Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

• Published in: Applied microbiology and biotechnology 2009, Vol.81 (5), p.875-886
• Main Authors: Ribitsch, D, Karl, W, Wehrschütz-Sigl, E, Tutz, S, Remler, P, Weber, H. J, Gruber, K, Stehr, R, Bessler, C, Hoven, N, Sauter, K, Maurer, K. H, Schwab, H
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Arthrobacter - enzymology; Arthrobacter - genetics; Arthrobacter - isolation & purification; Arthrobacter nicotianae; Bacteria; Betaine - analogs & derivatives; Betaine - metabolism; Biological and medical sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Carbon; Carbon sources; Chloride; Choline - metabolism; Choline oxidase; Cloning; Cloning, Molecular; Dehydrogenases; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Enzyme reaction kinetics; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic engineering; Heterologous expression; Hydrogen-Ion Concentration; Kinetics; Life Sciences; Magnetic Resonance Spectroscopy; Microbial Genetics and Genomics; Microbiology; Microorganisms; Molecular Sequence Data; Molecular Weight; NMR; Nuclear magnetic resonance; Oxidation; Plasmids; Proteins; Sequence Analysis, DNA; Soil contamination; Soil Microbiology; Studies; Substrate Specificity; Temperature; Yeast
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-008-1661-5

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15-70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 x 10⁻² U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 x 10⁻² U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K M = 1.51 ± 0.09 mM and V max = 42.73 ± 0.42 mU/min for choline chloride and K M = 4.77 ± 0.76 mM and V max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.