Purification, Crystallization and Properties of Primary Alcohol Dehydrogenase from a Methanol-oxidizing Pseudomonas sp. No. 2941

Abstract

A simple method for purification and crystallization of primary alcohol dehydrogenase (EC 1. 1. 99. 8) is reported. The purification procedures consisted of four steps: protamine sulfate treatment, ammonium sulfate fractionation, passage through a column of DEAE-cellulose at pH 8.0 and Sephadex G-200 gel filtration. Crystallization was performed by the addition of ammonium sulfate at 65% saturation with an overall yield of 39%. The crystalline enzyme had an isoelectric point of pH 7.38 and a sedimentation coefficient (s⁰_{20, W}) of 8.44 s. A molecular weight of 128, 000 was estimated, and the enzyme consisted of two subunits each having a molecular weight of 62, 000. The enzyme showed an affinity toward the lower primary alcohols, methanol to n-pentanol. Formaldehyde was also oxidized by the crystalline enzyme. The Km values for methanol and formaldehyde were found to be 20 μM and 70 μM, respectively. Ammonium ions were required for enzyme activity.