Characterization Of Polyphenol Oxidase Research Articles

Partial purification and characterization of polyphenol oxidase from Chinese parsley (Coriandrum sativum).

Purification and characterization of polyphenol oxidase (PPO) from Chinese parsley (Coriandrum sativum) were achieved. Crude PPO exhibited an enzyme activity of 1,952.24 EU/mL. PPO was partially purified up to 6.52x with a 10.89% yield using gel filtration chromatography. Maximal PPO activity was found at 35°C, pH 8.0 for 4-methylcatechol and at 40°C, pH 7.0 for catechol. PPO showed a higher affinity towards 4-methylcatechol, but a higher thermal stability when reacting with catechol. LCysteine was a better inhibitor than citric acid for reducing PPO activity at concentrations of 1 and 3mM in the presence of either substrate. Two 46 kDa isoenzymes were identified using SDS-PAGE. Isolation and characterization of Chinese parsley serves as a guideline for prediction of enzyme behavior leading to effective prevention of enzymatic browning during processing and storage, including inhibition and inactivation of PPO.

Characterization of polyphenol oxidase in two cocoa (Theobroma cacao L.) cultivars produced in the south of Bahia, Brazil

The reactions leading to the formation of precursors of chocolate flavor are performed by endogenous enzymes present in the cocoa seed. Polyphenol oxidase (PPO) presence and activity during fermentation of cocoa beans is responsible for the development of flavor precursors and is also implicated in the reduction of bitterness and astringency. However, the reliability of cocoa enzyme activities is complicated due to variations in different genotypes, geographical origins and methods of fermentation. In addition, there is still a lack of systematic studies comparing different cocoa cultivars. So, the present study was designed to characterize the activity of PPO in the pulp and seeds of two cocoa cultivars, PH 16 and TSH 1188. The PPO activity was determined spectrophotometrically and characterized as the optimal substrate concentration, pH and temperature and the results were correlated with the conditions during the fermentation process. The results showed the specificity and differences between the two cocoa cultivars and between the pulp and seeds of each cultivar. It is suggested that specific criteria must be adopted for each cultivar, based on the optimal PPO parameters, to prolong the period of maximum PPO activity during fermentation, contributing to the improvement of the quality of cocoa beans.

Isolation, Partial Purification and Characterization of Polyphenol Oxidase from Two Species of African Mango Seeds (<i>Irvingia gabonensis and Irvingia wombolu</i>)

Polyphenol oxidases are oxido-reductases that catalyses oxidation of phenolic compounds in the presence of oxygen to form corresponding quinone intermediates which spontaneously polymerize to form undesirable pigments. Polyphenol oxidase was isolated and characterized in Irvingia species (gabonensis and wombolu) kernel, a tropical African fruit. Extracts were partially purified with ammonium sulphate. The effects of optimum pH and temperature were investigated while the pH and thermal stability were carried out. The Km, Vmax and the activity on both monophenol and diphenol substrates were determined. The optimum pH and optimum temperature of activity were found out to be 7.0 and 60°C in both species. The enzyme was stable at pH 7.0 and pH 8.0, and also at temperatures of 25, 30, 40, 50 and 60°C, it however loses stability beyond 60°C. Km and Vmax for Irvingia gabonesis were 2.51mM and 0.0411 Unit/min while Km and Vmax for Irvingia wombolu were 2.55mM and 0.0415 Unit/min respectively. Higher activity was observed with diphenol substrate L-DOPA than with monophenol - tyrosine. It can therefore be deduced from this study that the storage of the seeds of African mango at a room temperature above 60°C will prolong its shelf-life.

Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana L.) fruit

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±0.07mM and Vmax=53.15±2.03UPPOmL−1min−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.

Characterisation of polyphenol oxidase and peroxidase and the role in browning of loquat fruit

Zhang X., Shao X. (2015): Characterisation of polyphenol oxidase and peroxidase and the role in brown

Purification and Characterization of Polyphenol Oxidase from Hemşin Apple (Malus communis L.)

The polyphenol oxidase (PPO) enzyme was purified and characterized from Hemşin Apple (Malus communis L.), which was organically grown in Hemşin, in the Rize province of Turkey. Enzyme (PPO) activation was determined with catechol substrate. Apples were homogenized with homogenate buffer (pH 8.5). This process was followed by precipitation with (20–80%) saturated solid (NH4)2SO4 and dialysis. Finally, purification with DE52-Cellulose ion-exchange and Sephadex G-25 columns was performed. Experiments were performed at an optimum pH (5.5) and optimum temperature (30–40°C). The kinetic and thermal parameters Km (3.40 mM), Vmax (333.3 EU/mL.min), Ea (3.57 kcal), ∆H (2.968 kcal/mol), Q10 (1.33), kcat (24.57 min−1) and V0 (7.2x103 mM−1.min−1) were assessed. Additionally, the effects of Mg2+, Pb 2+, Fe2+, Fe3+, Cd2+, Cu2+, Zn2+, Co2+, Al3+, Mn2+ and Na+ on enzyme activity was recorded, and the IC50 values, Kİ values and inhibition types were determined.

Characterization of Polyphenol Oxidase from Jipicao Yam

Polyphenol oxidase (PPO) was extracted from Jipicao yam grown in China, and its characteristic was studied. Among the various substrates employed, the Vmax/Km ratio indicated that PPO had the best affinity to catechol and the worst affinity to p-hydroxyphenylalanine. The optimum pH for this enzyme was 6.8, and the optimum temperature was 30C. When the temperature was lower than 60C, PPO was relatively stable. Fe3+ and Mg2+ improved the activity, while Ca2+, K+, Ba2+ and Mn2+ ions could significantly inhibit the activity. NaHSO3, Vc, L-cysteine, phytic acid, oxalic acid, citric acid and ethylenediaminetetraacetic acid-2Na had different inhibiting effects on the activity of PPO, and the former three were strong inhibitors of PPO activities. Practical Applications Our paper studied the properties of polyphenol oxidase (PPO) from Jipicao yam, including optimum temperature, optimum pH, thermal stability, substrate specificity and the effect of metal ions and inhibitors so as to select a reasonable method to restrain the activity of PPO, then providing a theoretical basis for inhibiting enzymatic browning that occurred during the process of manufacturing and storage to improve the nutritional value and exterior quality.

Purification and Characterization of Polyphenol Oxidase From Waste Potato Peel by Aqueous Two-Phase Extraction

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.

Characterization of polyphenol oxidase during three ripening stages of an eggplant (Solanum melongena L.) fruit: a local type in northeast Anatolia

Objective: A relatively new local type of eggplant, Solanum melongena L. ‘Kadife’ is widely consumed in Turkey because of its economic availability and good nutritional qualities. However, the high polyphenol content of eggplant renders it susceptible to unattractive oxidative browning catalyzed by polyphenol oxides (PPOs). Therefore, the main aim of this study was to characterize PPO in this eggplant cultivar at three stages of its development. Methods: In this study, we determined substrate specificity, optimum pH and temperature, and optimum substrate concentration of the partial purified eggplant fruits PPO during ripening. Results: L-DOPA was proved to be the preferred PPO substrate in all three stages of ripening. Optimum activity was observed at pH 7.0 for PPO in extracts of ripening and overly-ripe eggplant, while activity in extracts of immature eggplant exhibited a broad pH optimum between, pH 5.0 and 7.0. In general, at all ripening stages, PPO was most active at 30°C and was most sensitive to inhibition by sodium metabisulphite and ascorbic acid. The metal ions (Hg2+, Mn2+, Fe3+ and Co2+) mostly inhibited PPO activities. Conclusion: These data regarding the properties of PPO should enhance understanding of the browning reaction in eggplant and lead to the development of techniques for controlling this undesirable process.

Studies on polyphenol oxidase in pomegranate (Punica granatum L.)

The present investigation was undertaken to study properties of polyphenol oxidase and monitor changes in polyphenal oxidase activity in Ganesh cultivar of pomegranate during storage. The characterization of polyphenol oxidase in pomegranate fruit extract with respect to effect of enzyme concentration and substrate concentration was studied. For studying changes in polyphenol oxidase activity during storage, the pomegranate fruits at half yellow stage were freshly harvested and stored at ambient temperature and analyzed after 0, 2, 4, 6, 8 and 10 days of storage. The polyphenol oxidase activity has increased up to 8 th day of storage and thereafter, decreased slightly on 10 th day.

Partial purification and characterization of polyphenol oxidase and peroxidase from chestnut kernel

Presently there is very little information about the properties of polyphenol oxidase (PPO) and peroxidase (POD) from chestnut, which are both related with chestnut browning. The chestnut PPO and POD were partially purified by acetone extraction, 30–80 g/100 mL ammonium sulfate fractionation, DE-52 anion-exchange column. PPO and POD activity increased 4.6-fold and 9.52-fold, its yields were 2.03 U/100 U and 4.21 U/100 U, and its specific activity was 1375 and 16,500 U mg−1 protein after DE-52. SDS-PAGE results indicated that the molecular mass of PPO and POD was approximately 32.5 and 30.3 kDa, respectively. Its optima pH value for catalyzing catechol and 2-hydroxy phenol was both 7.0. The optimum temperature of PPO and POD for catalyzing catechol and 2-hydroxy phenol was found to be 40 and 50 °C, respectively. PPO showed the highest affinity to catechol among all our selected substrates. The Km and Vmax of PPO for substrate catechol were 92 mmol/L and 1.53 △OD/min. The Km and Vmax of POD for substrate 2-hydroxy phenol were 49 mmol/L and 0.2373 △OD/min. The investigation on the properties of PPO and POD is greatly important for minimizing the losses caused by fruit browning during chestnut processing.

Purification and Characterization of Polyphenol Oxidase from the Bud of L onicera confusa

A polyphenol oxidase (PPO), located in the bud of Lonicera confusa, was extracted, purified and characterized. Ammonium sulfate precipitation and diethyl-aminoethanol-cellulose were used to purify the PPO 20.27-fold. The molecular mass of PPO was 28.8 kDa, as determined by size exclusion chromatography combined with multiangle laser light scattering and refractive index. The optimum pH value and temperature for PPO activity in the presence of l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate were 8.5 and 15C, respectively. The enzyme was stable after 60 min at 10 and 20C, and the PPO activity remained above 90%. The Km for l-DOPA was determined to be 2.26 mM. The PPO exhibited both diphenolase and triphenolase activities. Using Vmax/Km as a specificity constant, pyrocatechol was the better substrate for PPO. Moreover, ascorbic acid was a strong inhibitor that inhibited more than 78% of PPO activity at 1 mM. Practical Applications Lonicera confusa is often used in the fields of medicine and drink production. L. confusa quickly turns brown during drying and storage. Our results may facilitate the characterization of the mechanism involved in this browning process and thus provide a strategy for controlling the browning reaction as this reaction is a serious limitation in the food industry.

Extraction, partial purification and characterization of polyphenol oxidase from Solanum lycocarpum fruits

• PPO from S. lycocarpum was extracted, partially purified and characterized. • PPO presented catecholase activity with a high affinity toward 4-methylcatechol. • The effects of inhibitors on Solanum lycocarpum PPO activity were investigated. • PPO from S. lycocarpum was characterized as a temperature-resistant biocatalyst. In this work a polyphenol oxidase (PPO) from Solanum lycocarpum ripe and unripe fruits was studied. The unripe fruits presented higher activity than ripe for both fresh fruits and dried pulp flours. The purification procedure was based on freezing precipitation and a sixfold purification factor was obtained. The SDS-PAGE of the partially purified PPO showed two bands around 47 and 68 kDa. Optimal conditions for enzymatic studies were determined to be pH 6.0–6.5 and 28 °C. The partially purified PPO presented high activity toward catechol ( V max 3.42 U mL −1 and K m 6.47 mM) and 4-methylcatechol ( V max 3.01 U mL −1 and K m 0.15 mM) and low activity toward phenol being classified as a catecholase type polyphenol oxidase. S. lycocarpum PPO was sensitive to inhibitors such as l -cysteine, sodium metabisulfite, ascorbic acid, thiourea and citric acid. l -Cysteine was the most effective inhibitor, presenting a competitive inhibition. The results from kinetic and thermodynamic parameters for the thermal inactivation evidenced that the partially purified PPO is a biocatalyst, whose inactivation process is related to aggregation of partially unfolded enzyme molecules.

Partial purification and characterization of polyphenol oxidase from persimmon

Activity of polyphenol oxidase (PPO) from “Rojo Brillante” persimmon (Diospyros kaki L.) fruits was characterized. Crude extracts were used for characterization of enzyme activity and stability at different temperatures (60, 70 and 80°C), pHs (from 3.5 to 7.5) and substrate concentrations (catechol from 0 to 0.5M). Maximum enzyme activity was reached at pH 5.5 and 55°C. Enzyme stability was higher than PPO activities found in other natural sources, since above pH 5.5 the minimum time needed to achieve an enzyme inactivation of 90% was 70min at 80°C. However, at pH 4.0 the enzyme stability decreased, reaching inactivation levels above 90% after 10min even at 60°C. Thus it was concluded that acidification can circumvent browning problems caused by PPO activity. Moreover, polyacrylamide gel electrophoresis of the enriched extract revealed the presence of at least four bands with strong oxidase activity, suggesting the existence of different PPO isoforms.

Purification and Characterization of Polyphenol Oxidase from Jackfruit (Artocarpus heterophyllus) Bulbs

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.

Purification and Characterization of Polyphenol Oxidase From Agaricus bisporus

A polyphenol oxidase was isolated from Agaricus bisporus using ammonium sulfate precipitation, combined with DEAE–Sepharose Fast Flow chromatography and Phenyl Sepharose CL-4B chromatography. The molecular weight of the purified polyphenol oxidase was determined to be about 43 kDa with N-terminal sequence Ala-Thr-Asn-Ser-Gly-Thr-Leu-Ile-Ile-Phe by Edman degradation. The optimum temperature and pH for the polyphenol oxidase activity was 20°C and a pH of 6.5–7.0. Moreover, it was stable at 30 and 40°C over a 60 min pre-incubation time period. The Km and Vmax values of this polyphenol oxidase for catechol were 0.67 mM and 3333 U/ml min−1, respectively. In addition, following the cross linking with polyphenol oxidase, the emulsifying activity index, foam capacity, and foam stability of the whey proteins were evaluated to be modified. This work was useful to better understand polyphenol oxidase from A. bisporus, and may lead to its practical use in the future.

Isolation and characterization of polyphenol oxidase from Sardinian poisonous and non-poisonous chemotypes of Ferula communis (L.)

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.

Purification and characterization of polyphenol oxidase from fresh ginseng

Polyphenol oxidase (PPO) was purified from fresh ginseng roots using acetone precipitation, carboxymethyl (CM)-Sepharose chromatography, and phenyl-Sepharose chromatography. Two isoenzymes (PPO 1 and PPO 2) were separated using an ion-exchange column with CM-Sepharose. PPO 1 was purified up to 13.2-fold with a 22.6% yield. PPO 2 bound to CM-Sepharose, eluted with NaCl, and was purified up to 22.5-fold with a 17.4% yield. PPO 2 was further chromatographed on phenyl-Sepharose. The molecular weight of the purified PPO 2 from fresh ginseng was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was about 40 kDa. The optimum temperature and pH were 20℃ and 7.0, respectively, using catechol as a substrate. Pyrogallol showed the highest substrate specificity. The effect of a PPO inhibitor showed that its activity increased slightly in the presence of a low concentration of citric acid. High concentrations of acidic compounds and sulfite agents significantly inhibited purified ginseng PPO 2.

Characterization of Polyphenol Oxidase from Feijoa Fruit

Characterization of Polyphenol Oxidase from Feijoa Fruit

Purification and Characterization of Polyphenol Oxidase from Cauliflower (Brassica oleracea L.)

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).