Production and characterization of a novel cold-active ß-glucosidase and its influence on aromatic precursors of Muscat wine

Abstract

ß-Glucosidases (ßGL)—widely used in the enological field—are enzymes that catalyze the liberation of aromatic volatiles from their glycosidic precursors during winemaking. In the present study, a ßGL obtained from the Antarctic yeast Mrakia sp. LP 7.1.2016 was produced on a bioreactor scale, purified, characterized, and the enzyme's properties studied for a potential enological application. Sodium-dodecylsulfate–polyacrylamide-gel electrophoresis indicated a high molecular weight for this enzyme, it having at least two subunits of 134 and 14 kDa. ßGL exhibited a pH optimum of 5.0 and retained substantial activity and stability at pH 4.0. The temperature range for optimal activity was 50–55 °C, with the enzyme demonstrating thermostability up to 50 °C and retaining 87% of residual activity at that temperature after 3 h. The respective kinetic constants determined with p-nitrophenyl-ß-D-glucopyranoside and cellobiose were 0.38 and 1.79 mM for Km and 20.1 and 5.65 μmol−1 mg−1 min.1 for Vmax, ß-Glucosidase manifested high activity in 10–25% (v/v) ethanol, 30.0 mg L−1 sulfur dioxide, and 10–200 g L−1 fructose; but it was strongly inhibited by glucose, retaining only 6% of residual activity in the presence of 20 g L−1. Upon investigating the influence of the enzyme on Muscat-wine glycosidic precursors, we found significant differences in terpene content after 14 days of ßGL treatment at an eightfold increase over control-wine levels. The enzyme was more active toward the precursors of the monoterpenes nerol and geraniol and oxides of trans- and cis-linalool. These findings contribute to our understanding of the potential of cold-active enzymes in advantageous biotechnological applications to enology.