Purification and Properties of Coenzyme-Independent Alcohol Dehydrogenase from the Membrane Fraction of Acetobacter aceti

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The alcohol dehydrogenase (coenzyme-independent) was solubilized from the membrane fraction of Acetobacter aceti subsp. aceti with Triton X-100 and separated into three fractions, namely, enzymes I, II and III, by chromatographies on SP-Sephadex C-25 (pH 5.0) and CM-cellulose (pH 4.5). These enzyme preparations did not penetrate the gel on conventional electrophoresis, but when treated with 0.3% Tween 20,they penetrated the gel and all showed the same electrophoretic mobilities.The alcohol dehydrogenase was purified approximately 40-fold from the cell-free extract, together with a cytochrome c-like component, by hydroxylapatite and DEAE-Sephadex chromatographies. Activity staining of the enzyme showed a wide, diffuse zone.The crude and purified preparations were active and relatively stable at pH 4,and sowed different substrate specificities, K_m values for ethanol and sensitivities toward inhititors. In particular, the K_m value of the crude enzyme preparation for ethanol was approximately 1/22 of that of the purified enzyme. The molecular weight of the purified enzyme could not be estimated by polyacrylamide gel electrophoresis by four methods, because their electrophoretic profiles differed from each other in the number of bands, in the relative mobilities, and in the staining strength.
公益社団法人日本生物工学会の論文
1982-02-25