Purification and some properties of cyclohexylamine oxidase from a Pseudomonas sp.
Cyclohexylamine oxidase was purified 90-fold from cell-free extracts of Pseudomonas sp. capable of assimilating sodium cyclamate. The purified enzyme was homogeneous in disc electrophoresis, and the molecular weight was found to be approximately 80,000 by gel filtration. The enzyme catalyzed the following reaction: cyclohexylamine+O2+H2O leads to cyclohexanone+NH3+H2O2. The enzyme thus can be classified as an amine oxidase; it utilized oxygen as the ultimate electron acceptor. The pH optimum of the reaction was 6.8 and the apparent Km value for cyclohexylamine was 2.5 X 10(-4) M. The enzyme was highly specific for the deamination of alicyclic primary amines such as cyclohexylamine, but was found to be inactive toward ordinary amines used as substrates for amine oxidases. The enzyme solution was yellow in color and showed a typical flavoprotein spectrum; the addition of cyclohexylamine under anaerobic conditions caused reduction of the flavin in the native enzyme. The flavin of the prosthetic group was identified as FAD by thin layer chromatography. The participation of sulfhydryl groups in the enzymic action was also suggested by the observation that the enzyme activity was inhibited in the presence of PCMB and could be recovered by the addition of glutathione.
D000588:Amines / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003494:Cyclamates / Q000378:metabolism; D003514:Cyclohexylamines; D005486:Flavin Mononucleotide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification / Q000378:metabolism*; D011549:Pseudomonas / Q000378:metabolism*
D000588:Amines; D002413:Cations, Divalent; D002729:Chloromercuribenzoates; D003494:Cyclamates; D003514:Cyclohexylamines; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D000587:Oxidoreductases Acting on CH-NH Group Donors; D005978:Glutathione