Purification and Characterization of Choline Oxidase from Arthrobacter globiformis

Abstract

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.

Acetylcholine in the crayfish optic lobe: concentration profile and cellular localization

“…The procedure is based upon the conversion of ACh to choline by acetylcholinesterase (AChE) and the conversion of choline to betain and hydrogen peroxide by choline oxidase (Ikuta et al, 1977). Two mol of H,O, are generated per mol of choline oxidized.…”

Synthesis of the osmoprotectant glycine betaine in Bacillus subtilis: characterization of the gbsAB genes

“…The ability to synthesize glycine betaine from choline is found in many cells of microbial, plant, and animal origin, and three different enzymatic systems can effect this synthesis. In Arthrobacter pascens, Arthrobacter globiformis, and an Alcaligenes sp., a bifunctional soluble choline oxidase that catalyzes the oxidation of choline and glycine betaine aldehyde to glycine betaine with the concomitant consumption of oxygen and the production of hydrogen peroxide has been found (23,40,47). In plants, glycine betaine synthesis occurs primarily in the chloroplasts and involves a soluble choline monooxygenase in combination with a soluble betaine aldehyde dehydrogenase (6,36,53).…”

Choline oxidase, a catabolic enzyme in Arthrobacter pascens, facilitates adaptation to osmotic stress in Escherichia coli

“…If choline is utilized as a carbon source, however, the presence of a betaine aldehyde dehydrogenase may be advantageous, given that the Km for betaine aldehyde of known betaine aldehyde dehydrogenases is 17-to 28-fold lower (22,29,31,34) than the Km of choline oxidase (8.7 mM [19]). As well, betaine aldehyde dehydrogenase activity can generate NADH or NADPH (22,29,31,34), which may serve in vivo as a source of reductant or be used for ATP synthesis, whereas choline oxidase, which uses 02 as the primary electron acceptor (20,33,49), does not provide this advantage.…”

“…The characteristics of choline oxidase, including it being a soluble and bifunctional enzyme that does not require any cofactors (19,20), make it a favorable choice to genetically engineer enhanced betaine biosynthesis in heterologous organisms. Although choline oxidase has a covalently attached FAD moiety (19,33,49), its assembly into a functional enzyme in the distantly related E. coli suggests that it can be modified appropriately in heterologous organisms.…”

“…Although choline oxidase has a covalently attached FAD moiety (19,33,49), its assembly into a functional enzyme in the distantly related E. coli suggests that it can be modified appropriately in heterologous organisms. Choline dehydrogenase of E. coli, the only other choline-oxidizing enzyme for which a gene has been cloned (1, 4a), is a membrane-bound protein that requires an as yet unidentified electron acceptor (22).…”