Isolation and characterization of polyphenol oxidase from Indian tea leaf (Camellia sinensis)

Abstract

Polyphenol oxidase (PPO) was purified and characterised from the acetone powder of tea leaves (Camellia sinensis). Substrate staining of the acetone powder extract indicated the presence of a maximum of three isozymic forms of this enzyme. The isozymes of PPO were separated on DEAE cellulose column. Two fractions were absorbed and the other was unabsorbed. The unabsorbed fraction was purified up to homogeneity in different chromatographic steps: gel filtration, hydroxyapatite, centricon-30, FPLC. SDS-PAGE data along with molecular mass data showed the active enzyme to be of 72 kD. The pH, temperature, and kinetic parameters were studied. The highly purified enzyme was unable to oxidize monophenols, p-quinol but could oxidize catechol and thus might be regarded as catechol oxidase. Catechin was the best substrate with a K_m of 0.49 mM. The enzyme was completely inhibited by 2 mM tropolone, suggesting it to be a copper-containing enzyme. The enzyme is localized in chloroplast and could be solubilized with Triton-X-100. Trypsinization experiment with freezethawed chloroplast further confirms the localization of the enzyme inside the chloroplast.