Prokaryotic expression and purification of Camellia sinensis polyphenol oxidase

BACKGROUND: Polyphenol oxidase (PPO) causes the postharvest loss of fruits and vegetables but is also a key factor in the quality development of tea. However, there are no reports on engineered active plant PPO purified from prokaryotic cells. RESULTS: In this study the ppo gene of about 1800 bp from Camellia sinensis cv. Yihongzao was successfully cloned and expressed in Escherichia coli. The PPOs purified from both the soluble fraction and the inclusion bodies showed activity. In addition, 1.0 × 10^?7 mol L^?1 Cu²⁺ and acidic conditions were found to be favourable for the engineered PPO catalysis of catechol oxidation. CONCLUSION: This paper represents the first report on C. sinensis ppo expression in E. coli and engineered active PPO purification. The results of the study provide a basis for the large‐scale preparation and application of PPO. Copyright © 2010 Society of Chemical Industry     

Polyphenol oxidase activity of two olive (Olea europaea L.) cultivars as an early criterion of Mn toxicity

BACKGROUND: The time course of polyphenol oxidase (PPO) activity in the leaves of two olive cultivars (Picual and FS‐17) irrigated with nutrient solutions differing in Mn concentration (0, 2 and 1280 µmol L^?1) was studied under hydroponic conditions to determine whether PPO activity could be used as an early criterion of Mn status of olive plants, and to elucidate whether genotypic differences exist between the two olive cultivars studied, concerning the effect of Mn concentration on PPO activity. RESULTS: In all the Mn treatments, PPO activity was greater in Picual than in FS‐17. Under excess Mn (1280 µmol L^?1), PPO activity gradually increased with time, starting from day 30 of the experiment in both cultivars, and this increase preceded the appearance of Mn toxicity symptoms. In contrast, in the other two Mn treatments (0 and 2 µmol L^?1) PPO activity increased and afterwards decreased during the experiment, but the trend was not clear. In the 1280 µmol L^?1 treatment, PPO activity linearly increased (R = 0.8836 for Picual and 0.943 for FS‐17) with the increase of Mn concentration in the leaves of both cultivars. In the 1280 µmol L^?1 Mn treatment, PPO activity was negatively related with Fe and Zn concentrations in the leaves, and positively in the 0 and 2 µmol L^?1 Mn treatments with the Ca, Mg and K concentrations. CONCLUSION: From the differential time course of PPO activity in the three Mn treatments (0, 2 and 1280 µmol L^?1), it is concluded that periodic measurements of PPO activity in the leaves of the olive cultivars Picual and FS‐17 can be used for the early detection of Mn toxicity (before the appearance of symptoms). Copyright © 2010 Society of Chemical Industry     

PARTIAL PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM FRESH-CUT CHINESE WATER CHESTNUT

The characteristics of polyphenol oxidase (PPO) from Chinese water chestnut (CWC) and its potential inhibitors for browning reactions were investigated. PPO was isolated from fresh‐cut CWC and was purified on a Sephadex G‐100 column, with a yield of total activity close to 10%. The molecular weight, Michaelis constant (K_m), substrate specificity, optimal pH and temperature of CWC PPO were examined. Kinetic studies indicated that the K_m and V_max values of CWC PPO for catechol were 10.32 mmol/L and 6.452 × 10⁴ U/min, respectively. The optimal pH and temperature for CWC PPO was 6.5 and 40C, respectively. Among the browning inhibitors tested, 4‐hexylresorcinol, at a concentration of 0.3 mmol/L, showed the strongest inhibition (70%) against the PPO activity of CWC, followed by 3.0 mmol/L N‐acetyl‐L‐cysteine with an inhibition of 53%.     

Changes in selected quality characteristics of minimally processed carambola (Averrhoa carambola L.) when treated with ascorbic acid

‘B 10’ carambola of ripening stage (RS) 3 and 4 were minimally processed (MP) and then dipped in 0, 15 and 30 mg L^?1 ascorbic acid (AA). The 1‐cm‐thick slices were then dried, packed into cling‐wrapped‐foam tray and stored at 7 °C for 0, 3 and 5 days. Skin colour (L*, C* and h°), flesh firmness, soluble solids concentration, vitamin C content, titratable acidity, pH, degree of browning, polyphenoloxidase (PPO) activity and sensory attributes of MP carambola treated with AA were determined. AA treatment had significant effect in decreasing cut surface browning degree but no significant effect on all the selected quality characteristics of the MP carambola. In the sensory evaluation, flesh colour, sweetness, flavour and overall taste were significantly affected by AA treatment especially at 15 mg L^?1. The RS of fruit significantly affected skin colour (C* and h°), pH and sensory attributes of colour and flavour of the MP carambola. As storage day (SD) progressed, skin colour (C* and h°), flesh firmness and vitamin C content, cut surface browning, PPO activity and all the sensory attributes of MP carambola decreased significantly. Flesh firmness of the MP carambola was affected by the interaction between AA × SD. Sensory attributes of MP carambola were affected significantly by AA × RS. All the sensory attributes of MP carambola positively correlated to each other but negatively correlated with browning degree. PPO activity positively correlated with browning degree. Copyright © 2007 Society of Chemical Industry     

PURIFICATION AND CHARACTERIZATION OF HOMOGENEOUS ACID PHOSPHATASE FROM NONGERMINATED BUCKWHEAT (FAGOPYRUM ESCULENTUM) SEEDS

A buckwheat acid phosphatase (orthophosphoric‐monoester phosphohydrolase, EC 3.1.3.2) was purified about 250‐fold from nongerminated buckwheat seeds to apparent homogeneity with a recovery of 4% from the acid phosphatase activity in the crude extract. It is the major acid phosphatase among eight different acid phosphatases identified in the crude extract. The purified enzyme behaved as a monomeric protein of molecular mass about 45 kDa. The purified enzyme exhibited a single pH optimum at 5.25. Optimum temperature for the degradation of p‐nitrophenyl phosphate was 50C. The kinetic parameters for the hydrolysis of p‐nitrophenyl phosphate were determined to be K_M= 76 μmol L^?1 and k_cat= 924 s^?1 at pH 5.25 and 37C. While the enzyme failed to act on phytate as a substrate, the enzyme exhibited a broad substrate selectivity. The purified enzyme showed no measureable carboxylesterase activity and no divalent metal ion requirement.     

PARTIAL PROPERTIES OF POLYPHENOL OXIDASE IN MANGO (MANGIFERA INDICA L. CV. "TAINONG") PULP

The properties of polyphenol oxidase (PPO, EC 1.14.18.1) from an extract of mango pulp were studied. PPO, with catechol as substrate, had an optimum pH at 7.0 and optimum temperature at 30C. PPO showed activity with dihydroxyphenols and trihydroxyphenols, but not with monohydroxyphenols. Kinetic parameters maximum velocity and Michaelis constant for PPO were 256.28 U/min and 6.30 mM with catechol, and 199.61 U/min and 47.81 mM with pyrogallol. The activity of PPO was well retained after heating the extract for 15 min at 50C, and 98% of the activity was lost after the extract was heated for 5 min at 80C. PPO was effectively inhibited by ascorbic acid as well as by β‐mercaptoethanol and L‐cysteine, and was enhanced by sodium dodecyl sulfate.     

Characterization of Polyphenoloxidase from Stanley Plums

Five plum cultivars were investigated for polyphenoloxidase. Stanley cultivar, which showed highest PPO activity, was selected for characterization of this enzyme. The activity of crude enzyme was 3.5 times greater in the flesh than in the skin of the fruit. The enzyme showed a K_m of 20 raM of catechol and V_max of 5.41 × 10^?1 O.D./ min at pH 6.0. Its pH and temperature optima were 6.0 and 20°C respectively. The enzyme lost activity below pH 4.5, but was stable even at 70°C. Among substrates, 4-methylcatechol was oxidized more rapidy, although catechol, dopamine, pyrogallol and caffeic acid were also good substrates. The enzyme was strongly inhibited by sodium metabisulfite (Na₂S₂O₅), L-cysteine and ascorbic acid.     

Inhibitory effect of rice bran extract on polyphenol oxidase of potato and banana

Rice bran was extracted with water and its effects on potato and banana polyphenol oxidase (PPO) were investigated. Rice bran extract (RBE), conc. 0.3 g mL^?1, exhibited PPO inhibition in potato and banana PPO with % inhibition of 69.31% and 47.63%, respectively (P ≤ 0.05). RBE showed a concentration‐dependent inhibition on potato and banana PPO. RBE (conc. 0.3 g mL^?1) inhibited potato PPO higher than ascorbic acid, citric acid, NaCl and EDTA (final conc. 20 mg L^?1); and it also inhibited banana PPO higher than citric acid, NaCl and EDTA (final conc. 20 mg L^?1), respectively. The combination of RBE with citric acid or ascorbic acid appeared to be additive inhibitory effect on banana and potato PPO. Kinetic study of the inhibition on potato and banana PPO by RBE showed that RBE was a mixed‐type inhibitor; however, RBE appeared to be able to act directly on enzyme structure rather than substrate structure.     

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     

Effect of nitric oxide on reactive oxygen species and antioxidant enzymes in kiwifruit during storage

BACKGROUND: The accumulation of reactive oxygen species (ROS) in kiwifruit can cause oxidative damage during storage. Little research has been carried out on the effects of nitric oxide (NO) on oxidative damage to kiwifruit. Therefore the aim of the present study was to evaluate the ability of 0.5, 1 and 2 µmol L^?1 NO aqueous solutions to alleviate oxidative damage to kiwifruit during storage. RESULTS: The most marked effect was obtained with 1 µmol L^?1 NO solution, which significantly reduced the accumulation of malondialdehyde, superoxide and hydrogen peroxide, delayed the decrease in vitamins C and E, maintained the content of soluble solids, inhibited the activity of lipoxygenase and peroxidase and increased the activity of superoxide dismutase and catalase in kiwifruit during storage. The 0.5 µmol L^?1 NO solution was too weak to significantly affect the content of ROS and the activity of enzymes. However, treatment with 2 µmol L^?1 NO solution promoted the accumulation of ROS, decreased the activity of antioxidant enzymes and accelerated peroxidation in kiwifruit during storage. CONCLUSION By increasing the activity of antioxidant enzymes and maintaining the content of vitamins C and E, treatment with 1 µmol L^?1 NO aqueous solution could protect kiwifruit against oxidative damage caused by ROS during storage. Copyright © 2008 Society of Chemical Industry     

Purification and biochemical characterisation of a novel glutamate decarboxylase from rice bran

BACKGROUND: Glutamate decarboxylase (GAD) is a useful enzyme whose main function is to catalyse the irreversible α‐decarboxylation of L ‐glutamate to produce γ‐aminobutyric acid. The cheap and abundant rice‐processing by‐product rice bran contains a high amount of GAD, the purification and characterisation of which have not yet been reported. In this study, research on rice bran GAD was initiated. RESULTS: Rice bran GAD was purified to homogeneity via a combined purification protocol of ammonium sulfate fractionation, ion exchange chromatography and two gel filtrations, with a purification fold of 128.6 and an activity recovery of 21.3%. The enzyme was active at pH 5.5 and 40 °C and retained 80% of its original activity in the pH range 5–9 and the temperature range 30–50 °C. GAD activity was significantly enhanced in the presence of Ca²⁺ but strongly inhibited by Ag⁺, Hg²⁺, sodium dodecyl sulfate and CH₃COOH. Kinetic determination of the apparent K_m for L ‐glutamate and pyridoxal 5′‐phosphate gave values of 27.4 mmol L^?1 and 1.16 µmol L^?1 respectively. CONCLUSION: Considering that rice bran is cheap and commercially available and that rice bran GAD is relatively stable, the development of cost‐effective rice bran GAD‐related functional foods would seem to be feasible. Copyright © 2010 Society of Chemical Industry     

Scirpusin A,a hydroxystilbene dimer from Xinjiang wine grape,acts as an effective singlet oxygen quencher and DNA damage protector

BACKGROUND: Grapes and red wines are rich sources of phenolic compounds such as anthocyanins, catechins, flavonols and stilbenes, most of which are potent antioxidants showing cardioprotective properties. We first isolated scirpusin A, a hydroxystilbene dimer, from a wine grape of Xinjiang, and studied its antioxidant activity. RESULTS: Reactive oxygen species scavenging effects and the protection against reactive singlet oxygen‐induced DNA damage of scirpusin A have been investigated in our experiments. The concentration of scirpusin A required to inhibit 50% of ¹O₂ generation was 17 µmol L^?1, while addition of scirpusin A at 140 µmol L^?1 caused complete inhibition. Further kinetic study revealed that the reaction of Scirpusin A with singlet oxygen has an extremely high rate constant (k_a = 4.68 × 10⁹ L mol^?1 s^?1). Scirpusin A (140 µmol L^?1) exhibited significant inhibition effects on pBR322 DNA breakage. However, scavenging effects of scirpusin A on superoxide anion O₂^?? and hydroxyl radical ·OH were not potent as the inhibitor rates at a concentration of 1400 µmol L^?1 were 28.83% and 19.5%, respectively. CONCLUSION: The present study shows that scirpusin A is a selective quencher of singlet oxygen and a protector against reactive singlet oxygen‐induced pBR322 DNA damage at very low concentrations. Copyright © 2010 Society of Chemical Industry     

Improvement of L(+)‐lactic acid production from cassava wastewater by Lactobacillus rhamnosus B 103

BACKGROUND: L (+)‐Lactic acid is used in the pharmaceutical, textile and food industries as well as in the synthesis of biodegradable plastics. The aim of this study was to investigate the effects of different medium components added in cassava wastewater for the production of L (+)‐lactic acid by Lactobacillus rhamnosus B 103. RESULTS: The use of cassava wastewater (50 g L^?1 of reducing sugar) with Tween 80 and corn steep liquor, at concentrations (v/v) of 1.27 mL L^?1 and 65.4 mL L^?1 respectively led to a lactic acid concentration of 41.65 g L^?1 after 48 h of fermentation. The maximum lactic acid concentration produced in the reactor after 36 h of fermentation was 39.00 g L^?1 using the same medium, but the pH was controlled by addition of 10 mol L^?1 NaOH. CONCLUSION: The use of cassava wastewater for cultivation of L. rhamnosus is feasible, with a considerable production of lactic acid. Furthermore, it is an innovative proposal, as no references were found in the scientific literature on the use of this substrate for lactic acid production. Copyright © 2010 Society of Chemical Industry     

Effects of different nitric oxide application on quality of kiwifruit during 20 °C storage

The effects of fumigating with 0, 10, 20, 30 μL L^?1 nitric oxide (NO) gas and dipping in 0.5, 1.0 and 2.0 μmol L^?1 NO aqueous solution on quality of kiwifruit (Actinidia Chinensis Planch cv Xuxiang) during storage at 20 °C were evaluated. It was found that fumigating with 20 μL L^?1 NO gas and dipping in 1.0 μmol L^?1 NO aqueous solution delayed firmness lost and increased SSC/TA ratios of kiwifruits. In comparison with the kiwifruits fumigated with 20 μL L^?1 NO, the kiwifruits dipped in 1.0 μmol L^?1 NO solution had slower ethylene production, lower contents of soluble solids and malondialdehyde (MDA), higher contents of vitamin C and E, but no significant difference existed in chlorophyll contents. The results suggested that dipping kiwifruits in 1.0 μmol L^?1 NO aqueous solution was more effective in maintaining kiwifruits quality during 20 °C storage.     

Characterization of polyphenoloxidase from 2 peach (Prunus persica L.) varieties grown in Argentina

Polyphenoloxidase was extracted from September peach (_SEPPO) and Summerset peach (_SUPPO) and its physicochemical characteristics were analyzed. The optimum pH was 6.5 for _SEPPO and 5.5 for _SUPPO. The optimum temperature was 35°C for _SEPPO and 39.4°C for _SUPPO. Activation energy (Ea) from thermal activation was 41.5 kJ/mol for _SEPPO and 37.5 kJ/mol for _SUPPO. Heating at 60°C by 5 min, _SUPPO was denatured whereas _SEPPO retained 2.6% of activity. Activation enthalpy (ΔH^#) and activation entropy (ΔS^#) for _SEPPO heat-inactivation were 69.9 J/mol and −83.5 kJ/mol·K for _SUPPO, ΔH^# was 91.8 J/mol while ΔS^# was −21.0 kJ/mol·K. Substrate specificity (V_max/K_M) was 4-methylcatechol>catechol>pyrogallol for _SEPPO and 4-mehtylcatechol>pyrogallol>catechol for _SUPPO. For both enzymes, the order of inhibition effectiveness using reductor agents was metabisulphite>ascorbic acid. Benzaldehyde, 4-hydroxybenzaldehyde, and dl-dopa were competitive inhibitors, and their K_I values were 38.86, 8.43, and 2.08 mM, respectively.     

Polyphenol oxidase activity,phenolic acid composition and browning in cashew apple (Anacardium occidentale, L.) after processing

This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Purification factor was 59 at 95% (NH₄)₂SO₄ saturation. For PPO activity, the optimal substrate was catechol and the optimum pH was 6.5. PPO K_m and V_max values were 18.8 mM and 13.6 U min⁻¹ ml⁻¹, respectively. Ascorbic acid, citric acid, sodium sulphite and sodium metabisulphite decreased PPO activity, while sodium chloride increased PPO activity. Wounding at 2 °C and 27 °C for 24 h increased PPO activity but storage at 40 °C reduced PPO activity. Gallic acid, protocatechuic acid and cinnamic acid (free and conjugate) were identified in cashew apple juice. Cutting and subsequent storage at 40 °C hydrolysed cinnamic acid. 5-Hydroxymethylfurfural content in cashew apple juice increased after injury and storage at higher temperatures, indicating non-enzymatic browning.     

Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape

Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera ‘Thompson Seedless’), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol l-Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent Michaelis–Menten constant (K _m) and maximum velocity of the reaction (V _max) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD_400 nm/min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD_478 nm/min for D, L-DOPA, respectively. The obtained similar specificity values of V _max/K _m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was l-cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg²⁺ and Cu²⁺ increased, while Zn²⁺ and K⁺ reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25 °C with 10 mM catechol as substrate. The enzyme was heat stable between 10 and 25 °C, but showed significant activity loss at temperatures higher than 40 °C and completely inactivation at 70 °C for 10 min. Thermal inactivation of PPO showed a first-order kinetic with an activation energy (E _a) of 146.1 ± 10.8 kJ/mol at pH 6.0.     

Biochemical studies of cocoa bean polyphenol oxidase

Polyphenol oxidase (EC 1.14.18.1) was isolated and partially purified from cocoa beans. The properties of the enzyme were studied. The Michaelis constant K_m for catechol was 1 × 10^?2 M . The pH optimum of polyphenol oxidase activity assayed with catechol as substrate occurred at pH 6.8 and was characterised by a relatively high thermal stability, 50% of its activity was lost after heating for 40, 25 and 5 min at 60, 69 and 80°C respectively. The optimum temperature for the enzyme activity with catechol as substrate was around 45°C. The enzyme was reactive towards 3-(3,4-dihydroxy phenyl)-DL -alanine, 3-hydroxytyramine hydrochloride and 4-methyl catechol but showed no activity towards tyrosine, p-cresol, and 4-hydroxy-phenol. A rapid deactivation of the enzyme was observed when catechol of concentration > 40 mM was used as substrate. The enzyme activity was inhibited by ascorbic acid, L -cysteine, sodium bisulphite and thiourea.     

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