Characterization Of Polyphenol Oxidase Research Articles

Partial purification and characterisation of polyphenol oxidase from celery root (Apium graveolens L.) and the investigation of the effects on the enzyme activity of some inhibitors

SummaryPolyphenol oxidase (PPO) of celery root was extracted and partially purified by (NH4)2SO4 fractionation and dialysis. Optimum pH and temperature were found at pH 7.0 and 30 °C, and Km and Vmax values were 29 mm and 5560 U mL−1 min−1 with catechol, respectively. The activation energy of the enzyme with catechol was 17.9 kJ mol−1 at pH 7.0. In electrophoretic seperation, six isoenzymes were detected with dl‐dopa substrate. PPO showed activity to catechol, 4‐methylcatechol, pyrogallol, gallic acid, dl‐dopa. l‐Tyrosine was also tested but was not oxidised by celery root PPO. β‐Mercaptoethanol was found to be the most effective inhibitor. (NH4)2SO4, NaCl, KCl and sucrose appeared to be protective agents of celery root PPO against thermal denaturation. Metal ions (Cu2+, Zn2+, Mn2+) were poor inhibitors of the celery root PPO at 1 mm. PPO activity was also inhibited by CaCl2, NaCl, BaCl2, FeSO4 and NiCl2.

Purification and Characterization of Polyphenol Oxidase from Edible Yam (Dioscorea opposita Thunb.)

Soluble polyphenol oxidase of edible yam (Dioscorea opposita Thunb.) was purified approximately 203-fold with a recovery rate of 15% by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration using dopamine as a substrate. The purified enzyme appeared as a single band on native PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be approximately 42kDa and 44kDa using gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized dopamine. The apparent Km value (Michaelis constant) of the enzyme was 1.5mM for dopamine (pH 7.0, 30°C). The optimum pH was 7.0 for dopamine oxidase. In the pH range from 6 to 10, the activity was quite stable at 5°C for 22h. The optimum temperature of enzyme activity was 25-30°C. The activity was stable up to 50°C after heat treatment for 20min. The browning reaction by the enzyme was completely inhibited by 1mM L-ascorbic acid, which reduced o-quinone to dopamine. The reaction was also completely inhibited by 1mM L-cysteine, which is a known quinone coupler. About 35% inhibition of edible yam PPO was observed using citric acid and acetic acid at 10mM in 0.1M citrate/ 0.2M sodium phosphate buffer (pH 7). In consideration of the observed results, L-ascorbic acid, L-cysteine, acetic acid, and citric acid are expected to be used as effective inhibitors of enzymatic browning in edible yam.

Characterization of polyphenol oxidase from Napoleon grape

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

Characterization of polyphenol oxidase from Uapaca kirkiana fruit

AbstractPolyphenol oxidase (PPO), the enzyme responsible for the postharvest spoilage of fruits, was extracted and purified from Uapaca kirkiana peel and pulp by ammonium sulfate precipitation and dialysis. Further purification of peel PPO was carried out by gel filtration chromatography. Optimum pH values were 7 and 8 for peel and pulp PPO, respectively. The optimum temperatures for peel and pulp PPO were 45 and 35 °C, respectively. Inhibition studies of the PPO enzyme were performed using citric acid, sodium azide, sodium metabisulfite and thiourea. The most effective inhibitors were sodium azide and citric acid for both peel and pulp PPO. Vmax and Km values were 13.63 units min−1 and 4.923 mmol L−1, respectively, for peel PPO and 14.03 units min−1 and 5.43 mmol L−1, respectively, for pulp PPO. Three isoenzymes of Uapaca kirkiana PPO were detected by polyacrylamide gel electrophoresis. One of the isoenzymes could be identified as having a molecular weight of 26 625 Da. Copyright © 2005 Society of Chemical Industry

Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity

Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH4)2SO4 precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30°C respectively and Km and Vmax values were 7.90 × 10−4 M, and 11290 EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, β-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a Ki value of 1.79 × 10−9 M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.

Purification and characterization of polyphenol oxidase from Ferula sp.

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

Partial purification and characterisation of polyphenol oxidase and peroxidase from marula fruit ( Sclerocarya birrea subsp. Caffra)

Marula fruit, native to sub-Saharan Africa, is of growing commercially importance. Polyphenol oxidase (PPO) and peroxidase from the fruit were partially purified by a combination of temperature induced phase separation in Triton X-114, DEAE-ion exchange and Sephadex G100 gel filtration. PPO activity was purified 58-fold with 75% recovery while the purification factor for peroxidase was 19% with 25% recovery. The enzymes were characterised for enzyme concentration–reaction rate relationship, thermal stability, pH activity and stability, molecular weight, isoelectric point (pI) and kinetic parameters. PPO and peroxidase shared the same molecular weight (71 kDa) and pI (5.43). Thermal deactivation curves were bi-phasic for both activities. Peroxidase displayed maximal activity at pH 4.0 with ABTS (2,2′-azino-(bis-3-ethylbenzthiazoline-6-sulfonic acid)) and a K M of 1.77 mM for hydrogen peroxide. The pH optimum for PPO was 7.0 with catechol. Marula PPO had K M values of 1.41, 1.43, 3.73 and 4.99 mM for catechin, 4-methylcatechol, 3,4-dihydroxyphenylpropanoic acid (DHPPA) and catechol, respectively.

Characterization of polyphenol oxidase from plants

Polyphenol oxidase (PPO) which can mediate browning reaction is a bifunctional copper-containing enzyme encoded by plant nucleolus gene. It usually leads to excessive browning reaction which reduces the commercial profits of fruits and vegetables. In this paper, PPO genes and enzymes in plants are characterized systematically, and the latest progress is reviewed. Some clonings of PPOs genes are reported; the specific temporal and spatial expression pattern of PPOs genes is described; the model of the structure of the precursor form of catechol oxidase is introduced; the possible functions of PPOs in defending against pathogen, wounding, surrounding stress and other inducing factors are demonstrated; the induction and activation of latent PPOs in some plants is elucidated; the scheme of browning inhibition by L-cysteine is clarified; the mechanism of suicide inhibition of latent PPO and kinetic synergism are established. Furthermore, the area for future study is also discussed.

Purification and characterization of polyphenol oxidase from Henry chestnuts (Castanea henryi)

Polyphenol oxidase from Castanea henryi nuts was partially purified. Some characteristics of the enzyme were then determined to help devise measures for the prevention of undesirable enzymatic browning during storage and processing of the nuts. Preparation of acetone powder extracts of the nuts, ammonium sulfate precipitation, dialysis, and gel filtration resulted in 37-fold purification of the enzyme with a yield of 13%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the excitation spectrum confirmed the enzyme preparation to be homogeneous. The approximate molecular weight of the enzyme was determined by gel filtration to be 69 kD. The enzyme catalyzed the oxidation of catechol and pyrogallic acid as substrates but did not affect cresol or tyrosine. Using catechol as substrate, p-nitrophenol, thiourea, orcinol, and naphthol showed strong inhibition. The optimal pH and temperature for the enzyme were pH 5.0 and 40°C, respectively. The enzyme proved heat labile. When the enzyme was incubated at 70°C for 30 min, the remaining activity of the enzyme was only 8%. Possible approaches to applying the results of this study to the prevention of enzymatic browning in the production of C. henryi nuts is also discussed.

Characterization of Polyphenol Oxidase from the Latex of Opium Poppy

Polyphenol oxidase from the latex of opium poppy was purified to the electrophoretic homogeneity by affinity chromatography using p-aminobenzoic acid as a ligand coupled to Sepharose CL-4B by divinyl sulphone activation method. The purified enzyme was used to prepare the polyclonal antibodies. The purified latex PPO exhibited high diphenolase activity in comparison with almost unmeassurable monophenolase activity. Both of these activities were sensitive to the activation with sodium dodecyl sulphate. Two isoforms (65 and 40 kDa) of latex PPO were separated by the gel filtration. There were no differences in substrate specifity (weak monophenolase and high diphenolase activity) and sensitivity to inhibitors between these isoforms, but they showed differences in electrophoretic mobility.

Purification and Characterization of Polyphenol Oxidase from Callus Cultures of Portulaca grandiflora.

Purification and Characterization of Polyphenol Oxidase from Callus Cultures of Portulaca grandiflora.

Partial purification and characterization of polyphenoloxidase from peppermint (Mentha piperita)

Abstract Polyphenoloxidase (PPO) of peppermint leaves ( Mentha piperita) was isolated by (NH4)2SO4 precipitation and dialysis. Its pH and temperature optima were 7.0 and 30°C, respectively. On heat-inactivation, half of the activity was lost after 6.5 and 1.5 min of treatment at 70 and 80°C, respectively. Sucrose, (NH4)2SO4, NaCl and KCl appeared to be protective agents of peppermint PPO against thermal denaturation. Km of this enzyme ranged from 6.25×10−3 M with catechol to 9.00×10−3 M with L-dopa. The I50 values of inhibitors studied on PPO were determined by means of activity percentage (I) diagrams. Values were 1.4×10−4 M, 1.7×10−4 M, 9.7×10−5 M, 2.45×10−4 M, 2.16×10−1 M, 1.83×10−5 M, 6.5×10−5 M, 1.4×10−2 M, 7.5×10−5 M, for potassium cyanide, glutathione, ascorbic acid, thiourea, sodium azide, sodium metabisulfite, dithioerythritol, β-mercaptoethanol and sodium diethyl dithiocarbamate respectively. Therefore, sodium metabisulfite was the most effective inhibitor.

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel.

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Characterization of polyphenoloxidase (PPO) extracted from ‘Jonagored’ apple

Polyphenoloxidase (PPO) was extracted from apple (cv. Jonagored) with addition of 2% PVP and 0.25% Triton X100 to the extraction buffer containing phenolic adsorbents. Experiments were performed to evaluate the affinity and specificity towards several substrates. ‘Jonagored’ apple PPO was found to have higher specificity (lower K m) towards L-dopa, 4-methylcatechol and (+) catechin than other phenols tested, but the highest activity level was obtained with p-cresol. The ratio V max/ K m indicates that p-cresol followed by L-dopa and 4-methylcatechol are the best substrates for ‘Jonagored’ apple PPO. The enzyme activity showed two pH optima, at 5.0 and 7.5, at room temperature, with the main peak at pH 7.5 and the secondary one at pH 5.0 when catechol was the substrate.

Characterization of polyphenol oxidase in coffee

Polyphenol oxidase (PPO) was characterized in partially purified extracts of leaves (PPO-L) and fruit endosperm (PPO-E) of coffee ( Coffea arabica L.). PPO activity was higher in early developmental stages of both leaves and endosperm of fruits. Wounding or exposure of coffee leaves to methyl jasmonate increased PPO activity 1.5–4-fold. PPO was not latent and was not activated by protease treatment. PPO activity was stimulated 10–15% with sodium dodecyl sulphate (SDS) at 0.35–1.75 mM, but at higher concentrations activities were similar to the control samples, without detergent. Prolonged incubation of extracts with trypsin or proteinase K inhibited PPO activity but pepsin had no effect. Inhibition of PPO with proteinase K was increased in the presence of SDS. PPO activity from both tissues was optimal at pH 6–7 and at an assay temperature of 30°C. Activity was highest with chlorogenic acid as substrate with a K m of 0.882 mM (PPO-L) and 2.27 mM (PPO-E). Hexadecyl trimethyl-ammonium bromide, polyvinylpyrrolidone 40, cinnamic acid and salicylhydroxamic acid inhibited PPO from both tissues. Both enzymes were inactivated by heat but the activity in endosperm extracts was more heat labile than that from leaves. The apparent M r determined by gel filtration was 46 (PPO-L) and 50 kDa (PPO-E). Activity-stained SDS–polyacrylamide gel electrophoresis (PAGE) gels and western blots probed with PPO antibodies suggested the existence of a 67 kDa PPO which is susceptible to proteolytic cleavage that generates a 45 kDa active form.

Purification and Characterization of Polyphenoloxidase from Guava Infected with Fruit-rot Disease

Purification and Characterization of Polyphenoloxidase from Guava Infected with Fruit-rot Disease

Characterization of polyphenol oxidase from aerial roots of an orchid, Aranda `Christine 130'

Four isoforms of polyphenol oxidase (PPO) were demonstrated in the aerial roots of a tropical orchid, Aranda `Christine 130'. They were extracted at neutral pH and purified to homogeneity as judged by SDS-gel electrophoresis. Purification was achieved by a combination of Triton X-114 treatment, temperature phase partitioning, gel filtration chromatography, ion-exchange separation and chromatofocusing. Two of the isoforms, designated PPO a and PPO d, differed in their N-terminal sequence, tryptic peptide map and substrate affinity for (+)-catechin, but exhibited similarity in their molecular mass under denaturing conditions, pH optimum and kinetic behaviour toward 4-methyl catechol. The other two isoforms, PPO b and PPO c, were identical to PPO a and PPO d, respectively, in terms of their N-terminal sequence, substrate preference and pH maximum, but were different with regard to their molecular mass under denaturing conditions. These four isoforms differed in their isoelectric point.

Characterization of PPO (polyphenol oxidase) cDNA in sweet persimmon (Diospyros kaki)

Polyphenol oxidases (PPOs) are copper metalloproteins which play a critical role in producing various derivatives of polyphenolic compounds. These enzymes are encoded in the nuclear genome and subsequently transported into the plastid. A partial cDNA clone encoding polyphenol oxidase was isolated from immature sweet persimmon leaves. Using two degenerate primers, a single band of PCR product was produced by the touchdown PCR method. In sequence analysis, this clone (define SPO; 1381 bp) revealed high similarities to the previously reported plant PPO genes. Moreover, this SPO clone was comprised of copper A and copper B-binding sites and a histidine-rich region, indicating that this clone contained the key conserved elements of PPO. To characterize the spatial and temporal expression of PPO, total RNAs were extracted from leaf and fruit peel tissues at different developmental stages. In northern analysis, transcripts of PPO were detected in all the samples tested, but the strongest expression levels were detected in mature fruit peels and leaves. Expression of PPO was inhibited in the transitional stage from chloroplast to chromoplast. In addition, the expression of PPO was highly induced in wounded immature fruit peel tissues.

Purification and characterization of polyphenol oxidase from Trametes sp. MS39401

Two polyphenol oxidases (EC 1.14.18.1), P-1 and P-2, were purified as electrophoretically homogeneous proteins from the culture filtrate of Trametes sp. MS39401 by acetone precipitation and column chromatographies on DEAE-Sephadex A-50, Sephadex G-150 and hydroxylapatite. P-1 was purified 34-fold with a yield of 4.2%, while P-2 was purified 37-fold with a yield of 20.7%. The molecular masses of P-1 and P-2 were estimated to be 61 kDa and 90 kDa, respectively, by gel filtration. The isoelectric points of P-1 and P-2 were 3.4 and 2.7, respectively. The optimum pH range of both enzymes was 4.5–5.0 at 45°C. The optimum temperature of both enzymes was 55°C at pH 5.0. P-1 was stable at pH 5.0–7.5 and temperatures up to 60°C. P-2 was stable at pH 3.0–7.5 and temperatures up to 50°C. The thermostability of P-1 was comparable to that of the PM1 laccase of basidiomycetes, which was reported to be the most stable among basidiomycete laccases. Both enzymes were active toward various phenolic compounds and aminophenols. However, they lacked activity toward l-tyrosine. The K m values for (+)-catechin were 0.19 mM for P-1 and 0.67 mM for P-2. Both enzymes were appreciably inactivated by Hg 2+ and Sn 2+. Significant activation of neither enzyme was observed in the presence of metal ions and reagents. Both enzymes were significantly inhibited by copper-chelating agents, reducing agents and N-bromosuccinimide. Carbon monoxide caused appreciable inactivation of neither enzyme, so it is suggested that P-1 and P-2 belong to the group of laccases.

Purification and Characterization of Polyphenol Oxidase From Muscat Bailey A Grape Juice

Polyphenol oxidase of Muscat Bailey A (Muscat Hamburg x Bailey) (MBA) was purified in a homogeneous form on SDS-PAGE and some of its properties were determined. The enzyme was extracted from the precipitate of MBA juice and purified by Sephadex G-100, hydroxyapatite, DEAE-Sepharose, and Ultrahydrogel 250 (HPLC) column chromatographies. The specific activity toward catechol increased 84-fold and the recovery was about 1%. The molecular weight of the enzyme was 40 kDa (SDS-PAGE) or 42 kDa (gel filtration). It had an optimum pH of 6.3 and was stable between pH 6 and 8. The optimum temperature for activity was 25°C to 30°C and the enzyme remained stable up to 30°C. When tested for substrate specificity, the enzyme catalyzed the oxidation of <i>o</i>-dihydroxy phenolic compounds but showed no activity toward monohydroxy phenols. The Km value toward catechol was 18.4 m<i>M</i>. Catecholase activity (browning activity) was well inhibited by ascorbic acid, potassium pyrosulfite, and other inhibitors, but the enzyme remained active in the presence of 1% SDS.