Characterisation of laccase activity of two strains of Botrytis cinerea

The purpose of the present study was the characterization of the polyphenol oxidase activity, which can mainly be ascribed to a fungal laccase, produced and released in grape juice by two different strains of Botrytis cinerea. This fungal laccase belongs to the group of polyphenol oxidases (PPO) and is mainly responsible for the oxidation of polyphenols contained in grape must. The analysis of the SDS-PAGE band profile of the proteins released in the grape must showed huge differences between the investigated strains. These differences regard both the proteins with a high molecular weight, among which presumably the laccase is present, and the proteins with a molecular weight below 40 kDa. The "laccase activity" of the enzymes, expressed and secreted by the two fungal strains in the growth medium, was tested at pH 5.0 by the syringaldazine test and at pH 3.5 by an oxygraphic method using three substrates present in the must: caffeic acid, caftaric acid and mal-vidin-3-glycoside. Also in this case significant differences among the apparent Michaelis-Menten constant values of the two strains for the various polyphenols were found. Although the activity behaviour cannot be unquestionably attributed to laccase, it could be of practical importance to wine producers since the kinetic data were obtained in experiments carried out at pH 3.5, i.e. under a condition typical of must.