J. Biochem. 82, 1741-1749 (1977)

Purification and Characterization of 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 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 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- 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 , 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. band on disc gel electrophoresis in the presence Isoelectric Point and Molecular Weight- and absence of SDS (Fig. 4). Isoelectric separation with Ampholine carrier am Absorption Spectrum-The absorption spec pholytes showed that the enzyme has a pI of 4.5 trum of the purified choline oxidase showed max (Fig. 6), although the recovery on isoelectric ima at 363 nm and 450 nm in the visible region, focusing was low (15%). The molecular weight having a shoulder at 480 nm (Fig. 5). This in- of the enzyme was determined to be about 83,000 by gel filtration on Sephadex G-150 (7) and about 71,000 by SDS-polyacrylamide disc gel electro- phoresis (Fig. 7-a & b).

Fig. 3. Column chromatography on Sephadex G-200. The enzyme solution (2 ml) from the second DEAE- cellulose column was fractionated with acetone and then Fig. 4. Polyacrylamide disc gel electrophoresis of the applied to a Sephadex G-200 column (2.7•~90 cm) at purified enzyme. The experimental conditions were as 20•Ž previously equilibrated with EDTA-KC1-Pi buffer. described in the text, except that 12 ƒÊg and 10 ƒÊg of the Fractions of 6 ml were collected at a flow rate of about enzyme protein (the "lyophilized powder" of Table I) 15 ml per h. Other experimental conditions are de were applied to columns in the presence and absence of scribed in the text. SDS, respectively.

TABLE I. Summary of the purification of choline oxidase from A. globiformis.

J. Biochem. PURIFICATION AND CHARACTERIZATION OF CHOLINE OXIDASE 1745

TABLE ‡U. Estimation of the reaction products of choline oxidation with choline oxidase. Experimental conditions are described in the text. The enzyme source was the "lyophilized powder" of Table ‡T.

Fig. 5. Absorption spectrum of choline oxidase. En with the enzyme (200 units), the resulting reaction zyme concentration, 9.2 mg protein per ml EDTA-KCI- mixture contained about 30 ƒÊmol of betaine and P1 buffer. Curve 1, oxidized enzyme; Curve 2, after addition of 0.2 mM choline chloride; Curve 3, after addi quinoneimine dye (equivalent to 60 ƒÊmol H2O2) tion of 2 mM choline chloride; Curve 4, after addition (Table ‡U). During the oxidation of choline to of sodium hydrosulfite. Aerobic conditions at 25•Ž. betaine, the transient intermediate, betaine alde Other experimental conditions are described in the text. hyde, accumulated in the reaction mixture and

disappeared at the end of the incubation time

Some Characteristics of Choline Oxidase- accompanied with the production of a stoichi

Identifzcation and determination of reaction products ometric amount of H202 per choline: using 0.1 of the oxidation catalyzed by choline oxidase: ƒÊ mol of choline and 0.25 units of enzyme, the

The methods used for the identification and deter aldehyde accumulated reaching a maximum of mination of betaine, betaine aldehyde, and hydro- about 0.04-0.05 ƒÊmol 3 to 8 min after the start of the reaction and after 30 min, when the aldehyde gen peroxide (quinoneimine dye) produced from the oxidation of choline and betaine aldehyde are could no longer be detected, about 0.2 ƒÊmol of

described under " MATERIALS AND METH- H2O2 was produced (Fig. 8-a). During the betaine

ODS." When 30 ƒÊmol of choline was incubated aldehyde oxidation with the enzyme (0.25 units),

Fig. 6. Isoelectric focusing of choline oxidase. The experimental conditions were as de scribed in the text, except that 12.5 units of the enzyme (the "lyophilized powder" of Table

‡T) was applied to the electrofocusing column. The total recovery of enzyme activity was 15%.

Vol. 82, No. 6, 1977 1746 S. IKUTA, S. IMAMURA, H. MISAKI, and Y. HORIUTI

Fig. 7-a & b. Determination of the molecular weight of choline oxidase by gel filtration (a) and SDS-poly- acrylamide gel electrophoresis (b). (a) The enzyme

(0.5 units) in EDTA-KC1-P1 buffer was applied to a Fig. 8-a & b. Time courses of oxidation of (a) choline Sephadex G-150 column (1•~100 cm) equilibrated with and (b) betaine aldehyde coupled with H202 generation the same buffer. Elution was performed with the same by choline oxidase. The reaction for H2O2 generation buffer at 20•Ž. The flow rate was adjusted to approx. was carried out for the indicated periods in the same 3 ml/h, and fractions of 0.9 ml were collected. The reaction mixture as for the assay of H2O2-generating reference proteins were run under similar conditions activity, except that 0.2 mM choline chloride or betaine

(Vo; void volume, Ve; elution volume). Other experi aldehyde was used. The reaction for formation or dis mental conditions are described in the text. (b) The appearance of betaine aldehyde was carried out in the enzyme protein (10 ƒÊg) was subjected to electrophoresis, same reaction mixture as for the assay of betaine alde- and the reference proteins were run under similar con hyde-forming activity, except that 0.2 mM betaine ditions. Other experimental conditions are described aldehyde, instead of choline chloride, was added to the in the text. The enzyme source was the "lyophilized mixture for disappearance of betaine aldehyde. The "lyophilized powder" of Table ‡T was used as the enzyme powder" of Table I. source, and 0.25 units enzyme were added for each reaction. the rate of disappearance of the substrate aldehyde

(0.1 ƒÊmol) corresponded to the rate of generation of H202 and by the end of the incubation period oxidations of choline and betaine aldehyde cor

(30 min), a stoichiometric amount (0.1 ƒÊmol) of responded quantitatively to those of oxygen con

H2O2 had been generated (Fig. 8-b). sumption (Table ‡V). These results indicate that the enzyme can Based on the above results, it is concluded catalyze two-step oxidation of choline to betaine that choline oxidase catalyzes the following reac aldehyde to betaine in reactions coupled with tions:

H202 generation, and that the first step is probably Choline+O2Betaine aldehyde+H2O2 faster than the second step.

The respective rates of H202 generation in the Betaine aldehyde+02+H20Betaine+H2O2

J. Biochem. PURIFICATION AND CHARACTERIZATION OF CHOLINE OXIDASE 1747

Substrate specificity and effects of substrate Effect of pH on activity: The maximal enzyme concentrations: The substrate specificity was ex activity was observed in the range of pHs 7-8, amined using derivatives of aminoethanol, alde when choline was used as a substrate (Fig. 10); hydes, and alcohols (Table ‡W). Choline and since the molar extinction of quinoneimine dye betaine aldehyde served as substrates for the en was significantly influenced by pH, the activity was zyme, but very low activities were found towards assayed by measuring the formation of betaine the other compounds: the activities relative to that aldehyde. of choline were about 50%. for betaine aldehyde Determinations of Choline and Betaine Alde- and below 6%. for the other compounds. The hyde with Choline Oxidase-The purified enzyme Michaelis constants (Km) were found to be 1.2 mM

for choline and 8.7 mM for betaine aldehyde, when

the rates of substrate oxidation were measured as

the formation of quinoneimine dye due to gener

ation of H2O2 (Fig. 9).

TABLE ‡V. Activities of H202 generation and oxygen consumption with choline oxidase. Experimental con ditions are described in the text. The enzyme source was the "lyophilized powder" of Table I.

Fig. 9. Lineweaver-Burk plot of the activity of choline oxidase. Experimental conditions were as described in the text, except that various concentrations of choline and betaine aldehyde were used. The enzyme source

TABLE ‡W. Substrate specificity of choline oxidase. was the "lyophilized powder" of Table I. Experimental conditions for the activity assay of H2O2

generation were as described in the text, except that various substrates were used. The enzyme source was the "lyophilized powder" of Table I.

Fig. 10. Effect of pH on the activity of choline oxidase. The enzyme activities at various pH values were assayed in the reaction mixture for the assay of betaine aldehyde forming activity described in the text. Dimethylgluta rate buffer was used for pH 4 to 6 (•œ); phosphate buffer for pH 6 to 8 (•ü); glycylglycine buffer for pH 8 to 9

(• ); and glycine buffer for pH 9 to 10 (ƒ¢). The enzyme source was the "lyophilized powder" of Table ‡T.

Vol. 82, No. 6, 1977 1748 S. IKUTA, S. IMAMURA, H. MISAKI, and Y. HORIUTI

acteristics of the enzyme reaction (NAD(P)-de- pendent or -independent and specific or nonspecific for betaine aldehyde), and the conditions necessary for oxidation of aldehyde to betaine. The NAD(P)-dependent betaine aldehyde dehydrogenase that was claimed to be present in the cytoplasm or soluble supernatant fraction (17-21) was partially purified from a liver homog enate (17) and completely from a cell-free extract of bacteria (20). On the other hand, the betaine aldehyde dehydrogenase which was clamined to be associated with mitochondria, as opposed to the above cytoplasmic enzyme (22-27), has not been isolated or characterized. Fig. 11. Estimation of choline and betaine aldehyde The choline oxidase purified and characterized with choline oxidase. Experimental conditions were as in this work was found to be a new type of flavo described in the text, except that 0.5 units of the enzyme and various concentrations of substrates (choline and protein enzyme, which can oxidize both choline betaine aldehyde) were used and the reaction period and betaine aldehyde using molecular oxygen as a was 20 min. The enzyme source was the "lyophilized primary electron acceptor and producing H202: powder" of Table 1. mitochondrial , a flavo protein enzyme (11, 28-30), is thought to catalyze was tested for the quantitative estimations of the oxidation of choline only and to mediate choline and betaine aldehyde by measuring the electron transfer from the substrate to oxygen via formation of quinoneimine dye (Fig. 11). The the electron transport chain involving cytrochromes formation of quinoneimine dye was proportional to (28, 31) and ubiquinone (14). In mitochondria or the amounts of these substrates up to 50 nmol. particulate fractions, it is still uncertin whether Thus, this enzyme can be used for the determina the oxidation of choline to betaine requires co tion of choline and betaine aldehyde. factors, such as NAD, and can generate H202 under suitable conditions. Then, our demonstra tion of the existence of choline oxidase might DISCUSSION provide clues for elucidating the mechanism of the Three kinds of that oxidize choline to enzymic oxidation of choline to betaine in mito betaine aldehyde or betaine aldehyde to betaine chondria or particulate fractions and also support have been found in animal tissues and bacterial the scheme we proposed for the oxidation of cells by various investigators. These three kinds choline to betaine in A. globiformis cells (1), which of enzymes are choline dehydrogenase [EC 1.1.99.1, has not yet been reported for other bacterial cells choline: (acceptor) ], NAD(P)-de- or animal tissues. pendent betaine aldehyde dehydrogenase [EC 1.2.1.8, betaine-aldehyde: NAD+ oxidoreductase] REFERENCES and NAD(P)-independent betaine aldehyde dehydrogenase. 1. Ikuta, S., Matsuura, K., Imamura, S., Misaki, H., Choline dehydrogenase has been demonstrated & Horiuti, Y. (1977) J. Biochem. 82, 157-163 to be localized in mitochondria or bacterial mem 2. Barabanov, M.I., Vil'chinskii, S.T., & Litvak, I.M. branes as a particulate enzyme and has been par (1962) Tr. Kievsk. Tekhnol. Inst. Pishchevoi Prom. 25,69-73 tially purified in a soluble form from a mammalian 3. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & liver homogenate (8-15) and from bacterial cells Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 (16). Unlike the findings on the choline dehy 4. Vesterberg, 0. (1971) Methods Enzymol. 22, pp. drogenase, those on the dehydrogenases for betaine 389-412, Academic Press Inc., New York aldehyde are conflicting with respect to the cellular 5. Davis, B.J. (1964) Ann. N.Y. Acad. Sci, 121, 404-427 location (mitochondria or cytoplasm), the char- 6. Weber, K., Pringle, J.R., & Osborn, M. (1972)

J. Biochem. PURIFICATION AND CHARACTERIZATION OF CHOLINE OXIDASE 1749

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