Abstract
Polyphenol oxidase was purified to homogeneity from Japanese wheat (Triticum aestivum cv. Tohoku 198) bran, using 4-methylcatechol as a substrate. The enzyme was purified 1670-fold with a recovery of 0.175% by alkalization, ammonium sulfate precipitation, anion exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The molecular mass was estimated to be 35kDa and 40kDa by gel filtration chromatography and SDS-PAGE, respectively. The N-terminal amino acid sequence matched well with the internal sequences of other plant PPOs. The pH optima for the purified enzyme and the crude enzyme were 4.5 and 5.3, respectively. The purified enzyme was stable in the range of pH 6.5-7.5 at 4°C for 24hr. The purified enzyme was stable after heat treatment at 60°C for 10min. The purified enzyme oxidized L-tyrosine, pyrocatechol, 4-methylcatechol, L-DOPA, chlorogenic acid, (+)-catechin and (-)-epicatechin. The Km values of the purified enzyme for 4-methylcatechol and L-DOPA were 3.3mM and 8.3mM, respectively. The purified enzyme was strongly inhibited by dietyldithiocarbamate, thiourea, sodium azide, cystein, idoacetic acid, sodium fluoride, trans-cinnamic acid, p-coumaric acid, ferulic acid and vanillic acid. The KI value of the purified enzyme for ferulic acid was 108μM.
