Characterization of polyphenol oxidase from broccoli (Brassica oleracea var. botrytis italica) florets

Polyphenol oxidase (PPO) from broccoli florets was extracted and purified through (NH₄)₂SO₄ precipitation, ion-exchange and gel filtration chromatography. The molecular weight was estimated to lie between 51.3 and 57 kDa by sodium dodecyl sulphate-polyacrylamide gel electophoresis (SDS-PAGE) and gel filtration. The effects of substrate specificity, pH, and sensitivity to various inhibitors: citric acid, ascorbic acid, sodium sulphate and EDTA (sodium salt of ethylenediaminetetraacetic acid) of partially purified PPO were investigated. Polyphenol oxidase showed the best activity toward catechol (K_M = 12.34 ± 0.057 mM, V_max = 2000 ± 8736 U/ml/min) and 4-methyl catechol (K_M = 21 ± 0.087 mM, V_max = 28.20 ± 0.525 U/ml/min). The optimum pH for broccoli PPO was 5.7 with catechol and 4-methylcatechol as substrates. The most effective inhibitor was sodium sulphate.     

Polyphenol oxidase from bayberry (Myrica rubra Sieb. et Zucc.) and its role in anthocyanin degradation

Polyphenol oxidase (PPO) was extracted from bayberry (Myrica rubra Sieb. et Zucc. cv. Biqi), and its partial biochemical characteristics were studied. Stable and highly active PPO extracts were obtained using insoluble polyvinylpolypyrrolidone (PVPP) in sodium phosphate, pH 7.0, buffer. The highest PPO activity was observed in the ripe fruits. Optimum pH and temperature for bayberry PPO activity were pH 6.0 and T = 30 °C with 0.1 M catechol as substrate. PPO showed activity using the substrates of catechol, gallic acid and protocatechuic acid, but no activity with the substrates (+)-catechin, p-coumaric acid or caffeic acid. K_m and V_max values were 313 mM and 3.26ΔOD/min/g FW, respectively, with catechol as the substrate. Bayberry PPO did not act directly on cyanidin 3-glucoside but the rate of anthocyanin degradation was stimulated by the addition of gallic acid.     

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.     

Purification and characterization of polyphenol oxidase from Ferula sp.

Polyphenol oxidase (PPO) of several Ferula sp. was extracted and purified through (NH₄)₂SO₄ 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.     

Purification and characterization of membrane-bound polyphenoloxidase (mPPO) from Snake fruit [Salacca zalacca (Gaertn.) Voss]

Membrane-bound polyphenoloxidase (mPPO) an oxidative enzyme which is responsible for the undesirable browning reaction in Snake fruit (Salacca zalacca (Gaertn.) Voss) was investigated. The enzyme was extracted using a non-ionic detergent (Triton X-114), followed by temperature-induced phase partitioning technique which resulted in two separate layers (detergent-poor phase at the upper layer and detergent-rich phase at the lower layer). The upper detergent-poor phase extract was subsequently fractionated by 40–80% ammonium sulfate and chromatographed on HiTrap Phenyl Sepharose and Superdex 200 HR 10/30. The mPPO was purified to 14.1 folds with a recovery of 12.35%. A single prominent protein band appeared on native-PAGE and SDS–PAGE implying that the mPPO is a monomeric protein with estimated molecular weight of 38 kDa. Characterization study showed that mPPO from Snake fruit was optimally active at pH 6.5, temperature 30 °C and active towards diphenols as substrates. The K_m and V_max values were calculated to be 5.46 mM and 0.98 U/ml/min, respectively, when catechol was used as substrate. Among the chemical inhibitors tested, l-cysteine showed the best inhibitory effect, with an IC₅₀ of 1.3 ± 0.002 mM followed by ascorbic acid (1.5 ± 0.06 mM), glutathione (1.5 ± 0.07 mM), EDTA (100 ± 0.02 mM) and citric acid (186 ± 0.16 mM).     

Inhibitory effects of p-alkylbenzoic acids on the activity of polyphenol oxidase from potato (Solanum tuberosum)

In order to prevent potato from browning during preservation and processing, the effects of p-alkylbenzoic acids (p-propylbenzoic acid, p-butylbenzoic acid, p-pentylbenzoic acid, p-hexylbenzoic acid, p-heptylbenzoic acid, p-octylbenzoic acid) on the activity of potato polyphenol oxidase (PPO) have been studied. The PPO was extracted with PBS (pH 6.8), fractionated with ammonium sulphate, concentrated, and purified with Sephadex G-100 (1.8 × 100 cm) filtration chromatography. The active fractions were pooled and the PPO activity was determined to be 79.83 U/mg. The results showed that p-alkylbenzoic acids could strongly inhibit the activity of potato PPO. The values of IC₅₀ of these six inhibitors were determined to be 0.213, 0.180, 0.152, 0.106, 0.075 and 0.047 mM, respectively. Kinetic analyses showed that p-alkylbenzoic acids used in this experiment were reversible and noncompetitive inhibitors to the enzyme. The inhibitory effects were potentiated with increasing lengths of the hydrocarbon chains, indicating that the inhibitory efficiency on the enzyme was influenced by the steric effect of the substituted groups.     

Purification and characterisation of a polyphenol oxidase from Boletus erythropus and investigation of its catalytic efficiency in selected organic solvents

Polyphenol oxidase (PPO) was purified from Boletus erythropus using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. Optimum pH and temperature were found to be 8.0 and 20 °C, respectively, using 4-methylcatechol as a substrate. The enzyme was extremely stable between pH 3.0 and 9.0 after 24 h incubation at 4 °C. B. erythropus PPO was also quite stable between 10 and 30 °C after 4 h incubation. The K_m and V_max values were calculated as 2.8 mM and 1430 U/mg protein by Lineweaver–Burk curve, respectively. The enzyme activity was inhibited by sodium metabisulfite, ascorbic acid, sodium azide and benzoic acid. It was seen that the mushroom PPO was an effective biocatalyst in selected organic solvents, such as dichloromethane, dichloroethane and toluene, when catechin was used as a substrate. All data support that B. erythropus has a highly active PPO, possessing similar biochemical and kinetic characteristics to other plant PPOs.     

Characterisation and tissue distribution of polyphenol oxidase of deepwater pink shrimp (Parapenaeus longirostris)

Characterisation and tissue distribution of polyphenol oxidase (PPO) was studied in deepwater pink shrimp (Parapenaeus longirostris) post mortem. PPO activity was the highest in the carapace, followed by that in the abdomen exoskeleton, cephalotorax, pleopods and telson. No PPO activity was found in the abdomen muscle and in the pereopods and maxillipeds using the enzymatic assay. Storage of whole shrimps and of the different organs showed that melanosis (blackening) required the presence of the cephalotorax to be initiated, indicating that its development depends on other factors in addition to the PPO levels. Further characterisation was carried out in extracts partly purified using 40–70% ammonium sulfate fractionation. The enzyme had the highest activity at pH 4.5 and was most stable at pH 4.5 and 9.0. No clear maximum was observed in the 15–60 °C range but the higher stability was achieved at 30–35 °C. Apparent kinetic constants in the partly purified PPO from carapace were K_M = 1.85 mM and V_max = 38.5 U/mg of protein, pointing to a high affinity and reactivity of the enzyme when assayed with DOPA. Electrophoretic mobility was studied in native PAGE and non-reducing SDS–PAGE followed by staining with DOPA. Approximate MW of 500 kDa and 200 kDa were observed, respectively. These two forms could correspond to aggregates of minor PPO subunits that could not be resolved in these electrophoretic systems. The peptide mass fingerprinting obtained by MALDI-TOF analysis showed some peptides whose homology with hemocyanins and different PPO subunit precursors has already been demonstrated in the same species.     

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.     

Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K⁺, other metal ions such as Zn²⁺, Cu²⁺, Fe³⁺, Ca²⁺, Fe²⁺ and Na⁺ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K_m and V_max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.     

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.     

Characterisation of lipoxygenase from pea seeds (Pisum sativum var. Telephone L.)

Lipoxygenase (LOX) from pea seeds (Pisum sativum var. Telephone L.) was extracted and studied of biochemical properties. The molecular mass of purified lipoxygenase was 93 kDa. The effects of substrate specificity, pH, and sensibility to various inhibitors: caffeic acid, ferulic acid, benzoic acid, catechin, quercetin and kaempferol of LOX were investigated. Lipoxygenase showed the highest activity toward linoleic acid and the lowest toward oleic acid as substrates. Kinetic studies indicated that V_max of the LOX activity was 151.5 U/min and corresponding K_m value of 0.44 × 10⁻³ M. Optimum pH of lipoxygenase was reported at 5.5. Caffeic acid was the most effective inhibitor and kaempferol was the least effective.     

Purification and partial characterization of polyphenol oxidase from the flower buds of Lonicera japonica Thunb.

The purification and partial enzymology characteristics of polyphenol oxidase from Lonicera japonica (LjPPO) were studied in this paper. The crude enzyme solution was purified in turn by ammonium sulfate, dialysis, and DEAE-cellulose ion-exchange chromatography after preliminary treatments. Purification resulted in 31-fold enrichment and its molecular weight was estimated to be ∼49 kDa exhibited on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The pH for optimal conditions of LjPPO was 7.5, and the temperature was 25 °C, in addition, the inhibitive effects of inhibitors were enhanced positively with increasing of the concentration. Moreover, crude enzyme solution showed diphenolase activity toward catechol, l-dopa and chlorogenic acid rather than monophenolase and triphenolase activity, and the best substrate was catechol because of the highest V_max/K_m value. However, the oxidation of diphenol related to browning significantly, so the data obtained in this research provided theoretical basis for the prevention of enzymatic browning of L. japonica during processing.     

Characterization of polyphenoloxidase (PPO) and total phenolic contents in medlar (Mespilus germanica L.) fruit during ripening and over ripening

Characterization of polyphenoloxidase (PPO) enzyme and determination of total phenolic concentrations during fruit ripening and over ripening in medlar (Mespilus germanica L.) were determined. During ripening, PPO substrate specificity, optimum pH and temperature, optimum enzyme and substrate concentrations were determined. Among the five mono- and di-phenolic substrates examined ((p-hydroxyphenyl) propionic acid, l-3,4-dihydroxyphenylalanine, catechol, 4-methylcatechol and tyrosine), 4-methylcatechol was selected as the best substrate for all ripening stages. A range of pH 3.0–9.0 was also tested and the highest enzyme activity was at pH 7.0 throughout ripening. The optimum temperature for each ripening stage was determined by measuring the enzyme activity at various temperatures over the range of 10–70 °C with 10 °C increments. The optimum temperatures were found to be 30, 20 and 30 °C, respectively, for each ripening stage. Optimum enzyme and substrate concentrations were found to be 0.1 mg/ml and 40 mM, respectively. The V_max and K_m value of the reaction were determined during ripening and found to be 476 U/mg protein and 26 mM at 193 DAFB (days after full bloom) – stage 1, 256 U/mg protein and 12 mM at 207 DAFB – stage 2, 222 U/mg protein and 8 mM at 214 DAFB – stage 3. For all ripening stages sodium metabisulfite markedly inhibited PPO activity. For stage 1 of ripening, Cu²⁺, Hg²⁺ and Al³⁺, for stage 2, Cu²⁺ and Hg²⁺, and for stage 3, Cu²⁺, Hg²⁺, Al³⁺ and Ca²⁺ strongly inhibited diphenolase activity. Accordingly, it can be concluded that as medlar fruit ripen there is no significant changes in the optimum values of polyphenoloxidases, although their kinetic parametres change. As the fruit ripening progressed through ripe to over-ripe, in contrary to polyphenoloxidase activity, there was an apparent gradual decrease in total fruit phenolic concentrations, as determined by using the aqueous solvents and water extractions.     

Partial purification and characterisation of endoglucanase from an edible mushroom, Lepista flaccida

A crude extract was prepared from the fruiting body of Lepista flaccida, an edible mushroom and endoglucanase activity of the extract was increased 14-fold with ammonium sulphate precipitation. Maximum enzyme activity was seen at pH 4.0 and 50 °C when carboxymethylcellulose was used as a substrate. K_0.5 and V_max values of the partially purified endoglucanase were 7.7 mg/ml and 25 ± 0.9 U/mg protein, respectively. The enzyme was quite stable over a broad range of pH (2.0–9.0) at 4 °C. When it was incubated at temperatures between 20 °C and 60 °C for 12 h, it conserved much of its original activity (over 40%). The activity of the enzyme increased by 234 ± 3.6% in the presence of 1 mM Mn²⁺. The endoglucanase was inhibited by EDTA, PMSF, β-ME and DDT. In conclusion, pH and thermal stability of the L. flaccida endoglucanase could make it useful for industrial purposes.     

Antioxidant, antiacetylcholinesterase and antimicrobial activities of Cymbopogon schoenanthus L. Spreng (lemon grass) from Tunisia

Aqueous extract, proanthocyanidin rich extract, and organic extracts of Cymbopogon schoenanthus L. Spreng (lemon grass) shoots from three different locations in South Tunisia were screened for their antioxidant, acetylcholinesterase and antimicrobial activities. In addition to the evaluation of these activities, the contents of flavonoids and total phenolic compounds were determined.Antioxidant activity measured by DPPH assay showed that the proanthocyanidin extract exhibited higher antioxidant activity than the aqueous extract. Extract concentration providing 50% inhibition (IC₅₀) ranged from 16.4 ± 6.8 μg/mL to 26.4 ± 6.8 μg/mL. The antioxidant activity was also determined using the β-carotene/linoleic acid bleaching test. The best results (IC₅₀ = 0.11 ± 0.10 mg/mL) were obtained with the proanthocyanidin extract of the plants collected from the desert region (Dhibat).The greatest acetylcholinesterase inhibitory activity (IC₅₀ = 0.23 ± 0.04 mg/mL) was exhibited by the ethyl acetate and methanol extracts of the plants collected from the mountainous region. It seems that extracts obtained with more polar solvents gave better results.The proanthocyanidin extracts showed a good antimicrobial activity against Streptococcus sobrinus at low concentration (MIC = 4 mg/mL). Therefore, these extracts could be used to prevent carious lesions by inhibiting S. sobrinus growth.     

Effect of various inhibitors on enzymatic browning,antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa)

In this study, polyphenol oxidase (PPO) was isolated from fresh lettuce. Its optimum temperature and pH were found to be 40 °C and 7.0, respectively. Lettuce PPO was shown to use catechin, catechol, chlorogenic acid, caffeic acid and gallic acid as substrates. Among the substrates used, the greatest substrate specificity was observed with chlorogenic acid. Lettuce PPO was sensitive to some inhibitors. Ascorbic acid, cysteine, oxalic acid and citric acid were tested as potential inhibitors of lettuce PPO. Cysteine was the most effective inhibitor. Total phenol and total antioxidant activity contents were also determined in the presence of these inhibitors at room and refrigerator temperatures. Ascorbic acid and cysteine increased the total antioxidant activity of lettuce while citric and oxalic acids had no effect on the total antioxidant activity. Lettuce phenolics were protected from oxidation by ascorbic acid and cysteine.     

Antioxidant potentials and rosmarinic acid levels of the methanolic extracts of Salvia verticillata (L.) subsp. verticillata and S. verticillata (L.) subsp. amasiaca (Freyn & Bornm.) Bornm

This study was designed to examine the in vitro antioxidant activities and rosmarinic acid levels of the methanol extracts of Salvia verticillata subsp. verticillata and S. verticillata subsp. amasiaca. The extracts were screened for their possible antioxidant activity by two complementary test systems, namely DPPH free radical-scavenging and β-carotene/linoleic acid systems. In the first case, S. verticillata subsp. verticillata was superior to the subsp. amasiaca with an IC₅₀ value of 14.5 ± 1.21 μg mg⁻¹. In the β-carotene/linoleic acid test system, inhibition capacity of S. verticillata subsp. verticillata was 74.4 ± 1.29%. Antioxidant activities of BHT, ascorbic acid, curcumin and α-tocopherol were determined in parallel experiments. Activity of rosmarinic acid was also screened for better establishing the relationship between rosmarinic acid level and antioxidant activity for the plant extracts. S. verticillata subsp. verticillata had the highest rosmarinic acid level with a value of 28.7 ± 0.89 μg mg⁻¹. There is a strong correlation between the rosmarinic acid level and antioxidant activity potential. Our results showed that rosmarinic acid and its derivatives are more likely to be responsible for most of the observed antioxidant activities of Salvia species.     

Giant Amazonian fish pirarucu (Arapaima gigas): Its viscera as a source of thermostable trypsin

A trypsin was purified from pyloric caeca of pirarucu (Arapaima gigas). The effect of metal ions and protease inhibitors on its activity and its physicochemical and kinetic properties, as well its N-terminal sequence, were determined. A single band (28.0 kDa) was observed by SDS–PAGE. Optimum pH and temperature were 9.0 and 65 °C, respectively. The enzyme was stable after incubation for 30 min in a wide pH range (6.0–11.5) and at 55 °C. The kinetic parameters K_m, k_cat and k_cat/K_m were 0.47 ± 0.042 mM, 1.33 s⁻¹ and 2.82 s⁻¹ mM⁻¹, respectively, using BApNA as substrate. This activity was shown to be very sensitive to some metal ions, such as Fe²⁺, Hg²⁺, Zn²⁺, Al³⁺, Pb²⁺, and was highly inhibited by trypsin inhibitors. The trypsin N-terminal sequence IVGGYECPRNSVPYQ was found. The features of this alkaline peptidase suggest that it may have potential for industrial applications (e.g. food and detergent industries).     

Evaluation of ability of ferulic acid to control growth and fumonisin production of Fusarium verticillioides and Fusarium proliferatum on maize based media

The aim of this study was to evaluate the efficacy of ferulic acid (1, 10, 20 and 25 mM) at different water activity (a_w) values (0.99, 0.98, 0.96 and 0.93) at 25 °C on growth and fumonisin production by Fusarium verticillioides and Fusarium proliferatum on maize based media. For both Fusarium species, the lag phase significantly decreased (p ≤ 0.001), and the growth rates increased (p ≤ 0.001) at the lowest ferulic acid concentration used (1 mM), regardless of the a_w. However, high doses of ferulic acid (10 to 25 mM) significantly reduced (p ≤ 0.001) the growth rate of both Fusarium species, regardless of the a_w. In general, growth rate inhibition increased as ferulic acid doses increased and as media a_w decreased. Fumonisin production profiles of both Fusarium species showed that low ferulic acid concentrations (1–10 mM) significantly increased (p ≤ 0.001) toxin production, regardless of the a_w. High doses of ferulic acid (20–25 mM) reduced fumonisin production, in comparison with the controls, by both Fusarium species but they were not statistically significant in most cases. The results show that the use of ferulic acid as a post-harvest strategy to reduce mycotoxin accumulation on maize needs to be discussed.