Purification and characterization of an alcohol dehydrogenase from Lithospermum erythrorhizon cell cultures.

An NAD-dependent alcohol dehydrogenase has been purified to apparent homogeneity from cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), using protamine sulphate and ammonium sulphate precipitation and chromatography on DEAE-Sephacel, Superdex 200, hydroxyapatite and HiTrap blue. The enzyme is a homodimer with a Mr of ca. 77,000. Each subunit with a Mr of 40,000 contains two zinc atoms. Its isoelectric point was found at pH 5.0. The best alcohol substrate of the enzyme is ethanol. The pH optimum for ethanol oxidation is at pH 8.7 and for acetaldehyde reduction at pH 4.6. The Michaelis constants for ethanol and NAD are 2.49 and 0.05 (pH 8.7), and for acetaldehyde and NADH 2.2 and 0.078 mM (pH 4.6), respectively. Partial amino acid sequences of the purified enzyme showed high homology to alcohol dehydrogenases from other plants.