J. Biochem. 82, 1741-1749 (1977) Purification and Characterization of Choline Oxidase from Arthrobacter globiformis Shigeru IKUTA, Shigeyuki IMAMURA, Hideo MISAKI, and Yoshifumi HORIUTI Research Laboratory, Toyo Jozo Co., Ltd., Mifuku, Ohito-cho, Tagata-gun, Shizuoka 410-23 Received for publication, June 7, 1977 Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate-polyacrylamide disc gel electro phoresis) and an isoelectric point (pl) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline+02betaine aldehyde+ H202, betaine aldehyde+02+H2O-betaine+H202. The enzyme was highly specific for choline and betaine aldehyde (relative reaction veloc ities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanol amine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5. In a previous report from this laboratory (1), the existence of choline oxidase was discussed in relation MATERIALS AND METHODS to the oxidative pathway of choline to betaine found in A. globiformis cells. The enzyme ap Culture of the Bacterium-Cells of A. glo peared to catalyze the oxidations of both choline biformis were grown aerobically in culture medium and betaine aldehyde coupled with H202 generation for 40 h, as described previously (1). and oxygen consumption. The present paper Assay-H202-generating activity, betaine reports on the purification and characterization of aldehyde-forming activity and oxygen-consumption this choline oxidase. were determined as described previously (1). Identification and Estimation of Betaine in the Incubation Mixture-The reaction mixture for the production of betaine contained 20 mM Tris-HC1 Abbreviations: pI, isoelectric point; SDS, sodium buffer (pH 8), 1.5 mM 4-aminoantipyrine, 2.1 mM dodecyl sulfate. phenol, 30 ƒÊmol of choline chloride, 40 units of Vol. 82, No. 6, 1977 1741 1742 S. IKUTA, S. IMAMURA, H. MISAKI, and Y. HORIUTI peroxidase, and 200 units of choline oxidase in a Co., Kyoto) at room temperature (25•Ž). final volume of 100 ml. The reaction was carried Materials-Choline chloride, betaine, 4-amino- out for 80 min at 37•Ž and stopped by adding antipyrine, phenol, 2,4-dinitrophenylhydrazine, sufficient conc. HC1 to give a final pH of 1.0. The monoethanolamine, diethanolamine, triethanol amount of betaine formed was determined by the amine, N-methylethanolamine, formaldehyde, method of Barabanov et al. (2) with some modi acetaldehyde, and propionaldehyde were obtained fications as follows. To the mixture, 5 g of char from Wako Pure Chemical Industries Co., Osaka. coal, previously washed with 0.1 N HC1, were N,N-dimethylethanolamine was from Tokyo added to remove the quinoneimine dye formed. Kasei Organic Chemicals Co., Tokyo, and perox The mixture was filtered, and the filtrate concen idase and Coomassie Brilliant Blue R were from trated to 5 ml with a rotary evaporator at 40•Ž. Sigma Chemical Co., St. Louis. Sephadex G-200 A portion (0.5 ml) of the concentrated solution and DEAE-cellulose were products of Pharmacia was mixed with I ml of reineckate solution: the Fine Chemicals, Uppsala, and Brown Co., Berlin, reineckate solution was freshly prepared by dis respectively. The reference proteins used for solving 1.5 g of the monohydrate in 100 ml of molecular weight determinations were aldolase, distilled water, adjusting the pH to 1.0 with conc. chymotrypsinogen A, ovalbumin, bovine serum HC1, and filtering the mixture. After addition of albumin, RNA polymerase, and trypsin inhibitor reineckate, the mixture was stood for 30 min at from Boehringer Mannheim GmbH, Mannheim. room temperature (25•Ž), and the resulting pre cipitate was collected by centrifugation (7,000•~g, RESULTS 5 min) and washed twice with 2 ml of ethyl ether. The washed precipitate was dissolved in 2.5 ml of Purification of Choline Oxidase from the Cells distilled water, and the absorbance of the solution -The bacterial cells were harvested from 2 liters at 525 nm was measured. 'The amount of betaine of culture medium and washed with 10 mM phos- was calculated from a standard curve obtained phate-2 mM EDTA-0.1 % KC1 buffer (pH 7) with authentic betaine. (EDTA-KCI-P1 buffer) by centrifugation. The Determination of Protein-Protein concen washed cells were suspended in 400 ml of the same trations were determined by the method of Lowry buffer containing 0.05 % lysozyme, and the sus et al. (3) with bovine serum albumin as a standard. pension incubated for 30 min at 37•Ž with stirring. I soelectrie Focusing-Isoelectric focusing was The resulting lyzed cell suspension was centrifuged carried out at 5•Ž for 40th With Ampholine carrier (7,000•~?, 20 min) to remove cell debris, and the ampholytes giving a pH gradient of 3.5 to 10 in a supernatant was mixed with 10 ml of 5 % protamine 110ml electrofocusing column, according to the sulfate solution (pH 7). The precipitate formed method of Vesterberg (4). was removed by centrifugation, and the clear Polyacrylamide Disc Gel Electrophoresis- supernatant mixed with an equal volume of cold Polyacrylamide disc gel electophoresis was carried acetone, stood for 20 min at 25•Ž and centrifuged out in 50 mM Tris-glycine buffer (pH 8.3) at a (7,000•~g, 10 min). The resulting supernatant was constant current of 2 mA per, column (5•~80 mM) mixed with acetone to 75 % (v/v), and the mixture for 150 min at 15•Ž, as described by Davis (5). stood for 20 min at 20•Ž and then centrifuged Disc gel electophoresis in the presence of sodium (7,000•~g, 10 min). The precipitate was dissolved dodecyl sulfate (SDS) was performed by the method in 50 ml of EDTA-KCI-P1 buffer and fractionated of Weber et al. (6) in 0.1 m phosphate buffer (pH by adding a saturated solution of ammonium 7.2) containing 0.1 % SDS on 5 % polyacrylamide sulfate (pH 8); the fraction which precipitated between 40% and 60% saturation was collected gel with 0.14% N,N•Œ-methylenebisacrylamide. Electrophoresis was carried 'out at 8 mA per by centrifugation (12,000•~g, 15 min) and dissolved column and at 25•Ž for 4 h. The gel was stained in 20 ml of EDTA-KCI-P1 buffer. The solution with Coomassie Brilliant Blue R (6). was desalted on a Sephadex G-25 column, mixed Absorption Spectrum-The absorption spec with acetone to 60% (v/v) and centrifuged (7,000•~g trum was measured with a Shimadzu double- , 10 min). The resulting supernatant was beam spectrophotometer UV-210 A (Shimadzu mixed with acetone to 75 % and the mixture stood J. Biochem. PURIFICATION AND CHARACTERIZATION OF CHOLINE OXIDASE 1743 for 20 min at 20•Ž. The precipitate formed was x g, 10 min). The precipitate was dissolved in collected by centrifugation (7,000•~g, 10 min) and 2 ml of EDTA-KC1-P1 buffer, and the solution dissolved in 2 ml of the buffer. This solution was rechromatographed on a column of DEAF-cellu- applied to a column of DEAE-cellulose (Fig. 1). lose in a similar manner to that described above The column was washed with 60 ml of EDTA- (Fig. 2). The fractions containing most of the KC1-Pi buffer containing 0.2 M KC1 and then activity (Nos. 59-75) were combined and again eluted with a linear gradient of KC1 (0.2-0.5 M) in subjected to acetone precipitation in the way the same buffer. Fractions showing the enzymic described above. The precipitate was dissolved in activity (Nos. 59-76) were combined, mixed with 2 ml of EDTA-KC1-Pi buffer, and chromato 2 volumes of cold acetone and centrifuged (7,000 graphed on a Sephadex G-200 column (Fig. 3). Fig. 1. Column chromatography on DEAE-cellulose. The enzyme solution (2 ml) after acetone fractionation (60-75%) was applied to a DEAE-cellulose column (2 •~ 15 cm) previously equilibrated with 10 mM phosphate buffer (pH 7) containing 2 mM EDTA and 0.1% KC1 (EDTA-KCI-Pr buffer). The column was washed with 60 ml of the same buffer containing 0.2 M KC1 and then eluted with 500 ml of a linear gradient of 0.2 to 0.5 M KC1 in the same buffer at a flow rate of about 30 ml per h, and fractions of 6 ml were collected. All procedures were carried out at 20•Ž. Other experimental conditions are described in the text. Fig. 2. Column chromatography on DEAE-cellulose. The enzyme solution (2 ml) from the first DEAF-cellulose column was fractionated with acetone and then applied to a DEAE- cellulose column (2•~15 cm). Other experimental conditions were the same as for Fig. 1. Vol. 82, No. 6, 1977 1744 S. IKUTA, S. IMAMURA, H. MISAKI, and Y. HORIUTI The fractions constituting the enzyme peak (Nos. dicates that the enzyme has a typical flavo-protein 46-54) were collected and lyophilized. The puri spectrum. The flavo-protein enzyme is easily fication procedure is summarized in Table I. The reduced by choline, the substrate, or by sodium lyophilized powder, having a specific activity of hydrosulfite as also shown in the absorption 12.5 units per mg protein, gave a single protein spectra: reduction is reversible.