PURIFICATION AND PROPERTIES OF ALCOHOL-DEHYDROGENASE FROM THE ACID-TOLERANT AND ETHANOL-TOLERANT YEAST CANDIDA-SOLICOLA

Two alcohol dehydrogenases (EC 1.1.1.1) from the acid- and ethanol-tolerant yeast Candida solicola WY-1 have been purified and characterized. The microbial strain cultured in a medium containing ethanol as a sole carbon source was disrupted in a Dyno-mill. From the cell-free extract obtained by centrifugation at 105,000 x g for 60 min, two alcohol dehydrogenases (ADH 1, ADH 2) were separated by DEAE-Toyopearl 650 M chromatography. ADH 1 was further purified by affinity chromatography using Matrex Blue A, ADH 2 was purified by chromatofocusing using Polybuffer Exchanger (PBE) 94 and affinity chromatography using Matrex Blue A. ADH 1 and ADH 2 had the same optimum pH, 7.0. ADH 1 was stable between pH 7.0 and 7.5, and ADH 2 at pH 7.0. The molecular weights of ADH 1 and ADH 2 were calculated to be about 160,000 and 162,000, while the isoelectric points were 5.3 and 5.25, respectively. The optimum temperature of ADH 1 was 30-degrees-C, while that of ADH 2 was 55-degrees-C. ADH 1 was stable at temperatures below 50-degrees-C, whereas ADH 2 was unstable at temperatures above 25-degrees-C.