Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Polyphenol oxidase (PPO) of garland chrysanthemum (Chrysanthemum coronarium L.) was purified approximately 32-fold with a recovery rate of 16% by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, and gel filtration. The purified enzyme appeared as a single band on PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be about 47000 and 45000 by gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized chlorogenic acid and (-)-epicatechin. The K(m) value (Michaelis constant) of the enzyme was 2.0 mM for chlorogenic acid (pH 4.0, 30 degrees C) and 10.0 mM for (-)-epicatechin (pH 8.0, 40 degrees C). The optimum pH was 4.0 for chlorogenic acid oxidase (ChO) and 8.0 for (-)-epicatechin oxidase (EpO). In the pH range from 5 to 11, their activities were quite stable at 5 degrees C for 22 h. The optimum temperatures of ChO and EpO activities were 30 and 40 degrees C, respectively. Both activities were stable at up to 50 degrees C after heat treatment for 30 min. The purified enzyme was strongly inhibited by l-ascorbic acid and l-cysteine at 1 mM.

Soluble polyphenol oxidase of edible yam (Dioscorea opposita Thunb.) was purified approximately ,*--fold with a recovery rate of +/ῌ by ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration using dopamine as a substrate. The purified enzyme appeared as a single band on native PAGE and SDS-PAGE. The molecular weight of the enzyme was estimated to be approximately .,kDa and ..kDa using gel filtration and SDS-PAGE, respectively. The purified enzyme quickly oxidized dopamine. The apparent Km value (Michaelis constant) of the enzyme was +./ mM for dopamine (pH 1.*, -*ῌC). The optimum pH was 1.* for dopamine oxidase. In the pH range from 0 to +*, the activity was quite stable at /ῌC for ,, h. The optimum temperature of enzyme activity was ,/ῌ-*ῌC. The activity was stable up to /*ῌC after heat treatment for ,* min. The browning reaction by the enzyme was completely inhibited by + mM L-ascorbic acid, which reduced o-quinone to dopamine. The reaction was also completely inhibited by + mM L-cysteine, which is a known quinone coupler. About -/ῌ inhibition of edible yam PPO was observed using citric acid and acetic acid at +* mM in *.+ M citrate/ *., M sodium phosphate bu#er (pH 1). In consideration of the observed results, L-ascorbic acid, L-cysteine, acetic acid, and citric acid are expected to be used as e#ective inhibitors of enzymatic browning in edible yam.

Occurrence of polyphenol oxidase (PPO, o-diphenol: oxygen oxidoreductase, EC.1.10.3.1) in the fruits of nine cultivars of banana (Musa spp.) commercially cultivated in China was investigated. All banana fruits peel and pulp tested had PPO activity, and all PPO strongly oxidized dopamine and 3-hydroxytyramine (tyramine). However, very weak and/or no oxidative activity was recognized on such trihydroxybenzenes as phloroglucinol and gallic acid. Similar substrate specificity of PPOs toward phenolic compounds was detected in the pulp and peel of all banana cultivars tested. The specific PPO activities toward dopamine for pulp were between 5.11 and 25.72, and those of peel were between 6.75 and 26.86 units/mg protein. However, PPO activities toward dopamine in the pulp and peel of AAA and AA genome group bananas were higher than those of ABB and AAB groups. The changes of pulp and peel PPO activities during fruit development were also determined in the three banana cultivars. During this development a remarkable decrement in pulp and peel PPO activities was found in all banana cultivars used.Keywords: banana (Musa spp.) cultivars, polyphenol oxidase, tyrosynase, development period Polyphenol oxidase (PPO, o-diphenol: oxygen oxidoreductase, EC.1.10.3.1) is a copper-containing enzyme which oxidizes phenolic compounds to produce undesirable browning during storage and processing of fresh fruits and vegetables. PPO has been widely studied in many fruits and vegetables to prevent the browning which results in the loss of their marketability (Kahn, 1977;Park et al., 1980;Park & Luh, 1985;Augustin et al., 1985;Tono et al., 1986;Fujita & Tono, 1979;Fujita & Tono, 1988;Fujita et al., 1991;Murata et al., 1992;Chilaka et al., 1993;Fujita et al., 1995;Castaner et al., 1996;Fujita et al., 1997;Ding et al., 1998). The PPO in banana (Musa sapientum L.) had been characterized (Palmer, 1963; Galeazzi & Sgabieri, 1981;Thomas & Janave, 1986;Yang et al., 2000;Yang et al., 2001), because a remarkable browning occurred in the pulp section. Many banana cultivars which fall into several genome groups are being cultivated in the world, however, little is known about the occurrence of PPO in them or on the change of PPO activity during development of the fruit. In the present study, determinations were made on the occurrence of PPO in nine banana cultivars commercially cultivated in China, and on the change of PPO activity during fruit development. Materials and MethodsFruit materials Banana fruits of the nine cultivars at the maturate stage for marketing were harvested from the experimental orchard of Guangxi Agricultural Vocation-Technical College and from a local farm in Nanning, China, placed in a plastic bag, and kept at room temperature to ripen the fruits for 2 to 3 days. The fruits at the same yellow-green and/or yellow-red stage were used as materials for our study.The cultivars, scientific name and genomic groups of bananas used were: Brazil Jiao (Musa cavendishii L.; AAA genome group), Nalong Jiao ( To determine the changes of PPO...

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