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Cloning, Expression, and Purification of Choline Dehydrogenase from the Moderate Halophile Halomonas elongata. Giovanni Gadda, 2003.Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate . Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures . To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro . In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase . The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose . The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 µmol of O2 min-1 mg-1 mM-1 at pH 7 and 25°C, respectively . With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent Vmax values for choline and betaine-aldehyde were 10.9 and 5.7 µmol of O2 min-1 mg-1, respectively . These Vmax values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor . Altogether, the kinetic data are consistent with the conclusion that H . elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available .
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