Heterologous Expression of aCandida molischianaAnthocyanin-β-glucosidase in a Wine Yeast Strain

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Abstract
A recombinant wine yeast strain expressing the Candida molischianabgln gene encoding a β-glucosidase/anthocyanase under the control of the Saccharomyces cerevisiae actin gene promoter has been constructed. The corresponding protein, BGLN, was mainly located on the cell wall. BGLN was purified in a single chromotagraphic step, and different physicochemical and kinetic properties have been determined. BGLN showed maximum activity against the artificial substrate p-nitrophenyl β-d-glucopyranoside. It also hydrolyzed salicin, p-nitrophenyl β-d-xyloside, cellobiose, and arbutin to a lesser extent. Fructose and SO2 did not affect enzyme activity, which was activated by ethanol, while glucose was a strong competitive inhibitor. The purified BGLN showed a novel anthocyanase decolorizing capability on red wines. This anthocyanase activity was readily observed during microvinification experiments. However, the physicochemical characteristics of the wines obtained with the recombinant wine yeast strain were indistinguishable from those obtained with the parental strain. Keywords: Wine aroma; anthocyanin-β-glucosidase; Candida molischiana; gene expression