Grape Alcohol Dehydrogenase. I. Isolation and Characterization

An alcohol dehydrogenase (ADH) was extracted from grape berries with dithiothreitol and glycerol present in the extraction medium. The enzyme was purified 297-fold with 81% yield by DEAE cellulose and affinity chromatographies (Blue Sepharose CL-6B). The enzyme so obtained, homogeneous in polyacrylamide gel electrophoresis, can be stored at least one year at -20°C. The optimum pH for grape ADH was found to be 9.2 to 9.3 for ethanol oxidation and 5.8 to 5.9 for acetaldehyde reduction. The iso-electric point was 5.5 ± 0.2. The molecular weight values determined by gel filtration, polyacrylamide gradient gel electrophoresis, and SDS polyacrylamide gel electrophoresis are indicative of a dimeric structure for this enzyme which is probably constituted by two identical subunits of MW = 45 000 ± 2000. Inhibition studies show that grape ADH is a metallo enzyme with SH groups essential for enzyme activity. The results obtained using ethanol as the inhibitor indicate that during carbonic maceration, the limitation of percent alcohol observed is not related to the inhibitor effect of the ethanol on grape ADH.