the optimum pH value varied from 5.0 to 7.0 depending on the substrate. The k cat /Km values exhibited that the enzyme presented the best activity towards catechol among the substrates used. Sodium metabisulfite, ascorbic acid, benzoic acid, l-cysteine, thiourea, β-mercaptoethanol, and glutathione prominently inhibited PPO activity. A remarkable decrease in PPO activity was observed at elevated concentrations of organic solvents, but in cases of the solvents with polarity indexes ≥5.1, the residual activity maintained more than 75% of its original activity up to 10% (v/v). Consequently, the current study suggested that fennel seeds could be used in various industrial sectors to produce low-cost polyphenol oxidase enzymes with an agricultural origin., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the literature, the polyphenol oxidase (PPO) enzyme has been purified a many times via Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity column. In order to study PPO purification efficiency, 2-aminophenol and 4-aminophenol were applied as a spacer arm to CNBr-activated Sepharose 4B. The effects of the spacer arm on specific activity, purification fold, and electrophoretic properties were investigated. The best performance with 11.7-fold purification and 23951 U/mg protein specific activity was achieved with the 4-aminophenol extension arm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with was done to check the purity of the potato PPO enzyme obtained from affinity columns. According to the results of SDS-PAGE and native PAGE, the molecular weight of the enzyme is 50 kDa. Furthermore, the inhibition effects of curcumin and quercetin on the enzyme activity were examined, and the IC 50 and K i values were computed for the mentioned substances. IC50 values were determined to be 0.018 and 0.029 mM for potato PPO with curcumin and quercetin inhibitors with catechol as a substrate, respectively. IC50 value was also determined to be 0.0086 mM for quercetin inhibitor with 4-methylcatechol as a substrate. K i constant was 0.0753 ± 0.0085 mM for curcumin using catechol as a substrate. No inhibition effect was observed for curcumin with the 4-methylcatechol substrate. The K i constant for quercetin was 0.0398 ± 0.00743 mM with the 4-methylcatechol substrate and 0.0109 ± 0.0021 mM with the catechol substrate., Competing Interests: Declaration of competing interest The author report no declarations of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Polyphenol Oxidase (PPO) belongs to the oxidoreductase enzyme family., Methods: Here, PPO was purified from potato using Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity chromatography. It determined the interactions between some phenolic acids and the enzyme., Results: The enzyme was obtained with a specific activity of 15333.33 EU/mg protein and 7.87- fold purification. It was found that phenolic acids exhibited inhibitory properties for PPO. The IC50 values of the phenolic acids were found in the range of 0.36-2.12 mM, and their Ki values were found in the range of 0.28± 0.07-1.72±0.32 mM. It was determined that all studied compounds displayed a competitive inhibition effect. Among these compounds, 3-hydroxybenzoic acid was found to be the most effective PPO inhibitor (Ki: 0.28±0.07 mM)., Conclusion: Investigating the inhibition kinetics of the enzyme will simplify the testing of PPO inhibitor candidates., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Apart from non-enzymatic browning, polyphenol oxidase (PPO) also plays a role in the browning reaction of orange ( Citrus sinensis Osbeck) juice, and needs to be inactivated during the processing. In this study, the protein with high PPO activity was purified from orange ( Citrus sinensis Osbeck) and inactivated by ultrasonic processing. Fluorescence spectroscopy, circular dichroism (CD) and Dynamic light scattering (DLS) were used to investigate the ultrasonic effect on PPO activity and structural changes on purified PPO. DLS analysis illustrated that ultrasonic processing leads to initial dissociation and final aggregation of the protein. Fluorescence spectroscopy analysis showed the decrease in fluorescence intensity leading to the exposure of Trp residues to the polar environment, thereby causing the disruption of the tertiary structure after ultrasonic processing. Loss of α-helix conformation leading to the reorganization of secondary structure was triggered after the ultrasonic processing, according to CD analysis. Ultrasonic processing could induce aggregation and modification in the tertiary and secondary structure of a protein containing high PPO activity in orange ( Citrus sinensis Osbeck), thereby causing inactivation of the enzyme.

Catechol oxidase (PPO) was extracted and purified from Tadela (Phoenix dactylifera L.) date fruit, by a procedure that included (NH 4 ) 2 SO 4 precipitation followed by dialysis, Q-Sepharose bb ion-exchange chromatography and HPLC gel filtration chromatography. Some of its biochemical characteristics were studied. The purification rate and the yield were 80% and 20%, respectively. The Tadela date fruit catechol oxidase exhibited a molecular weight of 90 kDa using SDS-PAGE. The catechol oxidase showed only o‑diphenolase and triphenolase activities while no monophenolase activity was detected. A better affinity was observed using catechol as substrate (Km = 35 mM) with thus, a higher Vmax/Km ratio (80 U/mM·mL). This enzyme is thermostable in the temperature range (30-60 °C) with optimum activity in acidic range of pH. Four inhibitors were used for the control of enzymatic browning, of which sodium metabisulfite was the most potent (IC 50  = 0, 11 mM). The values of K I and mechanism of inhibition were also determined. No significant change on enzyme activity was noticed in the presence of metal ion and detergents. Therefore, thermal inactivation was studied in the temperature range between 60 and 80 °C using catechol as substrate. Their kinetic (K, D, t 1/2 , Zt, Ea) and thermodynamic (ΔH, ΔG and ΔS) parameters were also estimated., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The soursop (Annona muricata L.) is a climacteric fruit that may undergo enzymatic browning during ripening, mainly by the activity of polyphenol oxidase (PPO). Soursop PPO was purified 160-fold by hydrophobic interaction and ion-exchange chromatography. The native structure has a molecular weight of 112 kDa corresponding to a dimeric structure. The protein has an optimum pH and temperature of 6.5 and 25°C, respectively; and activation energy of 40.97 kJ·mol -1 . The lowest K m value was observed for caffeic acid (0.47 mM); the best substrate was 4-methyl-catechol (1,067 U·mM -1  min -1 ). Inactivation assays showed that PPO was completely inactivated by tropolone, Na 2 S 2 O 5 and ascorbic acid, and thermally at 55°C for <5 min, microwave exposure reduced activity to 57% at 70 W in 30 s and ultrasound treatment diminished activity to 43% at 120 W in 220 s. This study allows a better understanding of soursop PPO behavior and provides inactivation information. PRACTICAL APPLICATIONS: The conservation of fresh fruits is complicated due to the enzymatic reactions that are present in fruits, such as enzymatic browning. The enzymes responsible for these reactions can be inactivated by, different chemical compounds as well as by the use of emerging technologies, such as microwaves and sonication, which seek to satisfy the consumer needs to obtain fresh products with good nutritional characteristics and adequate safety., (© 2019 Wiley Periodicals, Inc.)

This work describes a TLC-coupled bioautographic assay suitable for the separation and detection of apple polyphenol oxidase (PPO) inhibitors from natural extracts. PPO was immobilised in agar containing l-DOPA as substrate and 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) to enhance colour development. The inhibition was detected as white spots on reddish background. Minimum amount of PPO inhibitors detected was 0.0125 µg of 4-hexylresorcinol, 0.025 µg of ascorbic acid, 0.5 µg of cysteine and 1 µg of kojic acid. The assay was compatible with normal and reverse phase TLC systems and allows detecting compounds that directly had action on the enzyme as well as agents that could convert quinones back to their reduced form. The chromatographic run evidenced the different nature of enzymatic browning inhibitory compounds from garlic and onion extracts. Using natural enzymes will provide a fast and cheap alternative for target specific exploration of natural enzymatic inhibitors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Peroxidase (POD) from jackfruit bulb was purified using ammonium sulfate precipitation, hydrophobic interaction and gel filtration columns. The POD was a dimer with a molecular weight of 104kDa. The K m and V max values for guaiacol, gallic acid and o‑phenylenediamine (OPD) were estimated. OPD was the most suitable substrate. The enzyme showed its maximum activity at pH5.5 and 55-60°C. The activation energy (E a ) of heat inactivation was estimated to be 206.40kJ/mol. The enthalpy, free energy and entropy values for the thermal inactivation were also determined. The POD activity was enhanced by K + , Zn 2+ , Ba 2+ , citric acid, malic acid, benzoic acid and EDTA·Na 2 , but inhibited by Cu 2+ , Ca 2+ , glutathione, cysteine and ascorbic acid. Chemical modification indicated a histidine residue was located in the enzyme active site. The POD activity in fruit extracts significantly decreased when heated at 80°C and 90°C. The ferric-reducing antioxidant power, ABTS radical scavenging activity and total phenolics decreased with increasing heating temperature and time., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The industry discards generous organic wastewater in sweet potato starch factory and scrap tea in tea production. A simplified procedure to recover all biochemicals from the wastewater of sweet potato starch factory and use them to make health black tea and theaflavins from scrap green tea was developed. The sweet potato wastewater was sequentially treated by isoelectric precipitation, ultrafiltration and nanofiltration to recover polyphenol oxidase (PPO), β-amylase, and small molecular fractions, respectively. The PPO fraction can effectively transform green tea extracts into black tea with high content of theaflavins through the optimized fed-batch feeding fermentation. The PPO transformed black tea with sporamins can be used to make health black tea, or make theaflavins by fractionation with ethyl acetate. This work provides a resource- and environment-friendly approach for economically utilizing the sweet potato wastewater and the scrap tea, and making biochemical, nutrient and health products., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The present study evaluated the effect of high isostatic pressure (HIP) on the activity of peroxidase (POD) and polyphenol oxidase (PPO) from açaí. Açaí pulp was submitted to several combinations of pressure (400, 500, 600MPa), temperature (25 and 65°C) for 5 and 15min. The combined effect of HIP technology and high temperatures (690MPa by 2 and 5min at 80°C) was also investigated and compared to the conventional thermal treatment (85°C/1min). POD and PPO enzyme activity and instrumental color were examined after processing and after 24h of refrigerated storage. Results showed stability of POD for all pressures at 25°C, which proved to be heat-resistant and baro-resistant at 65°C. For PPO, the inactivation at 65°C was 71.7% for 600MPa after 15min. In general, the increase in temperature from 25°C to 65°C reduced the PPO relative activity with no changes in color. Although the thermal treatment and the HIP (690MPa) along with high temperature (80°C) reduced the PPO relative activity, and relevant darkening was observed in the processed samples. Thus, it can be concluded that POD is more baro-resistant than PPO in açaí pulp subjected to the same HIP processing conditions and processing at 600MPa/65°C for 5min may be an effective alternative for thermal pasteurization treatments., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Polyphenol oxidases (PPOs) catalyze browning reactions in various plant organs, therefore controlling the reactions is important for the food industry. PPOs have been assumed to be involved in skin browning of white grape cultivars; however, the molecular mechanism underlying PPO-mediated browning process remains elusive. We have recently identified a new PPO gene named VvPPO2 from "Shine Muscat" (Vitis labruscana Bailey × V. vinifera L.), and have shown that the gene is transcribed at a higher level than the previously identified VvPPO1 in browning, physiologically disordered berry skins at the maturation stage. In this study, we expressed VvPPO2 in Escherichia coli and, using the purified preparation, revealed unique physicochemical characteristics of the enzyme. Our study opens up a way to not only understand the berry skin browning process but also to elucidate the enzymatic maturation process of grape PPOs.

Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO 4 and low concentrations (≤ 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (K M = 2.0 mM) and chlorogenic acid (K M = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt O81103 ) by means of peptide mass fingerprinting.

Background: The extraction, purification, and characterisation of peroxidase (POD) and polyphenol oxidase (PPO) were studied for Kalipatti sapota fruit. The crude enzyme extract was partially purified by ammonium sulfate precipitation followed by BioGel P100 size exclusion and Unosphere Q anion-exchange chromatography., Results: Molecular weights of 20 kDa (POD) and 24 kDa (PPO) were indicated by SDS-PAGE. A single band was observed on SDS-PAGE with a fold purity of 10.38 and 7.42 for POD and PPO, respectively. Michaelis-Menten constants for POD and PPO were 22.3 and 23.0 mmol L -1 using guaiacol and catechol as substrates. Thermal inactivation kinetics was studied in the temperature range of 60-95 °C. The crude extract of POD and PPO showed D-values of 2.2-60.2 and 1.0-35.2 min; Z-values of 18.7 ± 0.4 and 16.0 ± 0.3 °C; and activation energies (E a ) of 128.6 and 151.0 kJ mol -1 , respectively., Conclusion: POD and PPO showed good stability over a wide range of pH and temperature. As reflected by Z and E a values, the fruit matrix had no significant influence towards enzyme stability. Designing of thermal process should take into consideration D- and Z-values of the enzymes along with D- and Z-values of microorganisms to obtain a product with better shelf life. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background: Polyphenol oxidase (PPO) mainly contributes to the browning reaction of fruits and vegetables and causes serious damage to the quality of sweet melon products. However, traditional methods to inactivate browning may induce more unexpected risks than ultrasonic processing. Meanwhile, there are no reports on the effect of ultrasound on PPO directly purified from sweet melon., Results: The PPO in the original juice was less inactivated than the purified form when treated with ultrasound. As for purified PPO, superior to thermal treatment, less heat was needed to inactivate the PPO with ultrasonic treatment. At intensity lower than 200 W, ultrasound did not significantly affect the structure and activity of PPO (P > 0.05), and latent PPO was activated. At intensity higher than 200 W, ultrasound inactivated PPO, induced aggregation and dissociation of PPO particles and significantly decreased the α-helix structure content., Conclusion: Low-frequency high-intensity ultrasound caused an inactivation effect and conformational changes of purified PPO from oriental sweet melons. Changes in the PPO structure induced by ultrasound eventually inactivated the enzyme. Ultrasound may be a potential method to inactivate PPO in oriental sweet melons. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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 Fe 3 O 4 /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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study a Polyphenol oxidase from borage plant was purified with 3.59-fold enrichment in the specific activity and 68.75% recovery of the total activity by using three-phase partitioning purification technique for the first time. Its molecular weight was found around 80kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH and temperature values of the enzyme for the used four substrates ranged between the pH 5.0-7.5 and 5-30°C. The kcat/K m values showed that the enzyme has the greatest reactivity toward caffeic acid among the substrates used. Ascorbic acid, l-cysteine and sodium metabisulfite markedly inhibited borage polyphenol oxidase activity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, polyphenol oxidase (PPO) from corn tassel  was extracted and partially purified through  (NH4)2SO4 precipitation and gel filtration chromatography. Optimal temperatures for subsrates catechol and 4-methyl catechol were 40 °C and 30 °C, respectively. The optimal pH values were 8.0 for catechol and 6.0 for 4-methyl catechol. Catechol was the most suitible substrate (Km: 3.48 mM, Vmax: 1.0 Abs./ min.). The moleculer mass of PPO was determined as 158 kDa. In this work, sodium azide, ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) were found to inhibit the enzyme activity as 26.6 %,  22.2 % and 12.2 % ratio, respectively. Besides, the effects of carbohydrates such as sucrose, fructose, ribose and glucose on PPO activity were investigated. The enzyme was found to be activated 17 % by fructose and ribose, 16 % by glucose and 4 % by sucrose.

A new strategy for the construction of a polyphenol oxidase carbon paste biosensor for paracetamol detection is reported. The eggplant (Solanum melongena) was processed to collect the polyphenol oxidase as an enzyme that was incorporated in the carbon paste sensor construction. The constructed sensor displayed high sensitivity and good selection for paracetamol detection and recognition. Optimized conditions included pH 6.0 (highest activity), pH 7.0 (highest stability), pulse amplitude of 50 mV, and 15% of vegetable extract per carbon paste. The sensor displayed a linear range from 20 to 200 µM, with a detection limit of 5 µM. Application of the sensor to paracetamol determination in tablet and oral solutions have shown satisfactory results. The efficiency of the method showed very good repeatability ranging between 1.26 and 1.72% relative standard deviation for interday analysis, while recoveries for paracetamol varied between 97.5 and 99.8% for the voltammetric determination. The strategy for a simple, low cost, and efficient eggplant polyphenol oxidase sensor showcased in this work provides an opportunity for the detection of other phenolic compounds in various matrices.

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was isolated and biochemically characterized. The enzyme was extracted and purified using acetone and aqueous two-phase systems. The data indicated that PPO had the highest substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid was the most suitable substrate (Km=0.56±0.07 mM and Vmax=53.15±2.03 UPPO mL(-1) min(-1)). The optimal pH values were 5.5 for catechol and 4-methylcatechol and 5.0 for chlorogenic acid. Optimal temperatures were 40°C for catechol, 25°C for 4-methylcatechol and 20°C for chlorogenic acid. In inhibition tests, the most potent inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This study shows possible treatments that can be implemented during the processing of Cape gooseberry fruits to prevent browning., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In the present study, we purified two prophenoloxidases (proPO) from haemolymph of green tiger shrimp, Penaeus semisulcatus by gel fermentation chromatography using blue Sepharose matrix. The two purified prophenoloxidase macromolecules are of about 76 and 75 kDa determined through SDS-PAGE and named as Penaeus semisulcatus prophenoloxidase I (PSproPO I) and Penaeus semisulcatus prophenoloxidase II (PSproPO II). It was further characterized by X-Ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and High Performance Liquid Chromatography (HPLC) analysis. The purified PSproPO I and PSproPO II showed the strongest agglutination titre against human erythrocytes compared to goat RBC. The PSproPO I and PSproPO II showed phagocytic activity against yeast Saccharomyces cerevisiae and encapsulation activity against Sepharose CL 6B beads compared to CM Sepharose and Sodium alginate beads. The functional analysis of purified PSproPO I and PSproPO II showed enhanced PO activity when added with the triggering molecules such as pathogen associated molecular patterns (PAMPs), metals and chemicals. In addition, eluted fraction containing PSproPO I and PSproPO II showed antibiofilm activity against Gram positive and Gram negative bacteria. The above results concluded that no significant differences were found between the purified PSproPO I and PSproPO II immune indices and functions. This study might provide a sensitive platform to understand more about the critical roles of PSproPO I and PSproPO II in crustacean immune system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented., (© 2015 Scandinavian Plant Physiology Society.)

Conventional three phase partitioning (TPP) and ultrasound assisted three phase partitioning (UATPP) were optimized for achieving the maximum extraction and purification of polyphenol oxidase (PPO) from waste potato peels. Different process parameters such as ammonium sulfate (NH4)2SO4 concentration, crude extract to t-butanol ratio, time, temperature and pH were studied for conventional TPP. Except agitation speed, the similar parameters were also optimized for UATPP. Further additional parameters were also studied for UATPP viz. irradiation time at different frequencies, duty cycle and, rated power in order to obtain the maximum purification factor and recovery of PPO. The optimized conditions for conventional TPP were (NH4)2SO4 0-40% (w/v), extract to t-butanol ratio 1:1 (v/v), time 40 min and pH 7 at 30°C. These conditions provided 6.3 purification factor and 70% recovery of PPO from bottom phase. On the other hand, UATPP gives maximum purification fold of 19.7 with 98.3% recovery under optimized parameters which includes (NH4)2SO4 0-40% (w/v), crude extract to t-butanol ratio 1: 1 (v/v) pH 7, irradiation time 5 min with 25 kHz, duty cycle 40% and rated power 150W at 30°C. UATPP delivers higher purification factor and % recovery of PPO along with reduced operation time from 40 min to 5 min when compared with TPP. SDS PAGE showed partial purification of PPO enzyme with UATPP with molecular weight in the range of 26-36 kDa. Results reveal that UATPP would be an attractive option for the isolation and purification of PPO without need of multiple steps., (© 2015 American Institute of Chemical Engineers.)

Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2(1)2(1)2(1) and P12(1)1 and diffracted to ∼ 1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3(1)21. The crystals of latent cgAUS1 belonged to space group P12(1)1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na6[TeW6O24] within the liquid-liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI).

Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr.) fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6). Three isoforms of polyphenol oxidase (PPO) were discovered and two isoforms (1-l and 1-2) were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6). Specfic activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule ofthe enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 °C, and at +70 °C the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content ofthe milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m- and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol) and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

The inhibition of Polyphenol oxidase (PPO) in plants has been widely researched for their important roles in browning reaction. A newly found germin-like protein (GLP) with high PPO activity in Satsuma mandarine was inactivated by low-frequency high-intensity ultrasonic (20 kHz) processing. The effects of ultrasound on PPO activity and structure of GLP were investigated using dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM), circular dichroism (CD) spectral measurement and fluorescence spectral measurement. The lowest PPO activity achieved was 27.4% following ultrasonication for 30 min at 400 W. DLS analysis showed ultrasound caused both aggregation and dissociation of GLP particles. TEM images also demonstrated protein aggregation phenomena. CD spectra exhibited a certain number of loss in α-helix structure content. Fluorescence spectra showed remarkable increase in fluorescence intensity with tiny blue-shift following ultrasonication. In conclusion, ultrasound applied in this study induced structural changes of GLP and eventually inactivated PPO activity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Polyphenol oxidase (PPO) has multiple functions, and the lack of commercially available enzyme sources limits its widespread application in various industries. An accurate PPO assay was developed by HPLC determination of the substrate oxidation. Resources screening indicated that sweet potato (Ipomoea batatas L.) wastewater in starch production has high PPO activity. A procedure was developed for separately recovering PPO, β-amylase, sporamins, and small molecular nutrients (SMNs) from sweet potato wastewater. The wastewater was adjusted to pH 3.5 to precipitate PPO, and then adjusted to 50 % acetone to precipitate β-amylase and further to 80 % acetone to precipitate sporamins. The SMNs were obtained after acetone recovery. Purified powders of 4.3 × 10(5) units of PPO, 4.0 × 10(6) units of β-amylase, 8.70 g sporamins, and 20.2 g SMNs were obtained from the wastewater of 1 kg sweet potato. More than 50 million tons of sweet potato is used for starch production annually around the world. Through this simple procedure, huge amount of biochemical resources can be recovered from the wastewater, which greatly increases the economic value of the crop and saves the environment.

Potato peel from food industrial waste is a good source of polyphenol oxidase (PPO). This work illustrates the application of an aqueous two-phase system (ATPS) for the extraction and purification of PPO from potato peel. ATPS was composed of polyethylene glycol (PEG) and potassium phosphate buffer. Effect of different process parameters, namely, PEG, potassium phosphate buffer, NaCl concentration, and pH of the system, on partition coefficient, purification factor, and yield of PPO enzyme were evaluated. Response surface methodology (RSM) was utilized as a statistical tool for the optimization of ATPS. Optimized experimental conditions were found to be PEG1500 17.62% (w/w), potassium phosphate buffer 15.11% (w/w), and NaCl 2.08 mM at pH 7. At optimized condition, maximum partition coefficient, purification factor, and yield were found to be 3.7, 4.5, and 77.8%, respectively. After partial purification of PPO from ATPS, further purification was done by gel chromatography where its purity was increased up to 12.6-fold. The purified PPO enzyme was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by Km value 3.3 mM, and Vmax value 3333 U/mL, and enzyme stable ranges for temperature and pH of PPO were determined. These results revealed that ATPS would be an attractive option for obtaining purified PPO from waste potato peel.

In this study, firstly, antioxidant and polyphenol oxidase (PPO) properties of Yomra apple were investigated. Seventeen phenolic constituents were measured by reverse phase-high-performance liquid chromatography (RP-HPLC). Total phenolic compounds (TPCs), ferric reducing antioxidant power (FRAP) and 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging activities were performed to measure antioxidant capacity. Some kinetic parameters (Km, Vmax), and inhibition behaviors against five different substrates were measured in the crude extract. Catechin and chlorogenic acid were found as the major components in the methanolic extract, while ferulic acid, caffeic acid, p-hydroxybenzoic acid, quercetin and p-coumaric acid were small quantities. Km values ranged from 0.70 to 10.10 mM in the substrates, and also 3-(4-hydroxyphenyl) propanoic acid (HPPA) and L-DOPA showed the highest affinity. The inhibition constant of Ki were ranged from 0.05 to 14.90 mM against sodium metabisulphite, ascorbic acid, sodium azide and benzoic acid, while ascorbic acid and sodium metabisulphite were the best inhibitors.

Polyphenol oxidase (PPO) is an enzyme widely distributed in the plant kingdom that has been detected in most fruits and vegetables. PPO was extracted and purified from Manila mango (Mangifera indica), and its biochemical properties were studied. PPO was purified 216-fold by hydrophobic interaction and ion exchange chromatography. PPO was purified to homogeneity, and the estimated PPO molecular weight (MW) by SDS-PAGE was ≈31.5 kDa. However, a MW of 65 kDa was determined by gel filtration, indicating a dimeric structure for the native PPO. The isolated PPO showed the highest affinity to pyrogallol (Km = 2.77 mM) followed by 4-methylcatechol (Km = 3.14 mM) and catechol (Km = 15.14 mM). The optimum pH for activity was 6.0. PPO was stable in the temperature range of 20-70 °C. PPO activity was completely inhibited by tropolone, ascorbic acid, sodium metabisulfite, and kojic acid at 0.1 mM.

Browning tends to occur in grape juice during processing and storage and decreases the commercial value of it. Thus, browning inhibition is an important objective for manufacturers. This study aims to investigate the efficacy of glutathione as a browning inhibitor for use on grape juice. Grape juice browning treated with glutathione was monitored during processing and accelerated browning. 0.04% of glutathione inhibited 99.4% of the polyphenoloxidase activity in the grape juice. Consequently, during processing at room temperature and accelerated browning at 80 °C, the browning in the grape juice treated with glutathione was significantly lower than that in the control (p<0.05). The results indicate that glutathione is a promising browning inhibitor used in grape juice., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The prophenoloxidase (proPO) cascade supplies quinones and other reactive compounds for melanin formation, protein cross-linking, hemolymph coagulation, and killing of microbial invaders as well as parasites. The high cytotoxicity of the generated compounds requires a strict control of the activation of the proPO system and phenoloxidase (PO) activity to minimize damage to host tissues and cells. The PO activity in hemolymph of Escherichia coli challenged Galleria mellonella larvae increased, with a temporal drop 1 h after the challenge, reaching the highest level 24 h after the challenge. In the present study, a potential role of G. mellonella defense peptides and lysozyme in controlling the proPO system was investigated. The effects of purified defense peptides (anionic peptides 1 and 2, cecropin D-like peptide, Galleria defensin, proline-rich peptides 1 and 2) and lysozyme were analyzed. Four compounds, namely lysozyme, Galleria defensin, proline-rich peptide 1, and anionic peptide 2, decreased the hemolymph PO activity considerably, whereas the others did not affect the enzyme activity level. Our results indicate that these hemolymph factors could play multiple and distinct roles in the insect immune response., (© 2014 Wiley Periodicals, Inc.)

Polyphenol oxidases (PPOs) catalyzing the oxygen dependent oxidation of phenols to quinones are ubiquitously distributed in plants and are assumed to be involved in plant defense against pests and pathogens. A protein with high PPO activity was identified in Satsuma mandarine, extracted with Tris-HCl buffer, purified by salt precipitation and column chromatography, and characterized by mass spectrometry as germin-like protein (GLP), which belongs to pathogenesis related protein (PR) family. In the present study, the structure and enzymatic properties of GLP were characterized using spectroscopy methods. Based on native PAGE analysis, the molecular weight of GLP was estimated to be 108 kDa and GLP was identified as a pentamer containing five subunits of 22 kDa. The optimum pH and temperature for PPO catalyzing activity of GLP was 6.5 and 65°C, respectively. Kinetic constants were 0.0365 M and 0.0196 M with the substrates catechol and pyrogallol, respectively. The structural characterization of GLP provided better insights into the regions responsible for its PPO activity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Polyphenol oxidase (PPO) from jackfruit bulb was purified through acetone precipitation, ion-exchange column, and gel filtration column. PPO was a dimer with the molecular weight of 130 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. The Km was 8.3 and 18.2 mM using catechol and 4-methylcatechol as substrates, respectively. The optimum pH was 7.0 (catechol as the substrate) or 6.5 (4-methylcatechol as the substrate). The optimum temperature was 8 °C. The enzyme was stable below 40 °C. The activation energy (Ea) of heat inactivation was estimated to be 103.30 kJ/mol. The PPO activity was activated by Mn(2+), SDS, Tween-20, Triton X-100, citric acid, and malic acid but inhibited by K(+), Zn(2+), Mg(2+), Ca(2+), Ba(2+), cetyl trimethyl ammonium bromide (CTAB), kojic acid, tropolone, glutathione (GSH), cysteine (Cys), and ascorbic acid (AA). Cys and AA were effective to reduce browning of jackfruit bulbs during the storage at 8 °C for 15 days.

The copper containing prophenoloxidase enzyme plays a crucial role in the defense system of arthropods, especially crustaceans and insects. In this study, we have reported a full length cDNA of prophenoloxidase identified from the constructed cDNA library of freshwater prawn Macrobrachium rosenbergii by genome sequence FLX technology. The identified full length M. rosenbergii prophenoloxidase (MrProPO) consists of 3378 base pairs (bp) with an open reading frame (ORF) of 2099 bp. This ORF encoded a polypeptide of 700 amino acids (aa) with an estimated molecular mass of 80 kDa and a predicted isoelectric point (pI) of 6.7. The motif analysis of MrProPO shows two copper binding sites (CuA and CuB) along with hemocyanin signatures and a thiol-ester like motif. MrProPO exhibited the maximum similarity (97%) with ProPO from Macrobrachium nipponense and is closely clustered with other crustacean ProPO in the phylogenetic tree. Bioinformatics analysis suggests that MrProPO is a member of the prophenoloxidase family, due to the conserved domains, motifs and similarity with other known ProPOs. The 3D structural analysis of MrProPO reveals that it has more random coils, moderate α-helices, few extended β-sheets and a very few β-turns. Among the 700 aa of MrProPO, 355 (50.71%), 206 (29.43%), 110 (15.71%) and 29 (4.14%) amino acids are responsible for random coils, α-helices, extended β-sheets and β-turns respectively. The gene expression results indicate MrProPO is widely distributed in all the tissues studied, but significantly (P<0.05) highest expression was observed in hepatopancreas. The relative expression of mRNA was quantified in hepatopancreas after being infected with virus [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacteria (Aeromonas hydrophila and Vibrio harveyi) using real-time PCR. MrProPO mRNA transcription significantly (P<0.05) increased at 24h post injection (p.i.) with subsequent decrease at 48 h p.i. in both viral and bacterial infected prawns. The highest enzyme activity was observed in hepatopancreas, which was also significantly higher (P<0.05) than detected in other tissues. Similar to gene expression results, the enzyme activity reached the peak at 24h p.i. and then the activity started decreasing. Overall results indicate that MrProPO is very likely to participate in the acute response against pathogen entry in prawns., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Ferula communis (L.), a plant belonging to Apiaceae, is widely present in Sardinia, Italy. Currently, interest in F. communis focuses on the presence of two chemotypes in the wild. One chemotype is poisonous to animals, whereas the other chemotype is non-poisonous. Polyphenol oxidase (PPO) has been extracted and partially purified from the two chemotypes of F. communis. The biochemical characterization of the enzymes showed significant differences. In particular, while the two PPOs were not able to use 6- and 7-hydroxycoumarin as substrates, they showed distinct specificity for 6,7- and 7,8-dihydroxycoumarin. Significant differences in the enzyme behavior towards common PPO inhibitors were also observed. In addition, activation energy and activation energy for denaturation were determined, showing significant differences between FP-PPO and FNP-PPO, particularly for denaturation kinetics. The possible roles of the two PPOs in determining differences in composition and toxicity of the two F. communis chemotypes are also discussed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hemocyanins are multi-subunit oxygen carrier proteins, found in select species of arthropoda and mollusca. Here, we have purified native hemocyanin from Pila globosa, a freshwater gastropod, verified using mass spectrometry and determined its molecular weight, secondary structure and the spectral properties, using Ultraviolet/visible, Fourier transform infra-red and Circular dichroism spectroscopy. Our results reveal the oligomeric and glycosylated nature of the protein, comprising of 400 kDa subunits, organized predominantly into a thermo-stable, alpha-helical conformation. Further, biochemical assays confirm catecholoxidase-like activity in hemocyanin, which has been used to develop a first-generation optical sensor, for the detection of phenols.

Tube-building sabellariid polychaetes have major impacts on the geology and ecology of shorelines worldwide. Sandcastle worms, Phragmatopoma californica (Fewkes), live along the western coast of North America. Individual sabellariid worms build tubular shells by gluing together mineral particles with a multipart polyelectrolytic adhesive. Distinct sets of oppositely charged components are packaged and stored in concentrated granules in separate cell types. Homogeneous granules contain sulfated macromolecules as counter-polyanion to polycationic Pc2 and Pc5 proteins, which become major components of the fully cured glue. Heterogeneous granules contain polyphosphoproteins, Pc3A/B, paired with divalent cations and polycationic Pc1 and Pc4 proteins. Both types of granules contain catechol oxidase that catalyzes oxidative cross-linking of L-DOPA. Co-secretion of catechol oxidase guarantees rapid and spatially homogeneous curing with limited mixing of the preassembled adhesive packets. Catechol oxidase remains active long after the glue is fully cured, perhaps providing an active cue for conspecific larval settlement.

The purification and partial enzymology characteristics of polyphenol oxidase from Lonicera japonica (LjPPO) were studied in this paper. The crude enzyme solution was purified in turn by ammonium sulfate, dialysis, and DEAE-cellulose ion-exchange chromatography after preliminary treatments. Purification resulted in 31-fold enrichment and its molecular weight was estimated to be ~49 kDa exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH for optimal conditions of LjPPO was 7.5, and the temperature was 25 °C, in addition, the inhibitive effects of inhibitors were enhanced positively with increasing of the concentration. Moreover, crude enzyme solution showed diphenolase activity toward catechol, l-dopa and chlorogenic acid rather than monophenolase and triphenolase activity, and the best substrate was catechol because of the highest V(max)/K(m) value. However, the oxidation of diphenol related to browning significantly, so the data obtained in this research provided theoretical basis for the prevention of enzymatic browning of L. japonica during processing., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Membrane-bound polyphenoloxidase (mPPO) an oxidative enzyme which is responsible for the undesirable browning reaction in Snake fruit (Salacca zalacca (Gaertn.) Voss) was investigated. The enzyme was extracted using a non-ionic detergent (Triton X-114), followed by temperature-induced phase partitioning technique which resulted in two separate layers (detergent-poor phase at the upper layer and detergent-rich phase at the lower layer). The upper detergent-poor phase extract was subsequently fractionated by 40-80% ammonium sulfate and chromatographed on HiTrap Phenyl Sepharose and Superdex 200 HR 10/30. The mPPO was purified to 14.1 folds with a recovery of 12.35%. A single prominent protein band appeared on native-PAGE and SDS-PAGE implying that the mPPO is a monomeric protein with estimated molecular weight of 38kDa. Characterization study showed that mPPO from Snake fruit was optimally active at pH 6.5, temperature 30°C and active towards diphenols as substrates. The K(m) and V(max) values were calculated to be 5.46 mM and 0.98 U/ml/min, respectively, when catechol was used as substrate. Among the chemical inhibitors tested, l-cysteine showed the best inhibitory effect, with an IC(50) of 1.3 ± 0.002 mM followed by ascorbic acid (1.5 ± 0.06 mM), glutathione (1.5 ± 0.07 mM), EDTA (100 ± 0.02 mM) and citric acid (186 ± 0.16 mM)., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We characterize umbelliferone, a derivative of 2,4-dihydroxycoumaric acid, as a substrate of polyphenol oxidase. This enzyme hydroxylates umbelliferone to esculetin, its o-diphenol, and then oxidizes it to o-quinone. The findings show that umbelliferone, an intermediate in one of the coumarin biosynthesis pathways, may be transformed into its o-diphenol, esculetin, which is also an intermediate in the same pathway. The activity of the enzyme on umbelliferone was followed by measuring the consumption of oxygen, spectrophotometrically and by HPLC. Kinetic constants characterizing the hydroxylation process were: kcat=0.09±0.02 s(-1) and Km=0.17±0.06 mM. The o-diphenol, esculetin, was a better substrate and when its oxidation was followed spectrophotometrically, the kinetic constants were: kcat=1.31±0.25 s(-1) and Km=0.035±0.002 mM. Both compounds therefore can be considered as alternative substrates to L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA), since both indirectly inhibit melanogenesis.

Prophenoloxidase (PPO) was purified from Galleria mellonella L. A 67-fold purification of the proenzyme with 352% yield was achieved by using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. The purified enzyme was migrated as a single band on SDS-polyacrylamide gel electrophoresis. K(m) and V(max) values were 0.017 M and 1430.45 EU for catechol. Inhibition of PPO was investigated with inhibitors such as p-aminobenzoic acid, etyleneglycol, and ascorbic acid. Among them, ascorbic acid showed the strongest inhibitory activity with IC(50) value of 2.94 μM. The current paper represents new strategies for the biological control of the Galleria mellonella L. insect.

Eggplant (Solanum melongena) is a very rich source of polyphenol oxidase (PPO), which negatively affects its quality upon cutting and postharvest processing due to enzymatic browning. PPO inhibitors, from natural or synthetic sources, are used to tackle this problem. One isoform of PPO was 259-fold purified using standard chromatographic procedures. The PPO was found to be a 112 kDa homodimer. The enzyme showed very low K(m) (0.34 mM) and high catalytic efficiency (3.3×10(6)) with 4-methyl catechol. The substrate specificity was in the order: 4-methyl catechol>tert-butylcatechol>dihydrocaffeic acid>pyrocatechol. Cysteine hydrochloride, potassium metabilsulphite, ascorbic acid, erythorbic acid, resorcylic acid and kojic acid showed competitive inhibition, whereas, citric acid and sodium azide showed mixed inhibition of PPO activity. Cysteine hydrochloride was found to be an excellent inhibitor with the low inhibitor constant of 1.8 μM., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A catechol oxidase from lemon balm (Melissa officinalis) moCO which only catalyzes the oxidation of catechols to quinones without hydroxylating tyrosine was purified. The molecular mass of the M. officinalis enzyme of 39,370 Da was obtained by MALDI mass spectrometry and the isoelectric point was determined to be 3.4. Addition of 2 eq. H(2)O(2) to the enzyme leads to oxy catechol oxidase. In the UV/Vis spectrum two new absorption bands occur at 343 nm (ε=8510 M(-1)cm(-1)) and 580 nm (ε=580 M(-1)cm(-1)) due to O(2)(2-)Cu (II) charge transfer transitions in accordance with the oxy forms of other type 3 copper proteins. The N-terminal sequence has been determined by Edman degradation to NPVQAPELDKCGTAT, exhibiting a proline at the second and sixth position conserved in other polyphenol oxidases., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Polyphenol oxidase (PPO) was examined from the extract of leaf, seed, and cell suspension of Hevea brasiliensis, a rubber plant. The defense-related isozyme from Hevea cell suspension induced by culture filtrate of Phytophthora palmivora or by agitation stress was isolated through anion exchange and affinity chromatography, respectively. A 104-purification fold, migrated as a single band of 70 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PPO, was obtained after further purified by the preparative gel electrophoresis. Based on reaction with catechol and dopamine but not with p-cresol and guaiacol, it is a diphenol-type PPO. The values of V(max)/K(m) ratio indicated that catechol was the most specific substrate. The optimal activity of the purified PPO was observed at pH 6.0. The PPO activity was retained at pH 4.0-10.0 and temperature 10-60 °C. The inhibitors which completely inhibited the activity were ascorbic acid, dithiothreitol, and β-mercaptoethanol while sodium azide was a poor inhibitor. The PPO obtained from Hevea cell suspension possesses high specific activity and is stable at wide range of pH and temperature. It is therefore suitable for extreme condition uses and may lead to an alternative source of PPO in various industrial applications.

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).

A new series of N,N'-diarylureas (1-9) was synthesized. These compounds were investigated as inhibitors of polyphenol oxidase (PPO) which had been purified from banana by an affinity gel comprised of Sepharose 4B-l-tyrosine-p-amino benzoic acid. K(i) values for (1), (2), (3), (5), (6), (7) and (8) were determined as 0.285, 17.97, 0.187, 0.108, 0.063, 0.044 and 0.047 mM, respectively. Thus (2) was by far the most effective inhibitor. Interestingly, (4) and (9) behaved as an activator of PPO in this study.