Purification and Characterization of an Alkaliphilic Choline Oxidase of Fusarium oxysporum

Abstract

A novel choline oxidase found in a fungus, Fusarium oxysporum strain V2, was purified to homogeneity as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme has a molecular mass of 128 kDa and consists of two identical subunits. The purified enzyme showed adsorption peaks at 340 nm and 450 nm. It showed alkaliphilic pH characteristics: its optimum pH was 9.0–10.0, and it was stable at pH 8.0–10.2. The Michaelis constant (K_m) values for choline and betaine aldehyde were 0.28 mM and 0.39 mM respectively. Trimethylamino-alcohols, dimethylamino-alcohols, and diethylaminoethanol were substrates for the enzyme, but the K_m values for them increased with decreasing numbers of methyl groups on the ammonium headgroup. A marked decrease in the maximum velocity (V_max) and V_max/K_m values was observed when N-replaced choline analogs were used as substrate instead of choline. The enzyme had a remarkably higher affinity for choline and betaine aldehyde than do previously reported enzymes. The enzyme oxidized these two substrates more quickly than a choline oxidase from Arthrobacter globiformis, and oxidation by the V2 enzyme was accompanied by an increase in the stoichometric amount of hydrogen peroxide.