Agric. Biol. Chem., 52 (3), 743~748, 1988 743

Purification and Characterization of Oxalate Oxidase from Pseudomonas sp. OX-53

Hirokazu Koyama Noda Institute for Scientific Research, Noda, Chiba 278, Japan Received September 14, 1987

Oxalate oxidase (EC 1.2.3.4) was purified to apparent homogeneity from Pseudomonas sp. OX- 53. The molecular weight of the was about 320,000 by Sephadex G-200 column chromatography and 38,000 by sodium dodecyl sulfate disc electrophoresis. The isoelectric point of the enzyme was pH4.7 by isoelectric focusing. This enzyme contained 1. 12 atoms of and 0.36 atoms of zinc per subunit. Besides oxalic acid, the enzyme oxidized glyoxylic acid and malic acid at lower reaction rates. The Michaelis constant of the enzymewas 9.5 mMfor oxalic acid at the optimal pH4.8. The enzyme was stable from pH 5.5 to 7.0. The enzyme was activated by flavins, phenylhydrazine, and o-phenylenediamine, and inhibited by I~, Br~, semicarbazide, and hydroxylamine.

Since oxalate oxidase (oxalate : oxi- acterization of this microbial oxalate oxidase. doreductase, EC1.2.3.4) was discovered in a mold by Houget and Mayer,1) oxalate oxidase MATERIALS AND METHODS has been found in various plants: barley seed- lings and roots,2'3) beet stems,4) and sorghum Materials. Ovalbumin, bovine serum albumin, yeast leaves.5) This enzymeproduces two moles of alcohol dehydrogenase, RNA-polymerase from E. coli, carbon dioxide and one mole of hydrogen aldolase, and ferritin were obtained from Boehringer Mannheim-Yamanouchi,Tokyo. Horse-radish peroxidase peroxide from one mole of oxalate and and beef liver catalase were purchased from Sigma oxygen. Chemical Co., St. Louis. DEAE-Sepharose CL-6B and (COOH)2 +O2 ->2CO2+H2O2 Sephadex G-200 were obtained from Pharmacia, Uppsala. Bio-Gel A-0.5m was obtained from Bio Rad Labora- tories, Richmond. Dye Matrex green A and Ampholine Oxalate oxidases from barley seedlings6) and pH 3.5~10.0 were purchased from Amicon Co., roots3) have been purified to homogeneity and Lexington and LKBProdukter, Bromma, respectively. All some of their properties have been reported. other chemicals were of spectral or analytical grade. However, oxalate oxidase has not yet been investigated in detail. Microorganism and cultivation. The bacterium, OX-53, In the microbial metabolism of oxalate, two was isolated from soil by an enrichment-culture technique types of enzyme are known. One is a fungal with a medium containing oxalate. The isolate was a Gram-negative, aerobic, non-spore forming short rod with oxalate decarboxylase (EC 4.1.1.2) producing polar flagella. It was classified in the genus Pseudomonas formate and carbon dioxide.7'8'9) The other is according to "Bergey's Manual."12) Cells were grown a bacterial oxalyl-CoA synthetase (EC aerobically for 20hr at 30°C in a medium (pH 8) contain- 6.2.1.8).10) Furthermore, oxalate oxidation in ing 0.5% potassium oxalate, 1%glycerin, 1 %yeast extract, teliospores of Tilletia contraversa has been 0.1% K2HPO4, and 0.05% MgSO4-7H2O. Cells were harvested by centrifugation and stored at 4°C. investigated by Vaisey et aLU) I have recently found an oxalate oxidase from a strain of the Enzymeassay. The standard assay was done polaro- soil bacterium Pseudomonas sp. OX-53. This graphically at 37°C with a Clark type oxygen electrode paper deals with the purification and char- YSI model 53 (Yellow Springs Instruments, Ohio). The 744 H. KOYAMA assay mixture (3ml) contained 300/miol of potassium removed by centrifugation and the super- citrate buffer, pH 5.0, 100/imol of potassium oxalate, natant with oxidase activity was put on a and enzyme. The reaction was initiated by the addition of the enzyme, and the initial velocity of oxygen con- DEAE-cellulose column (15x45cm) pre- sumption was measured. One unit of the enzyme was viously equilibrated with 20mM potassium denned as the amount of enzyme required to consume phosphate buffer, pH 7.0. The column was 1 /miol of oxygen permin. Both oxygen consumption washed with the same buffer containing 0.2 m and carbon dioxide formation were measured by the NaCland then the enzymewas eluted with the manometricmethod. Hydrogenperoxide formation was buffer containing 0.8m NaCl. Solid am- detected polarographically by addition of catalase. moniumsulfate was added slowly to the active Protein measurement.Protein was measured by the fraction to 35%saturation. After 60min, the method of Lowry et al.13) with bovine serum albumin as a precipitate was removed by centrifugation. standard. Elution patterns of protein upon column chro- Ammoniumsulfate was then added to the matography were monitored by the measurement of ab- supernatant to give 60% saturation. The re- sorption at 280nm. sulting precipitate was collected by centrif- Polyaerylamide gel electrophoresis. Analytical electro- ugation and resuspended in a minimal volume phoresis on polyacrylamide gel was done at 4°C by the of 20 mMpotassium phosphate buffer, pH 7.0. method of Davis.14) Gels were stained for protein with The active fractions were dialyzed overnight Coomassie brilliant blue and destained in 12.5% tri- against 20 mMpotassium phosphate buffer, pH chloroacetic acid. 7.0, and put on a DEAE-Sepharose CL-6B Molecular weight measurements. The molecular weight column (3.6 x 20cm) previously equilibrated of the enzyme was estimated by gel filtration on a with 20mMpotassium phosphate buffer, pH Sephadex G-200 column (2.3x90cm) at 4°C by the 7.0. The enzyme was eluted with a linear method of Andrews.15) The column was standardized with gradient of NaCl from 0.25m to 0.5m in the aldolase, yeast alcohol dehydrogenase, bovine serum al- same buffer. Active fractions were concen- bumin, ovalbumin, and ferritin. The molecular weight of the subunit of the enzyme was estimated by sodium trated by the addition of ammoniumsulfate dodecyl sulfate-polyacrylamide gel electrophoresis by the (60% saturation). The concentrated prepara- method of Weber and Osborn.16) Electrophoresis was tion was put on a column (2.6x100cm) of done using a 5%polyacrylamide gel column containing Bio-Gel A-0.5 m previously equilibrated with 0. 1 %sodium dodecyl sulfate. After electrophoresis for 4 hr 20mMTris-HCl buffer, pH 7.2. Gel nitration at 8 mAper gel at room temperature, the gel was stained and then destained as described in "Polyacrylamide gel was done with the same buffer at a flow rate of electrophoresis. " 20ml/hr. Active fractions were put on a Dye Matrex green A column (3.6x20cm) previ- Other analytical methods. Isoelectric focusing of the ously equilibrated with 20mMTris-HCl buff- enzyme in polyacrylamide gel was done by the method of er, pH 7.2. The column was washed with the Righetti and Drysdale17) with carrier ampholytes from pH 3.5 to 10.0 at 4°C. Absorption spectra were measured with same buffer and the enzymewas eluted with a a Hitachi 323 recording spectrophotometer (Hitachi, Ltd., linear gradient of KC1from zero to 1 m in the Tokyo) at 25°C. A Hitachi 180-80 atomic absorption buffer. Active fractions were concentrated and spectrophotometer was used for the metal measurement. dialyzed overnight against 20mM potassium phosphate buffer, pH 6.5. The overall purifi- RESULTS cation achieved was approximately 2000-fold with an activity yield of 23%. The purified Purification of oxalate oxidase enzyme preparation catalyzed the consump- All the procedures for isolation and purifi- tion of 629/imol of oxygen/min/mg of pro- cation of the enzyme were carried out at tein under the standard assay conditions. The 0~8°C. Harvested cells (260g as wet weight) results of the enzyme purification are sum- were suspended in 10 1 of 20niM potassium phosphate buffer, pH 6.5, and were treated marized in Table I. Disc electrophoresis of the with Triton X-100 (0.1%). The cell debris was purified enzyme in acrylamide gel showed only a single protein band (Fig. 1). Oxalate Oxidase from Pseudomonas 745

Table I. Purification of Oxalate Oxidase The activity was estimated by the polarographic method under the standard conditions.

Total Total Specific _..., Yield Step protein activity activity o/ (mg) (units) (units/mg) ^

Crude extract 24,700 7, 1 70 0.29 100 DEAE-Cellulose 2,980 5,950 2.0 83 Ammonium sulfate 1,360 5,570 4. 1 77 DEAE-Sepharose 1 47 4,980 34 69 CL-6B Bio-Gel A-0.5m 41.8 3,320 79 46 DyeMatrexgreenA 2.70 1,700 630 23

Fig. 2. Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis of Oxalate Oxidase. The purified Pseudomonas oxalate oxidase (#) was treated at 100°C for 5min in 1% sodium dodecyl sulfate, 2% 2-mercaptoethanol, and 25% glycerol. Standard proteins; a, trypsin inhibitor (20,100); b, bovine serum albumin (68,000); c, phosphorylase b (97,400).

Table II. Metal Content of Oxalate Oxidase The metal content of the purified oxalate oxidase (12.9 mg) was measured. The dialysates served as blanks. Content ent (atoms/38,000 daltons)

Mn 1.12 Zn 0.36 Fe 0.09 Fig. 1. Polyacrylamide Gel Electrophoresis of Cu 0 Pseudomonas Oxalate Oxidase. Mo 0 About 16/ig of the purified enzyme was put on the Co 0 column. The direction of migration is from the cathode Ni 0 (top) to the anode (bottom). An arrow represents the position of the tracking dye. Isoelectric point Molecular weight and subunit structure The isoelectric point of oxalate oxidase from The molecular weight of Pseudomonas oxa- Pseudomonas sp. OX-53was pH 4.7 by isoelec- late oxidase was about 320,000 by the tric focusing. Sephadex G-200gel filtration method. The subunit structure was examined by sodium Absorption spectra and metal analysis dodecyl sulfate polyacrylamide gel electro- Absorption spectra of the oxalate oxidase phoresis, which showed a single band of showed an absorption peak at 278nmand a stained protein. The molecular weight of shoulder at 288 nm, and no characteristic ab- the subunit was about 38,000 (Fig. 2). These sorption band was found from 350nm to results suggested that the enzyme was com- 700 nm. Before metal analysis, enzyme samples posed of eight subunits of identical molecular were dialyzed against doubly distilled water. weight. Atomic absorption spectrometry indicated that the purified Pseudomonas oxalate oxi- 746 H. Koyama dase contained 1.12atoms of manganese, 0.36 atoms of zinc, and only a trace of iron per subunit of molecular weight 38,000 (Table II).

Kine tics The initial reaction rates of oxygen con- sumption were measured at various concentrations under the standard assay con- ditions. The plots of the reaction velocity against oxalate concentration gave a typical Michaelis-Menten curve. The Michaelis con- stant of the enzyme was 9.5him for oxalate at pH 4.8. Fig. 3. Effects of pH on Oxalate Oxidase Activity. Substra te specificity Oxidase activities were measured polarographically under The relative activity of the enzyme with the standard conditions with 50 mMcitrate buffer at vari- other organic acids as substrate wasexamined ous pHs. (Table III). The enzyme was almost specific to oxalic acid. Glyoxylic acid, DL-malic acid, Table III. Substrate Specificity of maleic acid and citric acid were oxidized, but Oxalate Oxidase with extremely lower efficiencies. Other acids Oxygen consumption was measured polarographically. (succinic acid, formic acid and glycolic acid) Relative activity was calculated from the initial velocity of oxygen consumption. were not oxidized. Substrate Relative activity Stability and optimum pH (100 mM) (%) Whenthe enzyme was stored at -20°C in Oxalic acid 100 20mMpotassium phosphate buffer, pH 6.5, Glyoxylic acid 3.7 virtually no activity was lost for at least six DL-Malic acid 2. 1 Maleic acid 1.9 months. After thermal treatment for ten min Citric acid <0.01 remaining activities were measured. Re- Malonic acid 0 maining activities of 100%, 76%, and 9% of Succinic acid 0 the original activity were observed after heat- Formic acid 0 ing at 50°C, 60°C, and 65°C, respectively. Glycolic acid 0 a-Ketoglutaric acid 0 After the enzyme was incubated in 50mM Ascorbic acid 0 citrate buffer (pH 3-6.5) or 50mMpotassium phosphate buffer (pH 6-9) at 50°C for 10min, the activity of the enzymewas mea- o-phenylenediamine significantly activated the sured. The enzyme was stable from pH 5.5 to enzyme. Amongmetal ions tested, manganese 7.0. Figure 3 shows the effects of pH on the ion slightly stimulated the activity. On the activity of Pseudomonas oxalate oxidase. The contrary, /?CMB, KCN and NaN3 slightly optimum pH was about 4.8. inhibited the enzyme. Metal-chelating agents also inhibited the enzyme activity. Semi- Effects of activators and inhibitors carbazide and hydroxylamine strongly inhib- The effects of various reagents on the en- ited the enzyme activity. zyme activity were investigated (Table IV). Flavin compounds, such as riboflavin, FMN, DISCUSSION and FAD, and hydroquinone stimulated the oxalate oxidase activity. Phenylhydrazine and Some properties of oxalate oxidase from Oxalate Oxidase from Pseudomonas 747

Table IV. Effects of Various Reagents on Oxalate Oxidase Activity Oxygenconsumption was measured polarographically under the standard conditions. Unless otherwise noted, the reagent concentration was 0.1 him. _, Relative activity _ Relative activity Reagent ( \/o)O/. Reagent (%)

None 100 /7-Chloromercuribenzoate 96 Riboflavin 211 lodoacetamide 38 FMN 188 KCN 97 FAD 166 NaN3 90 Hydroquinone 223 Hydroxylamine 0 0-Phenylenediamine 605 Semicarbazide 43 Phenylhydrazine 380 KCl (l mM) 106 MgSO4 97 KBr (l mM) 42 MnSO4 175 KI (lmM) 6 HgCl2 64 EDTA 58 FeSO4 94 o-Phenanthroline 74 CuSO4 64 a,a '-Dipyridyl 69 ZnSO4 8-Hydroxyquinoline 59 barley seedlings have been reported by Sugiura sp. OX-53 required no additive co factor for its et al.6) The molecular weight of oxalate oxi- enzyme activity and the spectrum of the en- dase from barley seedlings was 150,000 and zyme showed no characteristic absorption the enzyme consisted of two identical subunits. band in the visible wavelengths. Pseudomonas The isoelectric point of the plant enzymewas oxalate oxidase contained 1.12 atoms of pH 2.8 and the Michaelis constant for oxalate manganese, 0.36 atoms of zinc, and a trace of was 0.42mM. Other differences between iron per subunit (molecular weight, 38,000). Pseudomonasand barley seedling oxalate oxi- Furthermore, some stimulation of the enzyme dases werefound in the substrate specificity activity by manganese ion was observed. These and effects of inhibitors. OptimumpHs of observations showed that Pseudomonas oxa- oxalate oxidase from barley seedlings,6) beet late oxidase is not an iron-flavoprotein but a stems,4) and sorghum leaves5) were pH 3.5, 5.7, metaloprotein (manganese). and 5.0, respectively. The optimum pH of Pseudomonas enzyme, pH 4.8, was similar with Acknowledgments.The author is very grateful to Dr. that of sorghum leaves. Thus, Pseudomonas N. Saito, the Research Division of the KikkomanCorp., and Dr. H. Suzuki, Kitasato University School of oxalate oxidase and the plant enzymesappear Medicine, for valuable suggestions and discussions. The to differ in many properties. technical assistance of Miss K. Moro is also gratefully Datta and Meeuse18) found that moss oxa- acknowledged. late oxidase required FMNor riboflavin to catalyze the reaction. The oxalate oxidase REFERENCES from barley seedlings was activated by the 1) J. Houget, A. Mayer and L. Plantefol, Compt. addition of flavins and quinones.2) On the Rend., 185, 304 (1927). other hand, mossoxalate oxidase activity was 2) J. Chiriboga, Biochem. Biophys. Res. Commun., ll, stimulated by Fe3+ 19) and Pundir and Nath 277 (1963). suggested the essentiality of Fe2+ for the en- 3) P. G. Pietta, A. Calatroni, D. Agnellini and M. zyme activity from sorghum leaves.5) How- Pace, Preparative Biochem., 12, 341 (1982). 4) D. M. Obzansky and K. E. Richardson, Clin. ever, no evidence was found that these are the Chem., 29, 1-815 (1983). native co factors of plant oxalate oxidase. 5) C. S. Pundir and R. Nath, Phytochemistry, 23, Purified oxalate oxidase from Pseudomonas 1871 (1984). 748 H. KOYAMA

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