Some properties of polyphenol oxidase from lily

A study of crude polyphenol oxidase (PPO) from lily bulbs was carried out to provide information useful for guiding food processing operations. Optimum pH for the enzyme activity in the presence of catechol, were 4.0 and 7.0 at room temperature(approximately 20 °C) and the enzyme was stable in the pH range from 5.0 to 6.5 at 4 °C for 10 h. Its optimum temperature was 40 °C and the heat inactivation of the enzyme followed first‐order kinetics. Lily PPO possessed a diphenolase activity toward catechol, catechin and gallic acid; catechin was the best substrate for the enzyme considering the V_max/K_m ratio. The most effective enzyme inhibitor was sodium sulphite, although ascorbic acid, l ‐cysteine and thiourea were also effective inhibitors at high concentration. But NaCl and citric acid were poor inhibitors of the enzyme. Data generated by this study might help to better prevent lily bulbs browning.     

CHARACTERIZATION OF POLYPHENOL OXIDASE FROM ROOSTER POTATO (SOLANUM TUBEROSUM CV ROOSTER)

The isolation and purification of polyphenol oxidase from potatoes ( Solanum tuberosum cv. Rooster) is described. A 64-fold purified preparation has been obtained with 10% yield by a procedure involving (NH₄)₂SO₄ precipitation, phenyl sepharose chromatography, ion exchange chromatography and hydroxyapatite chromatography. The partially purified enzyme has both cresolase and catecholase activity. Activity was lower toward monophenols than diphenols. Enzyme activity was optimal at pH 6.0–6.5 and at 30C. Greater than 50% activity was retained during storage for 72 h at pH 6.0–7.5. Residual activity was greater than 50% after incubation at 20C for 72 h, 30C for 48 h, 40C for 24 h, 50C for 2 h and 60C for 15 min. The most effective inhibitors tested were sodium metabisulfite and ascorbic acid. Sodium dodecyl sulphate appeared to activate the enzyme. The enzyme was capable of cross-linking casein but did not increase gel-strengths in acidified milk gels.

PRACTICAL APPLICATIONS

Rooster is the most important potato cultivar grown in Ireland and data on its isolation and characterization has not been reported previously. This work describes a method to isolate polyphenol oxidase and characterization of the enzyme. Information on characterization of the enzyme could be valuable in relation to control of enzymatic browning during current processing and in minimum processing. There is potential for use of the enzyme in the emerging cross-linking area, as the results show some success and there may be potential of more cross-linking as the field develops and as interest in natural methods of cross-linking for food texture grows. This could lead to an important use for potato waste. Food product applications are given.     

Purification and some properties of polyphenol oxidase of longan fruit

Polyphenol oxidase (PPO) was isolated from longan (Dimocarpus longan Lour.) fruit peel, with a 46-fold purification of PPO by ammonium sulfate, Sephadex G-200 and Phenyl Sepharose being achieved. Pyrogallol, 4-methylcatechol, and catechol were good substrates for the enzyme, and activity with chlorogenic acid, p-cresol, resorcinol, or tyrosine was not observed. The optimal pH for PPO activity was 6.5 with 4-methylcatechol. The enzyme had a remarkably temperature optimum (35°C) and was relatively stable, requiring a little more than 20 min at 50°C for 50% loss of activity. Reduced glutathione, l-cysteine, thiourea, FeSO₄ and SnCl₂ markedly inhibited PPO activity, whereas MnSO₄ and CaCl₂ enhanced PPO activity. Data obtained in this study might help to better understand longan fruit peel browning.     

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.     

Purification and enzymatic characteristics of a novel polyphenol oxidase from lotus seed (Nelumbo nucifera Gaertn.)

A polyphenol oxidase (PPO) from lotus seed was purified by the procedures including ammonium sulphate precipitation and affinity chromatography. The apparent molecular mass was 38.6 kDa by SDS‐PAGE. Kinetic studies showed that the K_m and V_max values for catechol were 6.04 mm and 416.67 U, respectively. The PPO performed optimal activity in 20 °C and pH 7.0. The enzymatic activity could be mainly maintained up to 50 °C and pH 4.0–7.0. The activity could be inhibited by various inhibitors including thiourea, urea, sodium hydrogen sulphite, EDTA·2Na, SDS, citric acid, guanidine hydrochloride, ascorbic acid, sodium sulphite and sodium thiosulphate. The metal ions Ba²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺ and Zn²⁺ could inhibit the activity of PPO, while Cu²⁺ performed obvious enhancement. The enzymatic properties of PPO could probably provide practical application in inhibiting the PPO activity and preventing enzymatic browning in the process of picking, transportation, processing and storage of fresh lotus seeds.     

Characterization of polyphenol oxidase from Uapaca kirkiana fruit

Polyphenol oxidase (PPO), the enzyme responsible for the postharvest spoilage of fruits, was extracted and purified from Uapaca kirkiana peel and pulp by ammonium sulfate precipitation and dialysis. Further purification of peel PPO was carried out by gel filtration chromatography. Optimum pH values were 7 and 8 for peel and pulp PPO, respectively. The optimum temperatures for peel and pulp PPO were 45 and 35 °C, respectively. Inhibition studies of the PPO enzyme were performed using citric acid, sodium azide, sodium metabisulfite and thiourea. The most effective inhibitors were sodium azide and citric acid for both peel and pulp PPO. V_max and K_m values were 13.63 units min^?1 and 4.923 mmol L^?1, respectively, for peel PPO and 14.03 units min^?1 and 5.43 mmol L^?1, respectively, for pulp PPO. Three isoenzymes of Uapaca kirkiana PPO were detected by polyacrylamide gel electrophoresis. One of the isoenzymes could be identified as having a molecular weight of 26 625 Da. Copyright © 2005 Society of Chemical Industry     

Lipoxygenase in Sweet Corn Germ: Isolation and Physicochemical Properties

Off-flavor and off-aroma development, which may be catalyzed by lipoxygenase (LPO), are common in frozen stored sweet corn. Lipoxygenase activity in the germ fraction of sweet com (Zea nruys L. cv. Jubilee) was determined and compared with that in the degermed fraction. Lipoxygenase activity/g germ was about three times greater than that of the degermed fraction, Optimized procedures for isolation of lipoxygenase from the germ fraction were developed. Lipoxygenase was isolated by preparation as an acetone powder, extraction with 0.2M TrisHCI, pH 8.0 (4°C), fractionation with 40–60% saturated ammonium sulfate and dialysis. Optimum pH was 6–7 and temperature 50°C for activity of partially purified lipoxygenase. The enzyme appeared stable at pH 5–8 and ～90% of original activity was inactivated after heating in pH 7 buffer at 70°C for 3 min.     

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.     

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.     

Polyphenoloxidase from Amasya Apple

Polyphenoloxidase (PPO) of Amasya apple was partially purified by (NH₄)₂SO₄ precipitation and dialysis. The sample was used for characterization of the PPO. Optimum pH were 7.0, 9.0, 8.6 and 6.6 on substrates catechol, 4-methyl catechol, pyrogallol and L-dopa respectively. Catechol was the most suitable for Amasya apple PPO. The optimum temperature for maximum PPO activity was 18°C with catechol. Of seven inhibitors tested, the strongest was L-cysteine. Effectiveness of inhibitors increased in the order: thiourea, glutathione, β-mercaptoethanol, sodium cyanide, ascorbic acid, sodium metabisulfide, and L-cysteine. The K_M was 34 mM of catechol. The activation energy with catechol was 107 cal/mol. In electrophoretic separation, three isoenzymes were detected with both catechol and L-dopa substrates.     

Polyphenol Oxidase from Bean Sprouts (Glycine max L.)

ABSTRACT: Polyphenol oxidase (PPO) was purified and characterized from bean sprouts by ammonium sulfate precipitation, DEAE‐Toyopearl 650M, CM‐Toyopearl 650M, SuperQ‐Toyopearl 650S and QAE‐Toyopearl 550C column chromatographies. Substrate staining of the crude extract on electrophoresis showed the presence of 2 isozymic forms of this enzyme. The molecular weight of the purified enzyme was estimated to be about 54 kDa. The optimum pH was 9.0 and optimum temperature 40 °C. Heat inactivation occurred about 30 °C. PPO showed activity to catechol, pyrogallol and dopamine. These compounds such as ascorbic acid, L‐cysteine, 2‐mercaptoethanol, and glutathione used was the effective inhibitor. Enzyme activity was maintained for 7 d at 4 °C but suddenly decreased after 8 d.     

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     

Pectin lyase and polygalacturonase of Aspergillus niger pathogenic for yam tubers (Diascorea rotundata)

Polygalacturonase and pectin lyase of Aspergillus niger partially purified by ethanol, ammonium sulphate precipitation, DEAE-cellulose and Sephadex G-150 column chromatography were characterized. Polygalacturonase gave optimum activity at pH 4–5, and at 35°C. It was stable at pH 3–7 and at 20–50°C. The molecular weight was 38020. For pectin lyase optimum activity occurred at pH 5 and 45°C. The enzyme was stable at pH 3–4 and at 40–50°C. The molecular weight was 30 900. Yam tissue was optimally macerated at pH 4–5 by the enzymes. At pH 4.5, potassium sorbate (0.6 mg/ml), benzoic acid (0.8 mg/ml) and sodium benzoate (1.0 mg/ml) caused complete inhibition of polygalacturonase activity. With pectin lyase, this effect was achieved with potassium sorbate and benzoic acid each at 0.9 mg/ml, but not with sodium benzoate.     

Secretagogue and bacteriostatic active fractions derived from a peptic hydro‐ lysate of alfalfa RuBisCO small purified subunit

For the first time a purification process for small RuBisCO (ribulose‐1,5‐bisphosphate carboxylase/oxygenase) subunit (SRS) was developed from an industrial by‐product of alfalfa, taking advantage of its solubility at low pH. Only one protein strip (14 kDa) was clearly detected in the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) profile of the supernatant at pH 2. The recovery of SRS was 48% by this method, with a purity estimated as 98% by densitometry and reverse phase high‐performance liquid chromatography (RP‐HPLC). Moreover, most polyphenolic compounds were discarded, as confirmed by spectrophotometry and RP‐HPLC. SRS hydrolysis was performed for 20 h at 37 °C using pepsin in ammonia/formic acid buffer at pH 3. The hydrolysate was fractionated on a Sephadex G25 column equilibrated with ethanolamine/HCl buffer. Biological activities were found in two fractions. The first fraction showed slight bacteriostatic properties against two pathogenic bacteria, Salmonella arizonae and Shigella sonnei. The second fraction, tested by radioimmunoassay (RIA), presented a secretagogue activity comparable to that of gastrin. Copyright © 2006 Society of Chemical Industry     

Enzymatic Production of Ribonucleotides from Autolysates of Kluyveromyces marxianus Grown on Whey

After 20h fermentation of medium containing 5% (w/v) dehydrated whey, at 30°C, pH 4.5, yeast cells were harvested, diluted in 0.1M KH₂PO₄, and autolyzed at different pHs (6.5–7.5) and temperatures (45–55°C). Phosphodiesterase (0.2–1.0% w/v, 65°C, pH 6.5, 6h) and adenyl deaminase (0.5-1.0% w/v, 60°C, pH 5.5, 4h) were added to the autolysates. After heat treatment (100°C, 15 min), samples were analyzed by RP-HPLC and LC/MS. Production of 5′-ribonucleotides was maximized at 50°C, pH 6.5. Yields of 5′-AMP (800 μg/g of biomass) and 5′-GMP (2000 μg/g) increased considerably after addition of 1.0% phosphodiesterase. 5′-IMP increased only after addition of 1.0% adenyl deaminase.     

Application of chemical and physical agents in model systems to controlling phenoloxidase enzymes

Several chemical and physical anti-browning agents are studied in different model systems in which caffeic acid (as substrate) and laccase from Trametes versicolor (LAC) and polyphenoloxidase from sunflower seeds (PPO) (as enzymes) are used to emulate the browning reaction. Temperature and low electric current were the tested physical agents, while acetic acid, sodium acetate, sodium chloride and, finally, sodium bisulfite were the chemical agents. Sunflower PPO was observed to be less heat sensitive than LAC that was fully inactivated after 1 and 3 min of exposure to 100 and 80 °C, respectively. Conversely, PPO required more than 3 min at 100 °C to be fully inactivated, and it still showed a significant activity (ca. 17%) after an exposure to 80 °C for 15 min. Both LAC and PPO were found to be active at frozen (−18 °C) and cool (+4 °C) temperature, and their activities were strengthened at 40 and 60 °C. As concerning chemical agents, inhibitory power of acetic acid on LAC was observed to be very weak. In the sodium acetate solution at the concentrations of 0.5, 1.0, 2.0 and 4.0%, LAC residual activity was equal to 81.5, 63.9, 61.1 and 35.2%, respectively. PPO is shown to be more sensitive to the NaCl than LAC and indifferent to the presence of NaHSO₃. A 28% residual activity of LAC was found in the solution with 200 mg L⁻¹ NaHSO₃. Finally, LAC activity was decreased to 72.3, 60.0, 16.7 and 8.4% after a low electric current (LEC) treatment of 30 s and 1, 3 and 6 min, respectively. Conversely, PPO activity was not affected under these conditions.     

Purification and properties of a new,commercial, thermostable Bacillus stearothermophilus α‐amylase

A rapid purification has been developed for Bacillus stearothermophilus α‐amylase starting with the commercial enzyme product. The two‐step procedure, using hydrophobic interaction chromatography and ion exchange, results in a 6.8‐fold increase in specific activity with an 86% recovery of starting activity. The molecular weight of the enzyme was 58,000 when measured by SDS‐PAGE The enzyme preparation consisted of three isozymes with pl values of 8.1, 8.0, and 7.8–7.7. The enzyme mixture had a broad pH optimum of pH 4.0–7.0 at 40° C and a narrower optimum of pH 5.2–6.5 at 90° C. The temperature optimum was 80° C when measured by the starch‐iodine assay; hydrolysis of maltodextrin indicated a constant, maximum rate between 70° C and 100° C. Calcium was shown to increase the half life of the enzyme at 90° C approximately 10‐fold; addition of 10% maltodextrin and calcium increased the half life of the enzyme 80‐fold.     

Extraction of Milk Clotting Enzyme from Sodom Apple (Calotropis procera)

A milk clotting enzyme with low proteolytic activity was extracted with ammonium sulfate, at 0.40-0.65 saturation, from sodom apple leaves. The enzyme with apparently a basic isoelectric point was activated by cysteine and was more active at 65°C than at 35°C. Milk clotting activity increased with pH at 65°C, with the enzyme being almost twice as active at pH 6.4 as at pH 5.4-5.7. Storage at 4°C for 15 days resulted in a 30-50% loss in enzyme activity.     

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     

Formation of acid‐precipitated soy protein–dextran conjugates by Maillard reaction in liquid systems

The conjugation reaction between soybean acid‐precipitated protein (SAPP) and dextran in liquid systems via the initial stage of the Maillard reaction was studied. Functional SAPP–dextran conjugates were prepared in 80% ethanol‐reacting system at 50 °C for 6 h, along with 95% ethanol‐reacting system at 60 °C for 24 h. The covalent attachment of dextran to SAPP was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and gel filtration chromatography.Compared to the classical dry‐heating, the reaction time of glycosylation in the two ethanol systems was largely shortened. Emulsifying activity of SAPP–dextran conjugates obtained by dry‐heating incubation and in ethanol was similar at pH 7.0 and10.0, significantly higher than that of SAPP–dextran mixture or SAPP alone. In addition, SAPP–dextran conjugates obtained in 80% ethanol‐reacting system for 6 h were completely soluble after heating at 90 °C for 20 min. The impact of various processing conditions on the formation of SAPP–dextran conjugates was investigated. This study provides important guidance to create protein–polysaccharide conjugates at mild temperatures in liquid systems.