The laccase/ABTS system oxidizes (+)-catechin to oligomeric products

Laccase (EC 1.10.3.2, p-diphenol dioxygen oxidoreductase)-catalysed (+)-catechin oxidation was investigated, both in the presence and absence of the mediator ABTS. The effect of ascorbic acid, a radical scavenger, on the reaction mechanism was also examined. Using reversed-phase HPLC chromatography, UV–vis and HPLC-ESI-MS spectroscopies, we identified the formation of dimers of the type dehydrocatechin B, which are relatively more hydrophilic than the parent substrate, and hydrophobic dimers of type-A, as well as previously not described oligomers, namely, a trimer, tetramer and their oxidized forms. Evidence is presented for the conversion of a hydrophilic dimer via a quinone intermediate to the hydrophobic dimers. The hydrophobic dimers were further oxidized by laccase to o-quinone, which upon condensation with catechin forms the reduced trimer. Both ABTS and ascorbic acid had effect on the overall rate of (+)-catechin oxidation and the proportions of the final oxidation products. ABTS favoured the formation of the hydrophobic products and decreased the hydrophilic dimers. In contrast, at low concentrations, ascorbic acid favoured the formation of hydrophilic products, whereas at higher concentrations the radical scavenger inhibited the laccase-catalysed catechin oxidation reaction, most probably, by reducing the semiquinone and o-quinone back to the parent polyphenol. A nucleophilic rather than radical mechanism accounts for the product formation.