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Purification and characterization of an NAD+-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotrophArthrobacter P1
Authors:Margaret M. Attwood  Nico Arfman  Ruud A. Weusthuis  Lubbert Dijkhuizen
Affiliation:(1) Department of Microbiology, University of Sheffield, UK;(2) Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands
Abstract:WhenArthrobacter P1 is grown on choline, betaine, dimethylglycine or sarcosine, an NAD+-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa±10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa±3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the Km values were 0.10 and 0.80 mM for formaldehyde and NAD+, respectively. The enzyme was heat-stable at 50° C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity.Abbreviation RuMP Ribulose monophosphate
Keywords:Arthrobacter P1  choline  formaldehyde dehydrogenase (NAD+-dependent) (EC 1.2.1.-)  methylamine
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