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Polyphenol oxidase (PPO) was purified to homogeneity from loquat (Eriobotrya japonica Lindl. cv. Mogi) fruit. The enzyme was purified 422-fold with a total yield of 35.6%. The molecular weight was estimated to be about 58 000 and 55 000 by SDS-PAGE and FPLC gel filtration chromatography, respectively, indicating that PPO is a monomer. The optimum pH and temperature of the enzyme activity were found to be pH 4.5 and 30 °C, respectively, and the enzyme was stable in the range of pH 4-8. In substrate specificity, a maximum activity was shown with epicatechin, followed by chlorogenic acid, neochlorogenic acid, 4-methylcathechol, and pyrocatechol, and no activity was apparent toward monophenol and p-diphenol. The K m values for chlorogenic and neochlorogenic acids were 0.105 and 0.425 mM, respectively. The enzyme activity was markedly inhibited by sodium ascorbate, diethyldithiocarbamate, metabisulfide, dithiothreitol, mercaptoethanol, NaF, NaN 3 , L-cysteine, and reduced glutathione.

Phenolic compounds in loquat fruit were identified as 5-caffeoylquinic acid (chlorogenic acid), neochlorogenic acid, hydroxybenzoic acid, 5-p-feruloylquinic acid, protocatechuic acid, 4-caffeoylquinic acid, epicatechin, o-coumaric acid, ferulic acid, and p-coumaric acid. Neochlorogenic acid was found to be dominant in the early stages of loquat fruit development. Both the concentrations and types of phenolic compounds were high in young fruit but then decreased steadily during growth. However, the concentration of chlorogenic acid increased during ripening and became predominant in ripe fruit. The large rise in chlorogenic acid concentration appears to be a characteristic of loquat fruit ripening. In all of the cultivars tested, the types of phenolic compounds were similar but the total phenolic content varied from 81.8 to 173.8 mg/100 g of fresh pulp. In the biosynthetic pathway of chlorogenic acid, the enzyme activities of phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (CL), and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (CQT) were high at the early stage of growth, diminished to low levels approximately 3 weeks prior to harvest, but then rose to a peak at 1 week before harvest. The changes of these enzyme activities seemed to be associated with variations in chlorogenic acid concentration during development, maturation, and ripening of loquat fruit.

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