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Applied and Environmental Microbiology, April 2003, p. 2126-2132, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2126-2132.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cloning, Expression, and Purification of Choline Dehydrogenase from the Moderate Halophile Halomonas elongata

Giovanni Gadda1,2* and Elien Elizabeth McAllister-Wilkins1

Department of Chemistry,1 Department of Biology and The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30303-30382

Received 23 September 2002/ Accepted 7 January 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.

* Corresponding author. Mailing address: Department of Chemistry, Georgia State University, Atlanta, GA 30303-3038. Phone: (404) 651-4737. Fax: (404) 651-2751. E-mail: ggadda{at}gsu.edu.

Applied and Environmental Microbiology, April 2003, p. 2126-2132, Vol. 69, No. 4
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.4.2126-2132.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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