Recovery of choline oxidase activity by in vitro recombination of individual segments.
Initial attempts to express a choline oxidase from Arthrobacter pascens (APChO-syn) in Escherichia coli starting from a synthetic gene only led to inactive protein. However, activity was regained by the systematic exchange of individual segments of the gene with segments from a choline oxidase-encoding gene from Arthrobacter globiformis yielding a functional chimeric enzyme. Next, a sequence alignment of the exchanged segment with other choline oxidases revealed a mutation in the APChO-syn, showing that residue 200 was a threonine instead of an asparagine, which is, thus, crucial for confering enzyme activity and, hence, provides an explanation for the initial lack of activity. The active recombinant APChO-syn-T200N variant was biochemically characterized showing an optimum at pH 8.0 and at 37 degrees C. Furthermore, the substrate specificity was examined using N,N-dimethylethanolamine, N-methylethanolamine and 3,3-dimethyl-1-butanol.
Heinze
Birgit
B
Department of Biotechnology and Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff-Str. 4, 17487, Greifswald, Germany.
Hoven
Nina
N
O'Connell
Timothy
T
Maurer
Karl-Heinz
KH
Bartsch
Sebastian
S
Bornscheuer
Uwe T
UT
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003642:Deanol / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004983:Ethanolamines / Q000378:metabolism; D000441:Hexanols / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011995:Recombination, Genetic; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D004983:Ethanolamines; D000441:Hexanols; D003642:Deanol; C007469:3,3-dimethylbutan-1-ol; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; C017500:N-methylaminoethanol