CHARACTERIZATION AND INHIBITION OF POLYPHENOL OXIDASE FROM PEARS (PYRUS COMMUNIS L. CV. BOSC AND RED)

Red pears had higher PPO activity, total phenolics and chlorogenic acid concentration than Bosc pears. PPO activity and phenolics both decreased in fruits held at room temperature. pH and temperature optima for Bosc and Red pears PPOs were 5.0 and 5.5, and 20 and 23C, respectively. 4-Methylcatechol, catechol and dopamine were good substrates for PPO from both pear cultivars; however, no activity was observed with any of the mono-hydroxy substrates studied. Higher K_m and lower V_max values were observed for Bosc pear PPO. Heating at 75C for 30 min completely inactivated the enzyme from both cultivars. Heating at 55 and 65C for the same duration resulted in partial inactivation (45–60%) of this enzyme. Ascorbic acid, L-cysteine, sodium metabisulfite and thiourea effectively inhibited browning due to pear PPOs.     

ISOLATION AND SOME PROPERTIES OF AN ACIDIC FRACTION OF POLYPHENOL OXIDASE FROM JERUSALEM ARTICHOKE(HELIANTHUS TUBEROSUS L.)

An acidic fraction of polyphenol oxidase (EC 1.14.18.1), representing the bulk activity, has been isolated from Jerusalem artichoke tubers by copper affinity chromatography, followed by DEAE-Sepharose ion-exchange chromatography. Isoelectric focusing analysis showed a cluster of activity bands (microheterogeneity) in the pH region of 4.5. The amino acid composition of the enzyme was also determined. The pH optimum for oxidation of chlorogenic acid, 4-methylcatechol and catechol was 6.0. Substrate inhibition was observed by high concentrations of chlorogenic acid. Thermal inactivation data indicated an apparent activation energy of 26.2 kcal/mol. Kinetics of inactivation, upon copper removal by KCN treatment, and reconstitution reactions revealed that the former is a much slower process than reactivation.     

PURIFICATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE FROM POTATO: I. PURIFICATION AND PROPERTIES

Polyphenol oxidase (Isozyme I) from potato was extracted and purified with ammonium sulfate, cation-exchange (Bio-Rad Bio-Scale S2) and Sephadex G-100 column chromatography. The enzyme was purified 11.8 fold resulting in a specific activity of 250.3 units/mg. Optimum pH of the enzyme was 6.6. Optimum temperature of the enzyme was 40C and its half-life was 0.8 min at 70C. The K_mfor catechol, pyrogallol, 4-methyl catechol, caffeic acid and L-DOPA were 4.11 mM, 0.61 mM, 0.78 mM, 0.50 mM and 32 mM, respectively. However, monophenols such as tyrosine, p-cresol and 1-naphtol did not show any activity. Data for V_max/K_m which represents catalytic efficiency show that 4-methyl catechol has the highest value. The molecular weight of the active enzyme was 86,000 Da, composed of two identical subunits. The number of Cu²⁺ ions bound was found to be 2 per enzyme molecule.     

PURIFICATION AND PROPERTIES OF POLYPHENOL OXIDASE FROM MANGO PEEL (MANGIFERA INDICA. VAR. RASPURI)

Polyphenol oxidase from the peel of ripe mango fruit (Mangifera indica. Var. Raspuri) was purifed 126-fold to homogeneity by ammoniun sulphate fractionation, column chromatography on Blue Sepharose CL-6B and gel chromatography on Sephadex G-200. It had an iso-electric point (pI) of 4.1±0.2 and molecular weight of 137,000 daltons as determined by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and gel filtration. It was more specific for catechol (than for other substatres tested) with a Km of 8.2 mM. 2-mercaptoethanol, L-cysteine, sodium diethyldithiocarbamate and thiourea were effective inhibitors of this enzyme. It had pH and temperature optima of 5.5 and 46⁰C, respectively. It lost 50% activity by exposure to 85⁰, 75⁰ and 65⁰C for 3, 16 and 25.5 min, respectively. Copper content was one atom per mole of enzyme and copper was essential for its activity. Unlike most of the polyphenol oxidases, multiple forms were not detected in the crude extract.     

PARTIAL PURIFICATION OF POLYPHENOL OXIDASE FROM PLUMS (Prunus domestica L., cv. STANLEY)

Polyphenol oxidase (PPO) was extracted and purified from Stanley plums (Prunus domestica L.) Crude PPO showed pH optima of 5.8 to 6.4 with different substrates. Heating for 5 min at 75C completely inactivated this enzyme. Plum PPO was stable at -20C for 16 weeks. K_mof this enzyme ranged from 17.5 mM with 4-methylcatechol to 31.2 mM with chlorogenic acid. The enzyme was purified 36-fold through (NH₄)₂SO₄ fractionation and chromatography on DEAE-cellulose and Sephadex G-100. PAGE of crude and purified plum PPO showed 7 and 3 bands, respectively, when stained for activity with catechol. The molecular weight of 3 subunits of purified PPO was estimated in the range of 45–66 kD.     

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 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 CHARACTERIZATION OF JERUSALEM ARTICHOKE (HELIANTHUS TUBEROSUS L) POLYPHENOL OXIDASE

Polyphenol oxidase (EC 1.14.18.1) has been purified from Jerusalem artichoke tubers by immobilized copper affinity chromatography. The enzyme is primarily an o-dihydroxyphenol oxidase with apparent K_m values of 1.9, 3.5 and 3.9 mM for chlorogenic acid, 4-methylcatechol, and catechol, respectively. Several compounds exhibited inhibitory action for the enzyme in the order of: sodium metabisulfite > sodium diethyldithiocarbamate > 2,3-naphthalenediol > thioglycollate. Multiple forms were identified by gel filtration and SDS-gradient polyacrylamide gel electrophoresis: two aggregates with apparent MW of 120 and 86 K and two monomeric subnits of 40–42 and 32–34 K, respectively. Concentration dependent association-dissociation phenomena most likely determine the multimeric state of this enzyme. While the aggregated forms exhibited specificity towards mono-, di- and polyhydroxyphenols, the low MW subunits were found active only with o-dihydroxyphenols. The isoelectric points of the various enzyme species were within the range of 4.0 to 10.0. The enzyme was found to contain appreciable amounts of associated carbohydrate material.     

PURIFICATION AND CHARACTERIZATION OF THREE POLYPHENOL OXIDASE ISOZYMES FROM LOBSTER (HOMARUS AMERICANUS)

ABSTRACT Three isozymes of polyphenol oxidase (PPO I, PPO II and PPO III) were purified from lobster (Homarus americanus) by ion-exchange chromatography and preparative isoelectric focusing using a Rotofor cell. The purified isozymes migrated as single protein bands in polyacrylamide gels with R_f values corresponding to molecular weights of 32,180, 35,480 and 39,300, respectively. The pI values of the PPO isozymes were 3.89, 4.26 and 4.54, respectively. PPO I was most active at pH 6.5 and most stable from pH 6.0 to 7.0; PPO II was most active within the pH range 6.0 to 7.0, and most stable within the pH range 4.0 to 9.0; while PPO III was most active at pH 7.0 and most stable in the pH range 6.0 to 8.0. The temperature optimum for the PPO-dihydroxyphenylalanine oxidation reaction was 35C with PPO I and 45C with PPO II or III. Lobster PPO I lost about 30% of its initial activity after 30 min incubation at 45C, while PPO II and II retained virtually all their activity after the same heat treatment. The catalytic specificities of the PPO isozymes were relatively higher with dihydroxyphenylalanine as substrate than with chlorogenic acid.     

PURIFICATION AND KINETIC CHARACTERIZATION OF POLYPHENOL OXIDASE FROM TOMATO FRUITS (LYCOPERSICON ESCULENTUM CV. MUCHAMIEL)

Two different polyphenol oxidase (PPO) fractions, soluble and particulate, were purified from unripe tomato fruits (Lycopersicon esculentum M. cv. Muchamiel). The PPO present in the soluble fraction was purified fivefold with a 43.5% yield after ammonium sulfate fractionation. PPO in the particulate was purified 4.56‐fold with a 23% yield using the nonionic detergent Triton X‐114. A strong correlation between tomato fruit PPO activity and the physiological disorder blossom‐end rot (BER) was found, with a large increase of the PPO activity in the particulate fraction. Kinetic characterization, including kinetic parameters, pH and temperature profiles, substrate specificity and inhibitors showed similarities in both the soluble and the particulate enzyme(s). However, thermal stability of the particulate enzyme was significantly higher than stability of the soluble PPO, thus indicating possible structural differences. Cupric ions were activators, probably because of their ability to reactivate PPO partly denatured during purification.     

GELATINIZATION BEHAVIOR OF GRAINS AND FLOUR IN RELATION TO PHYSICO-CHEMICAL PROPERTIES OF MILLED RICE (ORYZA SATIVA L.)

ABSTRACT

Physico‐chemical properties of milled rice grains of 13 japonica varieties were evaluated. The rice varieties differed in appearance. One sample had long grains (6.61 mm), two had short grains (5.27–5.32 mm) and the rest had grains of medium length (5.52–6.55 mm). The shape (length/width) of grains varied from bold (1.58–1.9) to medium (2.06–2.37). The protein content was high in all samples (7.82 to 9.72% on dry mass). Almost all, with exception of one cultivar (Osogovka), which had intermediate amylose content (23.3%), were low amylose varieties (12–19.4% amylose). Their minimum cooking time varied from 17.5 to 22.5 min. Significant correlations (P < 0.05) were established between some physico‐chemical characteristics of evaluated rice cultivars. Elongation during minimum cooking time was correlated with protein content (r = 0.6899), shape (?0.7137), thickness (0.7234) and width (0.9134) of grains. The rice flour paste viscosity correlated significantly with shape (0.6148) and length (0.7353) of grains. On the basis of the established results, it was concluded that appearance of milled rice could be used as indicator for predicting gelatinization behavior: elongation ratio (length after minimum cooking time/length of the grains) of the rice grains and viscosity of rice flour during cooking.

PRACTICAL APPLICATIONS

Results from this research constitute a useful tool to predict the gelatinization behavior of milled grains and flour of japonica rice varieties, on the basis of grain appearance. The appearance is critical in acceptability of rice varieties by the consumers. The possibility to predict rice grain behavior during cooking on the basis of its appearance could be an additional attribute or market value for consumer choice. The results could be used in selection of rice variety for rice grits or flour production and use in special applications.

     

PURIFICATION AND PROPERTIES OF TWO ACID PEROXIDASES FROM BRUSSELS SPROUTS (Brassica oleraceae L.)

An acid peroxidase isoenzyme (A1) from Brussels sprouts (Brassica oleraceae L.) has been purified while another isoenzyme (A2) has been partially purified. Studies of their properties leading to a potential application in immunoassays as an alternative to horseradish peroxidase were conducted. Isoenzyme A1 was purified 503 fold, through ammonium sulfate and acetone fractionation, and successive chromatography on DEAE-cellulose, Sephadex G-100 and Mono-S (FPLC system) columns. Isoenzyme A1 had a pI of 4.0, a molecular weight of 90 kDa, and contained two identical molecular weight subunits. Preliminary studies indicated a pI of 4.7 for isoenzyme A2. ABTS [2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid)] K_m values (0.2 mM) for both isoenzymes are 20 times lower than those reported for commercial horseradish peroxidase anionic isoenzymes. Optimum pH for activity of isoenzymes A1 and A2 were 4.3 and 4.5, respectively. Optimum temperature for isoenzyme A1 was 57C, with an activation energy for inactivation of 148.8 kJ/mol.