Purification and Characterization of β-Glucosidases from <i>Issatchenkia terricola</i> and <i>Pichia kudriavzevii</i> and Their Application in Aroma Enhancement of Red Wine

Non-<i>Saccharomyces</i> yeasts exhibit the potential for enhancing wine aroma through their β-glucosidase activity. In this study, intracellular β-glucosidases (A-3E and Y-5E) from <i>Issatchenkia terricola</i> (A-3) and <i>Pichia kudriavzevii</i> (Y-5) were purified by high-pressure homogenization, (NH₄)₂SO₄ salting out, DEAE-52 ion exchange, and Sephadex G-75 chromatography. Then, their enzymatic properties were characterized. Results showed that A-3E and Y-5E had optimum reaction temperatures of 55 and 50 °C and optimum reaction pH values of 5.5 and 5.5, respectively. They were inhibited by ethanol of > 3% (v/v) and activated by ethanol of less than 3% (v/v). Additionally, A-3E and Y-5E were glucose-tolerant with 80.90 and 85.45% relative activity maintained at 30.0% (w/v) glucose, respectively. Addition of A-3E and Y-5E could increase the contents of terpenes, higher alcohols, and esters in wines and produce distinct odor-active compound profiles. This is the first report of characterization and application of intracellular β-glucosidase from non-<i>Saccharomyces</i> yeasts for red wine aroma enhancement.