Purification and characterization of a polyphenol oxidase from the seeds of field bean (Dolichos lablab)

The polyphenol oxidase from field bean (Dolichos lablab) seeds has been purified to apparent homogeneity by a combination of ammonium sulfate precipitation, DEAE-Sephacel chromatography, phenyl agarose chromatography, and Sephadex G-200 gel filtration. The purified enzyme has a molecular weight of 120 +/- 3 kDa and is a tetramer of 30 +/- 1.5 kDa. Native polyacrylamide gel electrophoresis of the purified enzyme revealed the presence of a single isoform with an observed pH optimum of 4.0. 4-Methyl catechol is the best substrate, followed by catechol, and L-3,4-dihydroxyphenylalanine, all of which exhibited a phenomenon of inhibition by excess substrate. No activity was detected toward chlorogenic acid, catechin, caffeic acid, gallic acid, and monophenols. Tropolone, both a substrate analogue and metal chelator, proved to be the most effective competitive inhibitor with an apparent K(i) of 5.8 x 10(-)(7) M. Ascorbic acid, metabisulfite, and cysteine were also competitive inhibitors.     

A review on spectrophotometric methods for measuring the monophenolase and diphenolase activities of tyrosinase

Tyrosinase is a copper enzyme with broad substrate specifity toward a lot of phenols with different biotechnological applications. The availability of quick and reliable measurement methods of the enzymatic activity of tyrosinase is of outstanding interest. A series of spectrophotometric methods for determining the monophenolase and diphenolase activities of tyrosinase are discussed. The product of both reactions is the o-quinone of the corresponding monophenol/diphenol. According to the stability and properties of the o-quinone, the substrate is classified as four substrate types. For each of these substrate types, we indicate the best method for measuring diphenolase activity (among eight methods) and, when applicable, for measuring monophenolase activity (among four methods). The analytical and numerical solutions to the system of differential equations corresponding to the reaction mechanism of each case confirm the underlying validity of the different spectrophotometric methods proposed for the kinetic characterization of tyrosinase in its action on different substrates.     

Characterization of monophenolase activity of polyphenol oxidase from iceberg lettuce

Polyphenol oxidase (EC 1.14.18.1), a thylakoid membrane-bound enzyme, was isolated by sonication of osmotically shocked chloroplasts from iceberg lettuce (Lactuca sativa). The enzyme showed monophenolase activity when assayed on (p-hydroxyphenyl)propionic acid with 3-methyl-2-benzothiazolinone hydrazone in a reliable continuous spectrophotometric method, with high sensitivity, accuracy, and precision. The monophenolase activity showed a lag period before the steady-state rate (V(ss)) was reached. Both kinetic parameters, the lag period and the steady-state rate, depended on the pH, the enzyme and substrate concentrations, and the presence of catalytic amounts of o-diphenol. This activity shows inhibition by high substrate concentration. The experimental results correspond with the mechanism previously described for PPO from other sources. Kinetic constants K(m), V(max), and K(i) were determined.     

Characterization of catecholase and cresolase activities of eggplant polyphenol oxidase

In the present paper the catecholase and cresolase activities of eggplant polyphenol oxidase (PPO) are described. To preserve the latter activity, a partially purified enzyme was used. Peroxidase was removed from the preparation to avoid its interference with PPO during phenol oxidation. The partially purified eggplant PPO was fully active. The catecholase/cresolase ratio of 41.1 indicated that, in a pH close to the physiological, diphenol oxidation predominates over monophenol oxidation. The characteristic lag phase of the cresolase activity is modulated by the pH, the monophenol and diphenol concentrations, and the enzyme's concentration. The effect of several inhibitors was also tested, and the K(i) values of the two most effective (tropolone and 4-hexylresorcinol) were determined.     

Partial characterization of peroxidase and polyphenol oxidase activities in blackberry fruits

A partial characterization of peroxidase (POD) and polyphenol oxidase (PPO) activities in blackberry fruits is described. Two cultivars of blackberry (Wild and Thornless) were analyzed for POD and PPO activities. Stable and highly active POD and PPO extracts were obtained using insoluble poly(vinylpyrrolidone) and Triton X-100 in 0.05 M sodium phosphate, pH 7.5, buffer. Blackberry POD and PPO activities have a pH optimum of 6.5, in a reaction mixture of 0.2 M sodium phosphate. Optimal POD activity was found with 3% o-dianisidine. Maximum PPO activity was found with catechol (catecholase activity) followed by 4-methylcatechol. Polyacrylamide gel electrophoresis of blackberry extracts under non-denaturing conditions resolved in various bands. In the POD extracts of Wild fruits, there was only one band with a mobility of 0.12. In the Thornless POD extracts there were three well-resolved bands, with R(f) values of 0.63, 0.36, and 0.09. Both the Wild and Thornless blackberry cultivars produced a single band of PPO, with R(f) values of 0.1 for Wild and 0.06 for Thornless.     

Three polyphenol oxidases from red clover (Trifolium pratense) differ in enzymatic activities and activation properties

Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling process. The cDNAs encoding three red clover PPOs were expressed individually in alfalfa (Medicago sativa L.), which lacks detectable endogenous foliar PPO activity and o-diphenols. Several physical and biochemical characteristics of the red clover PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red clover PPOs exist in a latent state and are activated (10-40-fold increase in activity) by long incubations (>2 days) at ambient temperature or short incubations (<10 min) at > or =65 degrees C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. During incubation at ambient temperature, the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively, and latent PPO1 is slightly activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to approximately 7, and the activated PPOs retain substantial levels of activity as the pH increases above their optima. The latent and activated PPOs were surveyed for ability to oxidize various o-diphenols, and activation of the PPOs had little effect on substrate specificity. Activation increases the V max but not the affinity of the PPO enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and kinetic changes during activation and provide new insights to their effects in postharvest physiology.     

不同还田方式下拉巴豆秸秆腐解及养分释放特征

为明确拉巴豆秸秆的腐解和养分释放规律,采用网袋法模拟研究拉巴豆在覆盖还田、土埋还田和水淹还田方式下的腐解动态。结果表明:不同还田方式下,拉巴豆茎秆在0~20 d腐解速率较快,之后腐解缓慢;在100d时,覆盖还田、土埋还田和水淹还田方式下累计腐解率分别达42.4%、74.3%、66.9%。经过100 d的腐解,覆盖还田方式下碳、氮、磷、钾的累计腐解率分别为28.8%、14.3%、47.7%、86.2%;土埋还田方式下碳、氮、磷、钾的累计腐解率分别为68.0%、62.3%、85.2%、94.8%;水淹还田方式下碳、氮、磷、钾的累计腐解率分别为63.0%、55.0%、82.0%、91.1%,拉巴豆茎秆养分的释放速率表现为钾磷碳氮。3种还田方式下茎秆累计腐解率及碳、氮、磷、钾等养分的累计释放率均表现为土埋还田水淹还田覆盖还田。     

多酚氧化酶高强度脉冲磁场灭活及动力学模型

为了找到一种有效控制果蔬中多酚氧化酶（PPO）活性的方法,该文研究了高强度脉冲磁场（PMF）对PPO活性的影响,并进行了灭酶动力学模型的研究。结果表明,当PPO于磁场强度2.5、3.5和4.5特斯拉（T）分别处理5至40个脉冲时,酶的残余活性随着磁场强度和脉冲数的增加而逐渐降低。在4.5 T处理40个脉冲时,酶的灭活率最高达到93.10%。对灭活动力学曲线分别用Bigelow模型、Weibull模型和Hülsheger模型进行拟合,发现Weibull模型对PMF下PPO的灭活的拟合度最好。可见,高强度脉冲磁场可以作为一种有效杀灭果蔬中多酚氧化酶的非热技术,且酶的灭活过程符合Weibull模型,该模型可以为实际应用提供参考。     

Specific polyphenol oxidase activity of red clover (Trifolium pratense) and its relation with forage quality in field experiments

Abstract

In feeding studies, red clover (RC) influenced positively the N utilization by ruminants. A relationship between polyphenol oxidase (PPO) activity and forage quality has not been established. Our objective was to investigate seasonal, site, genotype, and management effects on specific PPO activity in RC in three experiments under field conditions, and relate the activity to forage-quality parameters. In Experiment 1, six RC genotypes at two study sites were submitted to a 3-cut system. Specific PPO activity, forage quality, and vegetation stage were determined. PPO activity varied between harvest and study sites, with genotypes differing up to 3-fold in PPO activity within harvests. The specific PPO activity, forage quality, and vegetation stage in RC subjected to 5-cut system and grazing (Experiment 2) were determined. Additionally, in Experiment 3, cutting frequency in RC swards including mechanical stress (rolling) was investigated. The induction of PPO activity in RC by grazing or mechanical stress (Experiments 2 and 3) increased the activity up to 2.5-fold compared with RC at similar vegetation stage submitted to the 5-cut system. Mechanical stress induced by grazing or rolling, and seasonal differences, seem to have a larger influence on specific PPO activity than does the genotype effect observed in Experiment 1. For forage quality, an increased specific PPO activity explained 29–46% of the reduction in protein fraction ‘A’ content (non-protein N) in the cutting systems in Experiments 2 and 3. Other CP fractions achieved a lower relation. Furthermore, the precipitation-to-temperature ratio preceeding a harvest explained 63% of the variation in the specific PPO activity. In conclusion, the PPO activity in RC is induced by grazing and rolling. Whereas weather conditions preceeding a harvest showed a large influence, genotype influence had only minor relevance. These results may have implications for regional harvest management towards efficient N utilization by ruminants.     

Differential activation of a latent polyphenol oxidase mediated by sodium dodecyl sulfate

A kinetic study of the activity of soluble and membrane-bound latent polyphenol oxidase (PPO) extracted from beet root (Beta vulgaris) was carried out. For the first time, two types of behavior (hyperbolic and sigmoid) are reported in the same enzyme for PPO activation by the surfactant sodium dodecyl sulfate (SDS), depending on substrate nature. A kinetic model based on cooperative systems is developed to describe the activation effect of SDS, enabling the determination of the number of surfactant molecules binding to the enzyme in the activation process. The results indicate that the active site of the enzyme is not affected by SDS and that a stepwise conformational change favors the access of hydrophobic substrates compared to hydrophilic ones. Differential activation of PPO mediated by SDS may be of relevance in the control of PPO activity since the enzyme is able to express activity toward a specific substrate while remaining latent to others.     

拉巴豆在不同土壤上的结瘤效应、饲用品质

选择重庆市4种具有代表性的土壤（灰棕紫泥两种、红棕紫泥和灰岩黄壤各一种）,接种促生根瘤菌,研究了拉巴豆的结瘤性能、生长情况、光合速率、营养品质及矿质养分等。结果表明,在微酸性和酸性灰棕紫泥土壤上,拉巴豆的生长、生理、光合、产量和品质显著优于灰岩黄壤和红棕紫泥。在微酸性灰棕紫泥（pH 6.14）和灰岩黄壤（pH 6.45）上,接种根瘤菌能形成根瘤,但在酸性灰棕紫泥（pH 4.09）和碱性红棕紫泥（pH 7.50）上,形成根瘤极少或不形成根瘤,说明土壤酸碱度过高和过低均抑制根瘤形成。接种根瘤菌形成根瘤之后,不同程度地改善了拉巴豆氮、磷营养,提高了其净光合速率,促进了生长,增加了生物量,改善了品质。拉巴豆生物量,氮、磷吸收量,收获后土壤有效氮、磷含量与根瘤重量呈显著正相关,故根瘤重量可反映根瘤活性。此外,在微酸性灰棕紫泥土壤中,拉巴豆的根瘤数量虽然比灰岩黄壤少53.32%,但根瘤体积较大,根瘤重量为灰岩黄壤上的1.66倍,接种效应总体上优于灰岩黄壤。所以,在重庆市的拉巴豆栽培中,选择微酸性和酸性灰棕紫泥,并接种根瘤菌有益于高产优质。     

柿叶提取物对马铃薯多酚氧化酶的抑制作用

多酚氧化酶（PPO）是生物体内黑色素合成的关键酶。研究了甜柿叶乙醇提取物、涩柿叶乙醇提取物、甜柿叶水提取物、涩柿叶水提取物对马铃薯多酚氧化酶活性的抑制作用。结果表明,上述4种柿叶提取物对马铃薯多酚氧化酶具有明显的抑制作用,使该酶活力下降50％所需的浓度（IG50）分别为0．21、0．26、0．37、0．45mg／mL。乙醇提取物较水提取物的作用效果更强,甜柿叶提取物的抑制作用比涩柿叶提取物的强,4种柿叶提取物对酶的抑制作用均为非竞争性可逆抑制,其抑制常数（KI=KIS）分别为0．18、0．23、0．34、0．45mg／mL。     

Inhibition of apple polyphenol oxidase activity by procyanidins and polyphenol oxidation products

The rate of consumption of dissolved oxygen by apple polyphenol oxidase in cider apple juices did not correlate with polyphenol oxidase activity in the fruits and decreased faster than could be explained by the decrease of its polyphenolic substrates. The kinetics parameters of a crude polyphenol oxidase extract, prepared from apple (Braeburn cultivar), were determined using caffeoylquinic acid as a substrate. Three apple procyanidin fractions of n 80, 10.5, and 4 were purified from the parenchyma of cider apples of various cultivars. Procyanidins, caffeoylquinic acid, (-)-epicatechin, and a mixture of caffeoylquinic acid and (-)-epicatechin were oxidized by reaction with caffeoylquinic acid o-quinone in order to form oxidation products. All the fractions were evaluated for their inhibitory effect on PPO activity. Native procyanidins inhibited polyphenol oxidase activity, the inhibition intensity increasing with n. The polyphenol oxidase activity decreased by 50% for 0.026 g/L of the fraction of n 80, 0.17 g/L of the fraction of n 10.5, and 1 g/L of the fraction of n 4. The inhibitory effect of oxidized procyanidins was twice that of native procyanidins. Oxidation products of caffeoylquinic acid and (-)-epicatechin also inhibited polyphenol oxidase.     

Characterization of polyphenol oxidase from litchi pericarp using (-)-epicatechin as substrate

Polyphenol oxidase (PPO) from litchi (Litchi chinensis Sonn.) pericarp was characterized using (-)-epicatechin, which was the major endogenous polyphenol in litchi pericarp as a substrate. The optimum pH for PPO activity with (-)-epicatechin was 7.5, and the enzyme was unstable below pH 4.5 and stable in the pH range of 6.0-8.0. Residual activities of PPO were 86.25, 86.31, and 80.17% after 67 days of incubation at 4 degrees C at pH 6.0, 7.5, and 8.0, respectively. From thermostability studies, the Ki value increased with temperature and the results suggested that the enzyme was unstable above 45 degrees C. Moreover, the results also provided strong evidence that the denaturalization temperature of PPO was near 70 degrees C. The inhibition studies indicated that l-cysteine and glutathione were strong inhibitors even at low concentrations while NaF inhibited moderately. In addition, the results also indicated that the inhibition mechanisms of thiol groups were different from those of halide salts.     

Oxidation of the flavonol quercetin by polyphenol oxidase

Because direct oxidation of flavonols by polyphenol oxidase (PPO) has not previously been reported and, given the importance of flavonols, the ability of broad bean seed PPO to oxidize the flavonol quercetin was studied. The reaction was followed by recording spectral changes with time. Maximal spectral changes were observed at 291 nm (increase) and at 372 nm (decrease). The presence of two isosbectic points (at 272 and 342 nm) suggested the formation of only one absorbent product. These spectral changes were not observed in the absence of PPO. The oxidation rate, which varied with pH, was highest at pH 5.0. The following kinetic parameters were also determined: V(m) = 11 microM/min, K(m) = 646 microM, V(m)/K(m) = 17 x 10(-)(2) min(-)(1). Flavonol oxidation was efficiently inhibited (K(I) = 3.5 microM) by specific PPO inhibitors such as 4-hexylresorcinol. The results obtained showed that quercetin oxidation was strictly dependent on the presence of PPO.     

Degradation of cyanidin 3-rutinoside in the presence of (-)-epicatechin and litchi pericarp polyphenol oxidase

The degradation mechanism of cyanidin 3-rutinoside in the presence of (-)-epicatechin and litchi pericarp polyphenol oxidase (PPO) was investigated using several model systems. The enzymically generated (-)-epicatechin o-quinone could induce cyanidin 3-rutinoside degradation. The results obtained in this study allowed us to propose a pathway for cyanidin 3-rutinoside degradation in the presence of (-)-epicatechin and litchi pericarp PPO. First, enzymatic oxidation of (-)-epicatechin produced the corresponding o-quinone, and then cyanidin 3-rutinoside and (-)-epicatechin competed for (-)-epicatechin o-quinone, resulting in degradation of cyanidin 3-rutinoside and regeneration of (-)-epicatechin. Moreover, the results of kinetic studies indicated this competition was influenced by both (-)-epicatechin concentration and cyanidin 3-rutinoside concentration in the model system.     

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).     

Study and characterization of polyphenol oxidase from eggplant (Solanum melongena L.)

In this study the catecholase and cresolase activities of eggplant polyphenol oxidase (PPO) were investigated. Enzyme activity was determined by measuring the increase in absorbance using catechol as substrate and 3-methyl-2-benzothiazolinone hydrazone (MBTH) as coupled reagent. The effects of substrate specificity, heat inactivation, temperature, pH, and inhibitors were investigated to understand the enzymatic alteration of ready-to-eat preparations. Browning of vegetables was determined through a colorimeter. Decrease of lightness (L*) and increase of color difference values (ΔE*) were correlated with tissue browning. Antibrowning agents were tested on PPO under the same conditions. The enzyme activity was strongly inhibited by 0.4 M citric acid. Under natural pH conditions, the enzyme was also inhibited by tartaric acid and acetic acid. All of the results were used to understand the best conditions for food transformation (ready-to-eat and grilled eggplant slices).     

Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.)

In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH(4))(2)SO(4) precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V(max)/K(m) values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic acid, gallic acid, d,L-dithiothreitol, tropolone, glutathione, sodium azide, benzoic acid, salicylic acid, and 4-aminobenzoic acid using 4-methylcatechol, pyrogallol, and catechol as substrate. The presence of EDTA, 4-aminobenzoic acid, salicylic acid, gallic acid, and benzoic acid did not cause the inhibition of artichoke PPO. A competitive-type inhibition was obtained with sodium azide, L-cysteine, and d,L-dithiothreitol inhibitors using 4-methylcatechol as substrate; with L-cysteine, tropolone, d,L-dithiothreitol, ascorbic acid, and sodium azide inhibitors using pyrogallol as substrate; and with L-cysteine, tropolone, d,L-dithiotreitol, and ascorbic acid inhibitors using catechol as a substrate. A mixed-type inhibition was obtained with glutathione inhibitor using 4-methylcatechol as a substrate. A noncompetitive inhibition was obtained with tropolone and ascorbic acid inhibitors using 4-methylcatechol as substrate, with glutathione inhibitor using pyrogallol as substrate, and with glutathione and sodium azide inhibitors using catechol as substrate. From these results, it can be said that the most effective inhibitor for artichoke PPO is tropolone. Furthermore, it was found that the type of inhibition depended on the origin of the PPO studied and also on the substrate used.