Partial characterization of lettuce (Lactuca sativa L.) polyphenol oxidase

Polyphenol oxidase (PPO) from garden lettuce (Lactuca sativa L.) was partially purified by ammonium sulphate ((NH₄)₂SO₄) precipitation and dialysis, and then some of its kinetic properties such as optimum pH and temperature, substrate specificity, thermal inactivation and inhibition were investigated. The total phenolic and protein contents of Lactuca sativa L. extracts were determined according to the Folin-Ciocalteu and Bradford methods, and found to be 304 mg/100 g on a fresh weight basis and 494 μg/mL, respectively. PPO activity was determined using 4-methylcatechol, catechol and pyrogallol as substrates. Kinetic parameters, K _m and V _max, were calculated from Lineweaver–Burk plots. According to V _max/K _m ratio, pyrogallol was the most suitable substrate, followed by catechol and 4-methylcatechol. The optimum temperature and pH values were 30, 40 and 30 °C; and 6.5, 8.0 and 7.5 for 4-methylcatechol, catechol and pyrogallol substrates, respectively. The thermal inactivation of PPO was investigated at 35, 55 and 75 °C. The enzyme activity decreased with increasing temperature. The effect of different inhibitors on partially purified Lactuca sativa L. PPO was spectrophotometrically investigated. For this purpose, tropolone, glutathione, ascorbic acid and 4-aminobenzoic acid were used to inhibit the activity of Lactuca sativa L. PPO at different concentrations. From the experimental results, it was found that glutathione was found to be the most potent inhibitor for Lactuca sativa L. PPO.     

苔干多酚氧化酶的酶学特性研究

研究了苔干（Lactuca sativa var.angustata）多酚氧化酶的酶学性质。结果表明：以邻苯二酚为底物,该酶的最适pH为7.4,最适温度30℃,70℃以上温度使酶迅速失活,动力学方程v=518.96[S]/（0.0166＋[S]）,VC、异VC钠、NaHSO3、L-Cys可完全抑制酶活性,饱和NaCl、10%蔗糖、SDS、EDTA-Na2均可显著抑制酶活性。该酶能催化邻苯二酚、焦性没食子酸氧化,但对邻苯二酚的亲和力更强。     

Purification and some properties of polyphenoloxidase in eggplant (Solanum melongena)

Polyphenoloxidase (EC 1.10.3.1) in eggplant (Solatium melongena L) was purified by ammonium sulphate fractionation, DEAE-Cellulofine and DEAE-Toyopearl chromatography and Sephadex G-100 gel filtration. The enzyme was purified about 110-fold with a recovery of 5%. The purified enzyme more quickly oxidised chlorogenic acid (5-caffeoylquinic acid, IUPAC) than 10 other substrates used. The K_m value for the enzyme was found to be 0·50 mM with respect to chlorogenic acid; the optimum pH of the enzyme was about 4 with enzyme stability between pH 5 and 8. The enzyme was completely inactivated after heat treatment at 75°C for 30 min or 80°C for 5 min. Sodium metabisulphite, potassium cyanide and sodium fluoride markedly inhibited the enzyme activity.     

Characterisation and variation of antioxidant micronutrients in lettuce (Lactuca sativa folium)

The main phenolic compound in six studied lettuce cultivars (five green and one red) was identified as dicaffeoyl tartaric acid. Quantitative but not qualitative differences were found in the phenolic profiles among green cultivars. The red oak leaf cultivar contained twofold more dicaffeoyl tartaric acid and 10‐fold more chlorogenic acid than the green cultivars. Total phenolics ranged from 8.4 to 12.9 mg g^?1 dry weight in green varieties and reached 27.8 mg g^?1 dry weight in red oak leaf. Carotenoids and vitamins E and C were also quantified. Lutein was the main carotenoid found in all cultivars, together with another xanthophyll. The period of harvesting had only a marginal influence on total phenolic levels, whereas carotenoid and vitamin E levels were higher at the second period of harvesting. Vitamin C levels ranged between 6.1 and 9.9 mg per 100 g fresh weight. We investigated the total antioxidant power in lettuce. Total phenolics accounted for more than 60% of the total antioxidant capacity. Dicaffeoyl tartaric acid accounted for 55%. Copyright © 2004 Society of Chemical Industry     

Purification and properties of polyphenol oxidase from oil bean (Pentaclethra macrophylla Benth) seeds

Polyphenol oxidase extracted from oil bean (Pentaclethra macrophylla) seeds was purified 165-fold over the crude enzyme extract. The apparent molecular weight of the enzyme by gel filtration was 110.8 k ± 9.0 k while SDS-PAGE indicated a single species of molecular weight 28.0 k. A copper content of 1.9 mg g^?1 corresponds to one copper atom for each of the four subunits. The purified enzyme oxidised pyrogallol, catechol, 4-methylcatechol and L-dihydroxyphenylalanine but had low activity towards tyrosine. p-Cresol, caffeic acid and cholorogenic acid were not oxidised. Thio-compounds were strong inhibitors of the enzyme. The phenolic compounds tyrosine, resorcinol and orcinol inhibited catechol oxidation but became oxidised in the process.     

莴苣籽油理化性质及脂肪酸组成研究

采用气相色谱-质谱联用法(GC/MS)对莴苣籽油中脂肪酸组成及相对含量进行了分析研究,同时按照国家标准对莴苣籽油的酸值、皂化值、碘值、折光率以及相对密度进行了测定。结果表明:莴苣籽油由亚油酸、油酸、棕榈酸等10种脂肪酸组成,不饱和脂肪酸达75%以上,其中亚油酸53.18%,油酸17.22%,棕榈酸11.58%。研究结果为莴苣籽在食品及药品领域的开发提供了一定理论依据。     

The formation of thearubigin-like substances by in-vitro polyphenol oxidase-mediated fermentation of individual flavan-3-ols

An enzymic in-vitro model tea fermentation system has been used in combination with gradient elution reversed-phase high-performance liquid chromatography to study the formation from individual fiavan-3-ols of resolvable and unresolvable thearubigin-like substances. It has been found that in 30 min at pH 5.6 tea polyphenol oxidase (EC 1.14.18.1) produces a distinctive range of resolvable thearubigin-like products from each flavan-3-ot substrate examined ((+)-catechin, (?)-epicatechin, (?)-epigallocatechin, (?)-epicatechin gallate and (?)-epigallocatechin gallate). However, (?)-epicatechin, and to a much lesser extent (+ )-catechin, were the only flavan-3-ols converted to an unresolvable thearubigin-like hump under these conditions. In addition, it was demonstrated that varying the pH of incubation in the range 5.0–6.0 had little effect on the profile of products obtained from the simple catechins, but a pronounced effect in the case of the gallocatechins. It is concluded that the combination of (?)-epicatechin and polyphenol oxidase is the simplest model of tea fermentation that can be used to generate in quantity thearubigin-like materials for structure elucidation.     

Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.     

Effects of light level, time of harvest and position within field on the variability of tissue nitrate concentration in commercial crops of lettuce (Lactuca sativa) and endive (Cichorium endiva)

Seven commercial crops of lettuce and one crop of endive were sampled in order to study the variability in plant tissue nitrate concentration (TNC). Assuming that an appropriate sampling pattern was employed, ten plants were sufficient to give an acceptable estimate of the mean TNC. Short-term shading (24-48 h) had no significant effects on mean TNC, unlike the increase in TNC known to occur following dull periods 10-14 days before harvest. The effect on TNC of time of day harvested was significant, but there was no obvious pattern of diurnal variation. Averaged over all experiments, the coefficient of variation for TNC was in the order of 35%. Increasing the sample size from ten to 40 plants would only be expected to decrease the standard error of measurement of TNC from 16 to 12% of the mean because of the underlying analytical error, which would remain constant.     

Identification of (−)-epicatechin as the direct substrate for polyphenol oxidase from longan fruit pericarp

Postharvest browning of longan fruit results in a short life and a reduced commercial value. The experiments were conducted to separate, then purify and finally identify the polyphenol oxidase (PPO) substrates that cause longan fruit to brown. PPO and its substrates were, respectively, extracted from longan fruit pericarp tissues. The substrate for longan PPO was separated and purified using polyamide column chromatography, Sephadex LH-20 column chromatography and silica gel column chromatography, respectively. The substrate was further identified by 0.5% FeCl₃ solution and enzymatic reaction with longan PPO. On the bases of UV, ¹H NMR, ¹³C NMR, and ESI-MS data, the direct substrate for the PPO from pericarp tissues of longan fruit was identified to be (−)-epicatechin. Furthermore, the contents of (−)-epicatechin of pericarp tissues of longan fruit of two major cultivars were determined by high performance liquid chromatography (HPLC). The HPLC analysis exhibited that the contents of (−)-epicatechin of fruit pericarp of ‘Shixia’ and ‘Chuliang’ were 0.26 and 0.56 mg/g on fresh weight (FW) basis at harvest and 0.15 and 0.09 mg/g FW after 3 days of storage. The more rapid decrease in the (−)-epicatechin content of ‘Chuliang’ was due to the oxidization catalyzed by PPO, which was in agreement with the higher browning index.     

Latent polyphenol oxidase from quince fruit pulp (Cydonia oblonga): purification,activation and some properties

Quince fruit polyphenol oxidase (PPO) was partially purified using a combination of phase partitioning in Triton X‐114 and PEG 8000/phosphate with a final ammonium sulfate fractionation between 30% and 75%, to avoid the deep browning of the enzyme due to the high amount of oxidizing substances present in the quince pulp. The clean and stable enzyme was partially purified in a latent form and could be optimally activated by the presence of 0.5 g dm^?3 sodium dodecyl sulfate (SDS) with an optimum pH of 5.0. In the absence of SDS, the enzyme showed maximum activity at acid pH. The apparent kinetic parameters of the latent enzyme were determined at pH 5.0, the V_m value being 15 times higher in the presence of SDS than in its absence, whereas the K_M was the same in both cases, with a value of 1.2 mmol L^?1. The effect of several inhibitors was studied, tropolone being the most active with a K_i value of 4.7 µmol L^?1. In addition, the effect of cyclodextrins was studied, and the complexation constant (K_c) between 4‐tert‐butylcatechol and cyclodextrins was calculated using an enzymatic method. The value obtained for K_c was 15 310 mol L^?1. Copyright © 2006 Society of Chemical Industry     

Nitrate content in lettuce (Lactuca sativa L) heads in relation to plant spacing,nitrogen form and irrigation level

A field study was conducted at two locations (Jordan valley and Al‐Jubeiha) with different rainfall levels, altitudes and temperature ranges. The study was established to evaluate the optimum planting density, nitrogen (N) form and irrigation level to attain the best quality of lettuce crop in terms of minimum nitrate (NO₃) content and to minimise the impact on the environment. Seeds of ‘Amar’ lettuce were sown 1 month before transplanting. Three forms of N fertiliser (Ca(NO₃)₂, (NH₄)₂SO₄ and CO(NH₂)₂) were applied at three times at a total rate of 100 kg N ha^?1. Three in‐row spacings (15, 20 and 25 cm) were assigned. Two irrigation levels were applied: level 1 had twice the amount of irrigation water as level 2, which was achieved by doubling the number of irrigation lines. The results indicated that N form significantly increased both N and NO₃ contents. Ca(NO₃)₂ was the most effective in increasing the N and NO₃ contents in lettuce leaf tissues, followed by CO(NH₂)₂ and then (NH₄)₂SO₄. The outer leaves (green colour) had about five times the NO₃ content of the inner leaves (pale yellow colour). However, the effect of N form on production, total N absorption and N recovery was significant in the following order: (NH₄)₂SO₄ > Ca(NO₃)₂ > CO(NH₂)₂. Increasing the plant spacing resulted in a significant increase in N and NO₃ contents in the lettuce leaves. Copyright © 2004 Society of Chemical Industry     

A study of variation in the tendency for postharvest discoloration in a lettuce (Lactuca sativa) diversity set

Lettuce as a component of processed salad packs often suffers from pinking or browning discoloration on leaf surfaces within a few days after harvest limiting product shelf life. A lettuce diversity set representing the primary gene pool was used to investigate phenotypic variation for postharvest discoloration. Discoloration of minimally processed lettuce was assessed using material harvested from a replicated field trial. Significant variation for pinking and browning was recorded (P < 0.001). Rates of discoloration were specific to lettuce type, however potential for pinking or browning within a type varied. Interestingly, accessions were significantly more susceptible to browning (P < 0.05) early postharvest (day 1) but significantly more susceptible to pinking (P < 0.05) during later stages (days 6, 9 and 13). The results indicate that there is a genetic basis for this phenotypic variation and this natural allelic variation could be exploited through breeding to develop discoloration resistant cultivars, consequently minimising pre‐ and postharvest treatments and reducing food wastage.     

Purification of diamine oxidase and its properties in germinated fava bean (Vicia faba L.)

Background: γ‐Aminobutyric acid (GABA) is a non‐protein amino acid with bioactive functions for human health. Diamine oxidase (DAO, EC 1.4.3.6) is one of the key enzymes for GABA formation. In the present study, this enzyme was purified from 5 day germinated fava bean and its properties were investigated in vitro. Results: The molecular mass of the enzyme estimated by Sephadex G‐100 gel filtration was 121 kDa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) displayed a single band at a molecular mass of 52 kDa. The enzyme had optimal activity at 40 °C and retained its activity after being incubated at 30 °C for 30 min. It showed higher activity at pH 6.5 than at other pH values. The enzyme was significantly inhibited by Mg²⁺, Cu²⁺, Fe³⁺, aminoguanidine, ethylene glycol tetraacetic acid (EGTA), ethylene diamine tetraacetic acid disodium salt (EDTA‐Na₂), L ‐cysteine and β‐mercaptoethanol. The K_m value of DAO was 0.23 mmol L^?1 for putrescine and 0.96 mmol L^?1 for spermidine. However, the enzyme did not degrade spermine. Conclusion: DAO from germinated fava bean was purified. The optimal reaction temperature and pH of the enzyme were mild. The enzyme had higher affinity to putrescine than to spermidine and spermine. Copyright © 2011 Society of Chemical Industry     

Purification and characterisation of polyphenol oxidase (PPO) from eggplant (Solanum melongena)

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 × 10⁶) 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.