Basic characterization and partial purification of β-glucosidase from cell-free extracts of Oenococcus oeni ST81

Abstract

This study was designed to characterize a β-glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra. The β-glucosidase of O. oeni ST81 seems to have a periplasmic localization into the cells. This activity was strongly inhibited by gluconic acid, partially inhibited by glucose and not inhibited by fructose, lactate, malate, mannitol or sorbitol. Ethanol increased the activity of this enzyme up to 147%. Among the several metal ions assayed, only Fe²⁺ (10 mmol l⁻¹) and Cu²⁺ (5 mmol l⁻¹) exhibited a partial inhibitory effect (40%). This enzyme was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. The single peak because of β-glucosidase in all chromatographic columns indicates the presence of a single enzyme with an estimated molecular mass of 140 kDa. The calculated K(m) and V(max) values for 4-nitrophenyl-β-D-glucopyranoside were 0·38 mmol l⁻¹ and 5·21 nmol min⁻¹, respectively. The enzyme was stable at pH 5·0 with a value of t(1/2) = 50 days for the crude extract. The β-glucosidase of O. oeni ST81 is substantially different from those characterized from other wine-related lactic acid bacteria (LAB), such as Lactobacillus plantarum and Lactobacillus brevis; however, it appears to be closely related to a β-glucosidase from O. oeni ATCC BAA-1163 cloned into Escherichia coli. The periplasmic localization of the enzyme together with its high tolerance to ethanol and fructose, the low inhibitory effect of some wine-related compounds on the enzymatic activity and long-term stability of the enzyme could be of interest for winemaking. Information regarding a β-glucosidase from O. oeni ST81 is presented. Although the release of aroma compounds by LAB has been demonstrated, little information exists concerning the responsible enzymes. To our knowledge, this study contains the first characterization of a native β-glucosidase purified from crude extracts of O. oeni ST81.