J. Biochem., 81, 851-858 (1977) Purification and Some Properties of Cyclohexylamine Oxidase from a Pseudomonas sp.1 Toshie TOKIEDA, Toshio NIIMURA , Fuminori TAKAMURA, and Tsutomu YAMAHA Department of Medical Chemistry , National Institute of Hygienic Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158 Received for publication, August 25 , 1976 Cyclohexylamine oxidase was purified 90-fold from cell-free extracts of Pseudomonas sp . capa ble of assimilating sodium cyclamate. The purified enzyme was homogeneous in disc elec trophoresis, and the molecular weight was found to be approximately 80 ,000 by gel filtration. The enzyme catalyzed the following reaction: cyclohexylamine+O2+H2O •¨ eyclohexanone+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 chro matography. The participation of sulfhydryl groups in the enzymic action was also suggested by the observations that the enzyme activity was inhibited in the presence of PCMB and could be recovered by the addition of glutathione. Sodium cyclamate (CHS-Na) was widely used as a Kojima and Ichibagase (2) reported that laboratory sweetening agent for various drugs and foods, but animals and humans receiving CHS-Na excreted an was banned for general use in 1969, due to possible appreciable amount of CHA in urine, many meta carcinogenicity (1) and metabolic conversion to bolic studies of CHS have been undertaken (3-6), cyclohexylamine (CHA), a toxic substance. Since and urinary excretion of other metabolites, includ ing cyclohexanone (CHnone) and cyclohexanol (CHnol), was observed in both amimals and man. 1A part of this work was presented at the Annual Meet- A series of studies in our laboratory (7-11) ing of the Kanto Division of the Agricultural Chemical Society of Japan, Tokyo, November 4, 1975. suggested that intestinal bacteria play an important Abbreviation: CHS-Na, sodium cyclamate; CHA, role in the metabolism of CHS, and this suggestion cyclohexylamine; CHnone, cyclohexanone; PCMB, was supported by results obtained in several other p-chloromercuribenzoate. laboratories (12-14). Moreover, the present Vol. 81, No. 4, 1977 851 852 T. TOKIEDA, T. NIIMURA, F. TAKAMURA, and T. YAMAHA authors (10) isolated cyclamate-assimilating bacte terms of the turbidity at 650 nm. Protein was ria from the feces of guinea pigs excreting CHA, determined by the method of Lowry et al. (15). and confirmed that a strain of Pseudomonas sp. Ammonia was determined colorimetrically with possessed an enzyme system forming CHnone from Nessler's reagent after microdiffusion in a Conway CHS via CHA. We also reported the partial unit (16). purification and some properties of CHS sulfamat Determination of CHA and CHnone-Two ml ase, which catalyzed the desulfation of CHS to of the reaction mixture was adjusted to pH> 13 CHA (11). with 6 N NaOH and CHA was extracted with 0.5 ml Our results on the enzymic conversion of CHS of dichloromethane containing 0.02% n-tridecane to CHnone by crude bacterial extracts led us to the as an internal standard. conclusion that amine oxidase might catalyze the Two drops of 6 N HCI and 0.5 g of NaCI were deamination of CHA to CHnone because the de added to 2 ml of the reaction mixture, then CHnone amination was dependent on oxygen (10). Al- was extracted with 0.5 ml of dichloromethane though many studies on amine oxidases have been containing 0.01 % n-dodecane as an internal stand reported, an enzyme deaminating alicyclic amines ard. such as CHA has not previously been obtained. CHA and CHnone were analyzed by gas The present paper describes the purification liquid chromatography on a Shimadzu 4APF unit and some properties of CHA oxidase, which is with dual flame ionization detectors using a 2 m x involved in the second step of the assimilation of 0.3 cm glass column packed with 10% Carbowax CHS by a Pseudomonas sp. 20 M plus 2.5 % NaOH on 60 to 80 mesh Chromo sorb G. Operating conditions were as follows; MATERIALS AND METHODS column temperature 110°C, injector temperature 200°C, detector temperature 230°C, and carrier gas Chemicals-FAD was obtained from Calbio (nitrogen) flow rate 50 ml/min. The retention times chem; FMN was from Tokyo Slats Inc.; crystalline of CHA, n-dodecane, n-tridecane, and CHnone bovine liver catalase [EC 1.11.1.6] was from Sigma were 3.5, 4.2, 7.0, and 7.2 min, respectively. Chemical Co. ; horse heart cytochrome c, bovine Enzyme Assay-Enzyme activity was de albumin, human gamma globulins and horse apo termined by measuring oxygen consumption mano ferritin were from Schwarz-Mann Co. Other metrically with a conventional Warburg apparatus chemicals were of the highest purity commercially at 30°C in air. The incubation mixture usually available. contained an appropriate amount of enzyme, Maintenance and Growth of the Organism- 120 ƒÊmol of Tris-maleate, pH 6.8, and 30 ƒÊmol of Pseudomonas sp. strain K was used in this work. CHA•EHCl in a total volume of 3 ml. The center This strain was isolated from the feces of guinea well contained 0.2 nil of 20 % (w/v) KOH. The pigs which had been given drinking water containing reaction was started by tipping in the substrate CHS-Na ad libitum, and which excreted CHA and from a side arm. One unit of the enzyme was CHnone in the urine (10). Stock cultures were defined as that amount which consumed 1 ƒÊmol of maintained in a CHS-Na-inorganic salts-vita oxygen per min under the standard assay condi mins medium (medium A) as described previously tions. Specific activity was expressed as units per (10). In order to obtain large quantities of cells, mg of protein. heavy inocula of the cells were transferred to 100 ml Preparation of Cellfree Extracts-In a typical of medium A containing 0.5 % CHS-Na in a 500 ml preparation, 5 g of dried cells was suspended in shaking flask. Sixty flasks were cultured for 7 to 0.04 M Tris-HCI, pH 7.2, to a level of 20 mg per ml, 9 days at 30°C with shaking. The cells were and disrupted with an ultrasonic disintegrator collected by centrifugation, washed three times with (Ohtake Sonic 5202) for periods of 2 min (total,- 0.02 M phosphate buffer, pH 7.0, and dried over exposure, 10 min). Unbroken cells and debris P2O5 in vacuo. The dried cells were stored at were removed by centrifugation at 20,000 x g for -20°C until use. 20 min, and the supernatant was used as the crude Determination of Bacterial Growth, Protein, and extract. Ammonia-Bacterial growth was measured in Purification Procedure-Step 1. Streptomycin J. Biochem. PURIFICATION AND PROPERTIES OF CYCLOHEXYLAMINE OXIDASE 853 sulfate treatment: Five percent streptomycin ƒÊ mol of CHA, 0.5 nil of the purified enzyme, 160 sulfate solution in 0.04 M Tris-HCI, pH 7.2, was ƒÊ mol of Tris-maleate, pH 6.8, and various electron added slowly to the supernatant with stiring to give acceptors in a total volume of 4 ml. The reaction a final concentration of 1 %. The mixture was was carried out at 30•Ž for 1 h in a Thunberg tube allowed to stand for 10 min, centrifuged at 20,000 under N2, then the reduction of the acceptor was x g for 20 min, and the supernatant was collected. determined spectrophotometrically. The enzyme activity was quite stable during storage Measurement of the Absorption Spectrum of for at least 3 months at -20•Ž. CHA Oxidase Reduced with CHA-Thunberg Step 2. (NH4)2SO4 fractionation: The cuvettes (Fujiwara Factory Co.) containing 6.5 mg supernatant was made up to 0.26 saturation by the of enzyme (specific activity 4,500 milliunits/mg), slow addition of solid (NH4)2SO4, then allowed to 120 ƒÊmol of KH2PO4-Na2HPO4, pH 6.8, and 30 stand at 5•Ž for 2 h. The precipitate formed was ƒÊ mol of CHA •E HCl in a total volume of 3 ml, were centrifuged off. The supernatant was then made made anaerobic by degassing the solution with a up to 0.84 saturation by further addition of solid vacuum pump and flushing with nitrogen. This (NH4)2SO4. The resulting precipitate, after stand- procedure was repeated 5 times, then the reaction ing as before, was collected by centrifugation and was started by the addition of CHA from a side redissolved in an appropriate amount of 0.04 M arm. After standing at room temperature for 5 Tris-HCI, pH 7.2. min, the absorption spectrum was measured in the Step 3. Gel filtration: The redissolved range of 340 to 659 nm. (NH4)2SO4 precipitate was applied to a column Isolation of Flavin in CHA Oxidase-Flavin (2.6 x 100 cm) of Sephadex G-200. The column was isolated according to the procedures of Tipton was eluted with 0.04 M Tris-HCl, pH 7.2, and 5-ml (19) and Burton (20). One ml of 50% (w/v) tri fractions were collected. The most active Sephadex chloroacetic acid solution was added to 4.0 ml of fractions (fractions 34-42) were combined.