Characteristic of β-glucosidase from Sicilian blood oranges in relation to anthocyanin degradation

Anthocyanin content in Sicilian sweet orange [Citrus sinensis (L) Osbeck] varieties known as blood oranges (Tarocco, Moro e Sanguinello) undergoes changes during the ripening process. Concentration increases during ripening, reaching a maximum in the fully ripe fruit. At latter stage of maturity, a decrease of these pigments is observed. This study aims to evaluate β-d-glucosidase activity (βG, EC 3.2.1.21) in Tarocco variety, the most common Sicilian blood orange, and to determine the main physicochemical and kinetic properties of this enzyme in order to underline its role on anthocyanins degradation during ripening. The enzymatic extract was assayed on both anthocyanins extract from Tarocco juice and a synthetic substrate (p-nitrophenyl-β-d-glucopyranoside, pNPG). It was observed a 400 times higher specificity of the synthetic substrate than the natural substrate. Kinetic studies and physicochemical characterization of βG such as Vmax and Km (Vmax 2.1 × 10−2 and Km 2.67 × 10−4 using pNPG as substrate; Vmax 3.3 × 10−3 and Km 2.1 × 10−1 using natural substrate), optimum conditions of pH (4.5) and temperature (60 °C), fructose and glucose inhibition (competitive inhibition by glucose) and thermal stability (decimal reduction time 3 min at 75 °C and decimal reduction temperature 9.5 °C) were also performed. β-d-Glucosidase activity seems involved on anthocyanins degradation during final ripening stage of fruit.