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.     

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.     

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.     

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     

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.     

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.     

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.     

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     

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     

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.     

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.     

The Inactivation Kinetics of Soluble and Membrane-Bound Polyphenol Oxidase in Pear during Thermal and High-Pressure Processing

Polyphenol oxidase (PPO) from pear was characterized with catechol as substrate. The Michaelis constant of soluble and membrane-bound PPO were 15.6 and 23.8 mM, respectively, and their optimum pH for activity were 6.0 and 6.5, respectively. The inactivation kinetics of soluble and membrane-bound PPO during thermal (45–75 °C) and high-pressure thermal processing (600 MPa, 40–80 °C) were studied. The inactivation kinetics of pear PPO were described by a first-order model at all processing conditions. Compared to soluble PPO, membrane-bound PPO was more sensitive to thermal and high-pressure inactivation. Both soluble and membrane-bound PPO displayed higher sensitivity towards thermal inactivation at pH 3.5 (pH of pear puree made from pears dipped in citric acid prior to blending) compared to pH 4.4 (pH of non-acidified pear puree). High pressure and temperature had synergistic inactivation effects on pear PPO at pH 4.4 while slight antagonistic effects were observed at pH 3.5.     

The Inhibitory Effects of Brans and Their Aqueous Extracts on the Lipolysis of Tributyrin Catalyzed by Calf Pregastric Lipase

The ability of red and white wheat brans and oat bran to inhibit calf pregastric lipase (CPGL)‐catalyzed hydrolysis of tributyrin has been studied in vitro at pH 6.5, 37 °C. Brans that had been presoaked in Bis‐Tris buffer at pH 6.5, 4 °C reduced CPGL activity by 13% to 20% after being soaked for 0.25 h and by 17% to 30% after being soaked for 24 h. Maximum reduction of CPGL activity was seen within 2 to 5 min incubation between coarse red wheat bran and CPGL. The results are discussed in terms of likely inhibitory components within the brans and their water‐soluble components and their roles in inhibiting pregastric and other lipase‐catalyzed hydrolysis of dietary lipids.     

Characterization of Sultaniye grape (Vitis vinifera L. cv. Sultana) polyphenol oxidase

Polyphenol oxidase (PPO) was extracted from Sultaniye grapes grown in Turkey, and its characteristics in terms of pH and temperature optima, thermal inactivation, kinetic parameters and potency of some PPO inhibitors were studied. Optimum pH and temperature for grape PPO were found to be 3.4 and 30 °C, using catechol as substrate. K_m and V_max values were found to be 44.5 ± 5.47 mm and 0.695 ± 0.0353 OD₄₁₀ min^?1, respectively. Four inhibitors were tested in this study and the most potent inhibitor was sodium metabisulphite, followed by ascorbic acid. From the thermal inactivation studies in the range of 65–80 °C, the half‐life values of the enzyme ranged between 2.6 and 49.5 min. Activation energy (E_a) and Z values were calculated to be 208.5 kJ mol^?1 (r² = 0.9544) and 10.95 °C (r² = 0.9517), respectively.     

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, ion‐exchange chromatography on Q‐Sepharose (Fast flow), gel filtration chromatography on Sephadex G‐100 and hydrophobic interaction chromatography on Phenyl Sepharose CL‐4B. The enzyme was purified 5‐fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg^?1 protein. SDS‐PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag⁺, Ca²⁺, Co²⁺, Fe²⁺, Mg²⁺, iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu²⁺, Sr²⁺, phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn²⁺ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K_m of 18 mg·mL^?1 and a V_max of 11.1 μmol · mL^?1 · min^?1 with casein as substrate.     

Effect of Soluble Dietary Fibers on Lipase-catalyzed Hydrolysis of Tributyrin

The ability of soluble fibers to inhibit calf pregastric lipase (CPGL)‐catalyzed hydrolysis of tributyrin (TBG) has been measured at pH 6.5, 37 °C (CPGL 0.02 mg/mL). Fibers tested were pectin, carboxymethylcellulose (CMC), carrageenan, and gum arabic. The fibers (5 g/L), solubilized in Bis‐Tris buffer (50 mM, pH 6.5) at 4 °C for either 15 min or 24 h, were added to an emulsion containing TBG, before initiating the reaction by addition of CPGL. An immediate inhibitory effect of 8% to 19% was observed. The value of the CPGL–pectin dissociation constant, K_i, was 0.76 to 2.34 mM. Dialysis of pectin solution reduced the inhibitory effect by 14% and the concentration of galacturonic acid by 15%.