Characterisation of ‘Starking’ apple polyphenoloxidase

Experiments were performed to optimise the extraction conditions of ‘Starking’ apple fruit polyphenoloxidase (PPO), to evaluate the affinity and specificity towards several substrates and to study the stability of enzyme extracts from apple samples stored under different conditions. Sodium phosphate buffer (0·2 M , pH 6·5) plus 0·25% Triton X₁₀₀ and 1% or 2% PVPP was found to be the most efficient extraction medium. Chlorogenic acid, dopamine and 4-methylcatechol showed similar specificity towards PPO, and chlorogenic acid was found to be the best substrate for the enzyme. Enzyme extracts from frozen cut apple stored at −4°C, and extracts from lyophilised apple samples stored at 4°C were more stable than extracts obtained from fresh-cut or acetone powder samples. © 1998 SCI.     

Purification and properties of polyphenol oxidase in head lettuce (Lactuca sativa)

Polyphenol oxidase (EC 1.10.3.1) in head lettuce (Lactuca sativa L) was purified by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The enzyme was found to be homogeneous by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 56 000 amu by Sephadex G-100 gel filtration. The purified enzyme quickly oxidised chlorogenic acid (5-caffeoyl quinic acid) and (—)-epicatechin. The K^m values for the enzyme, using chlorogenic acid (pH 4·5, 30°C) and (—)-epicatechin (pH 7·0, 30°C) as substrate, were 0·67 mM and 0·91 mM, respectively. The optimal pH of chlorogenic acid oxidase and (—)-epicatechin oxidase activities were 4·5 and 7·8, respectively, and both activities were stable in the pH range 6–8 at 5°C for 20 h. Potassium cyanide and sodium diethyldithiocarbamate markedly inhibited both activities of the purified enzyme. The inhibitory effect of metallic ions such as Ca²⁺, Mn²⁺, Co²⁺ and Ni²⁺ for chlorogenic acid oxidase activity was stronger than that for (—)-epicatechin oxidase activity.     

Convenient partial purification of polyphenol oxidase from apple skin by cationic reversed micellar extraction

Polyphenol oxidase (PPO) was selectively extracted from reconstituted freeze-dried apple skin using reverse micelles formed by a cationic surfactant, dodecyl trimethyl ammonium bromide (DTAB). An optimum forward extraction was achieved with sodium phosphate buffer (pH 6, 100 mM, no added KCl) and an organic phase (isooctane:hexanol at a ratio of 5:1) containing 100 mM DTAB. The solubilised PPO was efficiently recovered by a stripping solution (pH 6, 1 M KCl) containing 10% ethanol. Under the optimised conditions, the purification fold and recovered activity of PPO were 12.6% and 71%, respectively. This purification fold and recovery were maintained when the extraction volume increased from 10–200 ml. Overall, reversed micellar extraction can be used as an efficient first step for the purification of PPO from apple skin.     

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.     

Polyphenoloxidase activity from strawberry fruit (Fragaria ananassa,Duch., cv Selva): characterisation and partial purification

In this work, polyphenoloxidase (PPO) from Selva strawberry fruit (Fragaria × ananassa, Duch) was extracted, characterised and partially purified. The activity of PPO was analysed in crude extracts obtained from either fresh fruits or acetone powder. The presence of NaCl and Triton X‐100 in the extraction buffer caused a marked increase in enzyme extractability. The enzyme showed an apparent K_m value of 11.2 mM with pyrocatechol as substrate. The maximum enzyme activity was observed at 50 °C and pH 5.3–6.0 without SDS and pH 7.2 in the presence of SDS. The presence of SDS increased PPO activity at pH 7.2 but diminished it at pH 6.0. The enzyme showed high thermal stability and maintained activities equal to or greater than 50% of its maximum activity in the 2.6–9.3 pH range. One polyphenoloxidase isoenzyme was detected in crude extracts of all ripening stages, showing an isoelectric point of 7.3. The specific activity of PPO decreased continuously through fruit ripening. Maximum specific activities were found at the ‘small green’ and ‘large green’ ripening stages. A total enzyme extract was partially purified by means of (NH₄)₂SO₄ precipitation and cationic exchange chromatography in an FPLC system. The purification grade achieved was near 25. The partially purified enzyme showed an isoelectric point equal to 7.3 and a molecular mass of 135 ± 4 kDa for the native protein. © 2000 Society of Chemical Industry     

Polyphenoloxidases from Williams Pear (Pyrus communis L,cv Williams): Activation,Purification and Some Properties

Activation of a crude polyphenoloxidase (PPO) preparation extracted from Williams pears was investigated. Comparison between several activation agents led to the hypothesis of a limited conformational change involved in the activation process. Using ammonium sulphate precipitation followed by hydrophobic and ion exchange chromatography, two PPO fractions, F1 and F2, were 124- and 36-fold purified. F1 contained two forms characterised by isoelectric point equal to 4·2 and 4·5 while F2 contained two other forms associated with isoelectric point of 3·8 and 4·0. The molecular mass determined by gel filtration and confirmed after SDS-page was unique (c 43 kDa). F1 and F2 showed similar apparent Km values, in the 5–11 mM range for the three main phenolic substrates in pear cortex. Chlorogenic acid appeared to be a better substrate than catechins for pear PPO. © 1997 SCI.     

Mechanism of Browning in Fresh Highbush Blueberry Fruit (Vaccinium corymbosum L). Role of Blueberry Polyphenol Oxidase,Chlorogenic Acid and Anthocyanins

Browning reactions which occurred after crushing fresh blueberries (Vaccinium corymbosum L) were followed by determination of chlorogenic acid colour density, polymer colour and the level of polymers. A polyphenol oxidase (PPO) activity plays a dominant role in enzymatic browning. Peroxidase (POD) does not contribute to brown colour formation. The loss of 29% of the colour in a model system containing PPO and blueberry anthocyanins indicated that PPO could act directly on these pigments. However, the rate of anthocyanin degradation was stimulated by the addition of chlorogenic acid. © 1997 SCI.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-β1,3-GLUCANASE FROM GREEN MALT

An endo-β1,3-glucanase from a green malt extract was purified by DEAE- and CM-cellulose ion exchange chromatography followed by molecular sieve chromatography on BioGel P-100. A final enzyme preparation had two protein components on disc electrophoresis, one of which was in-active. The enzyme had a pH optimum of 5·0 for activity on laminarin and 5·8 on carboxymethyl pachyman. The activity was stable up to 60°C and was stimulated by NaCl. The isoelectric point of the enzyme was 9·8.     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

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.     

Ultrathin-layer isoelectric focusing of partially purified peroxidase from tomato fruit

Partially purified peroxidase from tomato fruits was obtained by gel filtration on Ultrogel AcA 34 of a pH 5·5 extract prepared by homogenising the fruits with 0·4m McIlvaine buffer containing 0·2% Triton-X 100, subsequent centrifugation at 2000 × g and ultrafiltration of the supernatant. This fraction, with a molecular weight of 43 000 daltons, showed an optimum activity of pH 5·3 with guaiacol as the hydrogen donor, a K_m for H₂O₂ of 0·8 mm at optimum guaiacol concentration, a K_m of 9·0 mm for guaiacol at optimum H₂O₂ concentration and competitive cyanide inhibition with a K_i of 1·0 μm. Ultrathin-layer isoelectric focusing in 50 μm polyacrylamide gels resulted in the separation of eight peroxidase isozyme bands, detected mainly in the range pH 2 to 4. Parallel experiments carried out with horseradish peroxidase (Boehringer, Mannheim) exhibited the main enzyme activities in the range pH 7 to 9.     

Heat inactivation and kinetics of polyphenoloxidase from palmito (Euterpe edulis)

Polyphenoloxidase (PPO, EC 1.14.18.1)was extracted from palmito (Euterpe edulis Mart) using 0.1 M phosphate buffer, pH 7.5. Partial purification of the enzyme was achieved by a combination of (NH₄)₂SO₄precipitation (35–90% saturation) and Sephadex G-25 and DEAE-cellulose chromatography. The purified preparation gave five protein bands on polyacrylamide gel electrophoresis, three of them with PPO activity. The K_mvalues for chlorogenic acid, caffeic acid, catechol, 4-methylcatechol and catechin were 0.57, 0.59, 1.1, 2.0 and 6.25 mM , respectively. PPO has a molecular weight of 51 000 Da, maximum activity at pH 5.6 with chlorogenic acid as substrate, and was stable between pH 5.0 and 8.0. The enzyme was heat stable at 50–60°C and inactivated at 75°C. The heat stability of palmito PPO was found to be pH dependent; at 50°C and pH 4.0 the enzyme was fully inactivated after 30 min. The pH/activity studies showed two groups with pK values c 4.6 and 6.7 involved in PPO catalysis.     

Effect of buffered acidulant systems on the survival of some food poisoning bacteria in medium acid media

The use of buffered food acidulant systems in medium acid media with pH higher than the minimum pH for growth of pathogenic bacteria in unbuffered acidulant systems has been studied. The buffered aqueous phase of the media contained 1% acetic acid, 1% other acidulant (lactic acid, malic acid, tartaric acid or citric acid), 0·05% sorbic acid, 0·05% benzoic acid and 3·5% salt, and was buffered to pH 4·8 with sodium hydroxyde. The buffered food acidulant systems showed a bactericidal activity on Salmonella blockley, Escherichia coli and Staphylococcus aureus and a bacteriostatic activity on Streptococcus faecalis.     

Partial purification and characterisation of banana [Musa (AAA Group) 'Gros Michel'] polyphenol oxidase

Polyphenol oxidase (PPO) from pulp of banana [Musa (AAA Group) 'Gros Michel'] was extracted and precipitated with 80% saturated ammonium sulphate followed by conventional column chromatography on Sephacryl S-200 HR and fast protein liquid chromatography on Mono Q column. The lyophilised PPO obtained from Sephacryl S-200 HR column was used for characterisation and inhibition studies. The partially purified PPO obtained from the Mono Q column exhibited at least three isoenzymes. The banana PPO had optimum pH for activity at 7 and it was stable around the same pH. Only 48% of initial enzyme activity was lost after heating at 70 °C for 30 min. The enzyme was completely inhibited by 2 m m sodium metabisulphite, 2 m m l -cysteine, 4 m m ascorbic acid, and 100 m m 4-hexylresorcinol. The K _m and V _max of banana PPO for dopamine were 2.08 m m and 0.124 m m  min⁻¹ respectively.     

Formation of mutagens in an amino acid-glucose model system and the effect of creatine

Heating of amino acids and glucose in a water solvent system produces substantial amounts of mutagens only at alkaline pH (10·5). Positive response was demonstrated with the Salmonella tester strains TA 98, TA 1538, TA 97, TA 1537 and TA 100. When creatine was added to this system prior to heating, mutagens were formed at all pHs tested (pH 4, 7, 9 and 10·5) and highest mutagenic activity was recorded at pH 9·0 and pH 7·0. The amount of mutagen produced was dependent on the creatine concentrations. A linear increase of mutagenic activity was recorded in the threonine-glucose system when creatine concentration increased to 0·5m. Similar results were also demonstrated in the glycine-glucose system up to 0·2-0·3m creatine. The increased amount of mutagenic activity found in the presence of creatine was demonstrated to be active only against the frame shift tester strains (TA 98 and TA 97) and not when tested with the TA 100 strain.HPLC analyses indicated that the mutagenic activity found in the pure amino acid glucose system at alkaline pH was also found in the presence of creatine; however, the major peak of mutagenic activity in the creatine containing system was not found in the absence of creatine.     

Purification and Some Properties of Glutaminase fromActinomucor taiwanensis,Starter of Sufu

Glutaminase of Actinomucor taiwanensis was purified approximately 96-fold with a yield of 18%, by sequential fractionation with ammonium sul-phate, anion exchange with DEAE-Sepharose CL-6B and gel filtration with Sephacryl S-200. The pH and temperature optima of purified glutaminase were 8·0 and 45°C, respectively. Glutaminase was stable at a temperature up to 35°C and at pH values of 6·0–8·0. The molecular weight was 80000 as determined from SDS-PAGE. The enzyme activity was markedly inhibited by HgCl₂. In the presence of 100 g litre⁻¹ NaCl, the enzyme activity was inhibited 50%.     

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.     

茶叶多酚氧化酶三相分离纯化及酶学性质研究

为研究一种快速高效的茶叶多酚氧化酶(polyphenol oxidase,PPO)制备方法,本试验采用三相分离法(three phase partitioning,TPP)获取茶叶PPO,并研究其酶学特性.结果表明,通过两次三相分离纯化后可获得酶比活力210.46 U/mg、纯化15.76倍、回收率8.04％的茶叶PP...     

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.     

Purification and properties of three albumins from Triticum aestivum seeds

Three albumins were isolated from bread wheat seeds by gel filtration on Sephadex G-100 and differential preparative disc-electrophoresis on polyacrylamide gel. The proteins were obtained in sufficient yield and purity to carry out characterisation studies. The electrophoretic mobilities of the three albumins in polyacrylamide discs in glycine—Tris medium, pH 9·5, were 0·28, 0·34 and 0·39 (referred to that of bromophenol blue taken as 1). The isoelectric points were 6·40, 6·40 and 5·35 and the molecular weights 17, 700, 18, 200 and 18, 900, respectively. The amino acid compositions of the purified albumins were very similar. The correlation of the purified albumins with those isolated from bread wheats by other authors is discussed.