Agric. Biol. Chem., 44 (9), 2039•`2045, 1980 2039

Properties of New Glycerol Oxidase from Aspergillus japonicus AT 008

Takayuki UWAJIMA and Osamu TERADA

Tokyo Research Laboratory, Kyowa Hakko Co., Machida-shi, Tokyo 194, Japan Received February 5, 1980

Purified glycerol oxidase from Aspergillus faponicus AT 008 was homogeneous by ultracen- trifugation and acrylamide gel electrophoresis. The molecular weight was determined to be 400,000 by sedimentation equilibrium, and the isoelectric point was found to be 4.9 by isoele- ctric focusing. The enzyme showed spectral characteristics of a heme protein. The reduced form possessed absorption maxima at 557 and 430 nm and the oxidized one at 557, 530, 420, 280, and 238 rim. The heme in the enzyme was identified as protoheme IX (one mol per mol of enzyme protein). Glycerol was the best for the enzyme, and the Km value for glycerol was deter- mined to be 10.4 mal. Dihydroxyacetone was oxidized at 59 % of that for glycerol, but glycerol 3-phosphate, dihydroxyacetone phosphate, methanol, and ethanol were not oxidized at all. The enzyme had an optimal pH at 7.0 with glycerol as substrate, and the enzymatic activity increased by treatment in alkaline pH. The enzyme was also activated by addition of several divalent metal ions including Zn2+, Nit+, and Mgt+.

During the investigation on glycerol metabo- Sephadex G-200. The final preparation lism by microorganisms, the authors found a achieved was homogeneous in polyacrylamide new enzyme which oxidized glycerol to glyceral- gel electrophoresis and ultracentrifugation. dehyde without requirement of an exogeneous The present paper describes molecular and .'' The amount of hydrogen peroxide catalytic properties of the enzyme from generated was equivalent to that of Aspergillus japonicus AT 008 as well as its consumed, and the reaction was shown21 characteristics as a hemoprotein. to proceed according to the following scheme: CH2OHCHOHCH2OH+O2 -> MATERIALS AND METHODS CHOCHOHCH2OH+H202. This glycerol-oxidizing enzyme was quite Preparation of glycerol oxidase. Purified glycerol oxidase (specific activity 142.2) was prepared from different from any previously reported Aspergillusfaponicus AT 008, as described previously.2r which participate in the metabolism of glycerol, and thus we have designated tentatively the Materials. Peroxidase, c, lactate de- enzyme as "glycerol oxidase (glycerol: oxygen hydrogenase, and urease were pur- chased from Sigma Chemical Co., St Rouis, U.S.A. 1-)." Glycerol oxidase was oxidase was prepared from Brevibacterium inducibly formed in the mycelia of some strains sterolicum according to our methods.3) Sephadex belonging to Ascomycetes, grown on glycerol as G-200 was obtained from Pharmacia Fine Chemicals, a sole carbon source. The enzyme was purified Uppsala, Sweden. All other reagents were of the from the cell-free extract of Aspergillus japoni- highest grade available from commercial sources. cus AT 008 by a procedure involving column Spectrophotometric determinations. Spectrophoto- chromatographies on DEAE-Sephadex A-25 metric determinations were carried out with a Schima- and hydroxylapatite, and gel filtration on dzu Model MPS-50L multipurpose recording spectro- photometer or with a Hitachi Model 139 Perkin-Elmer Abbreviations: TES, N-tris (hydroxymethyl)- spectrophotometer. methyl-2-aminoethane sulfonic acid; EDTA, ethylene- diamine tetraacetic acid; SDS, sodium dodecyl sulfate. Assay of glycerol oxidase activity. Enzyme assay 2040 T. UWAJIMA and 0 . TERADA

was based on the measurement of hydrogen peroxide •@ as standard.

generated during the oxidation of glycerol. The hydro Sedimentation analysis. Sedimentation velocities gen peroxide was couplingly oxidized with 4-amino- antipyrine and phenol in the presence of peroxidase to were measured at twelve 3-min intervals by a Hitachi model UCA-1 ultracentrifuge equipped with Schlieren form a quinoneimine dye by the method of Attain et al.4) The amount of quinoneimine formed was me optics and a double-sector cell, operating at 51,200 rpm at 20•Ž. The solvent employed was 0.01 M TES- asured spectrophotometrically at 500 nm. The assay NH40H buffer, pH 7.0. system in a final volume of 3.0 ml contained 50 ƒÊmol

of glycerol, 100 ƒÊmol of TES-NH4OH buffer (pH 7.0), Determination of molecular weight. Molecular 21 timol of phenol, 1.2 ƒÊmol of 4-aminoantipyrine, weight was determined by the centrifugal sedimentation 100 units of horseradish peroxidase, and a suitable equilibrium method of Yphantis.6) The equilibrium amount of glycerol oxidase. The incubation was per ultracentrifugation was performed at 20•Ž with the formed at 37•Ž for 10 min with shaking. The control Hitachi model UCA-1 analytical ultracentrifuge and an run contained all components except substrate. Gly eight-channel cell. Four of the channels served for cerol oxidase activity determined by this assay method, four samples of different concentrations ranging from was found to be a linear function of both incubation 1.2 to 4.8 mg/ml in 0.01 M TES-NH4OH buffer, pH 7.0, time and protein concentration with the purified enzyme and the other four for the buffer. The equilibrium was preparation, at least up to the absorbance of 1.0 at established in 20 hr at a speed of 3,230 rpm. The 500 nm (Fig. 1). initial concentration of the enzyme was determined by centrifugation at 12,290 rpm at 20•Ž with a synthetic

boundary cell. All Schlieren observations were made at a constant phase-plate angle of 65•Ž.

Molecular weight was also estimated by the gel filtra tion method of Andrews.7) Sephadex G-200 was

packed to a column (2 •~ 50 cm), and was equilibrated with 0.05 M TES-NH40H buffer, pH 7.0, containing 0.1 M NaCl. The flow rate was adjusted to 30 ml per

hr, and 3.0 ml-fractions were collected. Five protein standards were used: horse cytochrome c (mol. wt. 12,400), cholesterol oxidase (mol. wt. 33,000), lactate

dehydrogenase (mot. wt. 115,000), glucose oxidase (mol.

wt. 153,000), and urease (mol. wt. 470,000). FiG. 1. Enzyme Activity as a Function of Incuba tion Time and of Enzyme Concentration. Isoelectric focusing. Glycerol oxidase was subjected

The enzyme activity was determined by measuring to isoelectric focusing with Carrier Ampholites of pH hydrogen peroxide formation as described in the text. 3.5•`10.0 (LKB-Produkter AB, Stockholm, Sweden).

In Fig. 1A, the purified enzyme of 5 ƒÊg protein was, One percent of the Ampholites in a sucrose density

used. In Fig. III, the incubation was carried out for gradient (20•`60 %) was layered in a LKB 8102 column 10 min. (vol. 440 ml) with a gradient mixer. Electrofocusing was run at 4•Ž for 48 hr with a constant potential of

1,200 volts. The column was then carefully drained Enzyme activity was also assayed by measuring oxy through the bottom tubing, and 2.0 ml-fractions were gen uptake with a Bioxygraph Oxygen Electrode (Kyu collected. Each fraction was individually tested for the sui Kagaku Kenkyusho Co., Ltd.) placed in a thermo pH value, protein concentration, and enzyme activity. statically controlled vessel at 37•Ž. The reaction mix ture contained 50 ƒÊmol of glycerol, 100 ƒÊmol of TES-

NH40H buffer (pH 7.0), and a suitable amount of gly RESULTS cerol oxidase in a final volume of 3.0 ml. Molecular properties of glycerol oxidase One unit of enzyme activity was defined as the amount of enzyme which catalyzes the formation of Homogeneity. Purified glycerol oxidase

mol of H202 per min at 37•Ž in the standard assay 1 ƒÊ sedimented as a single, symmetric Sehlieren conditions. Specific activity was expressed as units per peak on ultracentrifugation in 0.01 M TES- mg of protein. NH,OH buffer, pH 7.0 (Fig. 2). Assuming a

partial specific volume of 0.74, the sedimenta Determination of protein concentration. Protein tion coefficient in water at 20•Ž (s // w) was concentration was determined by the method of Lowry et al.,5) using crystalline bovine serum albumin calculated to be 9.98 Svedberg units, when the Properties of New Enzyme Glycerol Oxidase 2041

FIG. 3. Determination of Molecular Weight of Glycerol Oxidase by Sephadex G-200 Gel Filtration at pH 7.0. A, cytochrome c; B, cholesterol oxidase; C, ; D, glucose oxidase; E, glycerol oxi FIG. 2. Sedimentation Patterns of Glycerol Oxidase from Aspergillus japonicus AT 008. dase; F, urease.

The progress of ultracentrifugation at 20•Ž is shown from A to D at 6, 15, 21, and 27 min after reaching

51,200 rpm. Protein concentration was 0.48 % in 10 mM TES-NH4OH buffer, pH 7.0. Pictures were taken at a bar angle of 65•Ž. protein concentration was varied from 1.9 to 7.5 mg per ml of 0.01 M TES-NH4OH buffer, pH 7.0. The enzyme gave a single band on acrylamide gel electrophoresis carried out at pH 8.3.2) FIG. 4. Isoelectric Focusing of Glycerol Oxidase in

a pH 3.5•`10.0 Gradient. Molecular weight. The molecular weight Purified enzyme (15 mg of protein, specific activity of glycerol oxidase was determined by the 142.2) was used. Fractions of 2.0 ml were collected sedimentation equilibrium method of Yp- and tested for pH (------), absorbance at 280 nm (•›•\•›) hantis8) as described above. Extrapolation of and enzyme activity(•œ•\•œ). the values obtained in four different enzyme concentrations (1.2, 2.4, 3.6, and 4.8 mg/ml) to zero concentration, led a value of 400,000 for the molecular weight of the enzyme, assuming a partial specific volume of 0.74.

The molecular weight was also estimated to be 395,000 by measurement of the relative rate of migration on a Sephadex G-200 column at pH 7.0 (Fig. 3).

Isoelectric point. The isoelectric point was FIG. 5. Absorption Spectra of Purified Glycerol found to be 4.9 by isoelectric focusing with Oxidase. Carrier Ampholites of pH 3.5•`10.0 (Fig. 4). A, the native enzyme (protein concentration 3.0 mg/

ml, specific activity 142.2); B, the reduced enzyme after

Absorption spectra. The purified enzyme the addition of 5 ƒÊmol of sodium dithionite to A under showed typical absorption spectra of a heme anaerobic conditions. 2042 T. UWAJIMA and O . TERADA

TABLE I. EFFECT OF INHIBITO

Enzyme activity was assayed by // measu // g oxygen uptake as described in the text. Glycerolerol oxidase

(10 ƒÊg of protein, specific activity 142.2) was incubated with glycerol in the presence of variable concentra tions of inhibitors under the standard assay conditions.

FIC. 6. Absorption Spectra of the Reduced Pyridine Ferrohemochrome of Glycerol Oxidase (A) and

Authentic Hematin (B).

To 1.0 ml of the enzyme (3.6 mg of protein, specific

activity 142.2) and hematine (2.4 ƒÊM) were added 0.1 ml of pyridine, 0.02 ml of 5 N sodium hydroxide,

and 5 ƒÊmol of sodium dithionite to obtain the pyridine ferrohemochromes.

protein. The absorption peaks of the di- thionite-reduced form were found at 557 nm and 430 nm, whereas those of the oxidized

(native) form were at 557 nm, 530 nm, 420 nm, 280 nm, and 238 nm (Fig. 5). The absorption ratios A557 nm/A280 nm, A530 nm/A280 nm, and

A420 nm/A2s0 nm in the oxidized form were 0.078, 0.083, and 0.41, respectively. The extinction coefficient (El% lem) of glycerol at

280 nm in 0.01 M TES-NH4OH buffer of pH 7.0 was determined to be 14.70. Figure 6 shows the absorption spectrum of the pyridine ferrohemochrome of the enzyme as well as that of the authentic hematin. The coincidence of the absorption maxima and troughs indi •@copper-chelating agent diethyldithiocarbamate cated that the iron porphyrin in the enzyme is was observed to cause about 40 % inhibition protoheme IX. The content of heme was of the activity at a concentration of 1.0 mM. determined from the extinction coefficient of Neither flavin nucleotide nor pyridoxal pho the pyridine ferrohemochrome (E mM=32 sphate may be involved in the enzyme activity, at 557 nm).8) The enzyme was found to con since quinacrine, chlortetracycline , hydrazine, tain 0.94 mot of heme per mol of enzyme pro or NaBH4 showed no effects on the enzyme tein, assuming the molecular weight of 400,000. activity. The enzyme was resistant to sulf hydryl reagents such as p-chloromercuric

Effect of inhibitors. The effects of a number benzoate, heavy metal ions, and . iodoacetic of inhibitors on the enzyme reaction are given acid. On the other hand, the enzyme lost in Table I. The enzyme activity was potently considerable activity on modification with N - inhibited by potassium cyanide, hydroxyl bromosuccinimide, which is // reactive amine, and sodium azide, common inhibitors with tryptophan residues. of iron porphyrin enzymes. But the enzyme was not inhibited by EDTA, ƒ¿, ƒ¿•Œ-dipyridyl, Catalytic properties of glycerol ox o-phenanthroline, or 8-hydroxyquinoline. A Stability. The purified enzyn // // be Properties of New Enzyme Glycerol Oxidase 2043

TABLE II. SUBSTRATE SPECIFICIT

OF GLYCEROL OXIDASEY

Enzyme activity was assayed by measuring hydro

gen peroxide formation as described in the text. Glycerol oxidase (2 ƒÊg of protein, specific activity 142.2) was incubated with 50 ƒÊmol of each substrate under the standard assay conditions.

FIG. 7. Effect of pH on Enzyme Activity.

Enzyme assays were carried out by measuring oxygen uptake (•œ•\•œ) and hydrogen peroxide formation

(•›•\•›) as described in the text. The enzyme (5 Fag of protein, specific activity 142.2) was incubated with

glycerol in 0.03 M TES-NH40H of pH as described, under the standard assay conditions.

preserved at 5•Ž in 0.05 M TES-NH4OH buffer or boric acid-potassium chloride-sodium carbonate buffer of pH over the range of 8.0

to 10.0 for a week. The enzyme could be stored at -80•Ž in the above buffers for at least 6 months. On heating, the enzyme was stable up to 45•Ž and lost about 40 % of activity

at 50•Ž at pH 7.0 for 15 min.

Effect of pH. The effect of pH on the enzyme activity is shown in Fig. 7. When the enzyme activity was measured by hydrogen

peroxide formation and oxygen uptake, the tacked at all. Among the saccharides, galac pH optima were found to be 7.0 and 7.5, respectively. The assay of hydrogen peroxide tose and fructose showed slight reactivities, but formation showed somewhat higher values in glucose and sucrose were inert not reactive. acid media and lower ones in alkaline media, than the assay of oxygen uptake. Effect of substrate concentration. The effect of substrate concentration on the initial oxida

Substrate specificity. Table II illustrates tion rate was tested with glycerol as substrate. relative activities of the enzyme with a variety The saturation curve obeyed Michaelis-Menten of hydroxyl compounds. The enzyme assay kinetics, and the Lineweaver-Burk plots gave was carried out by following the formation of a Kin value of 10.4 mM (Fig. 8). hydrogen peroxide. Glycerol was oxidized most rapidly. Dihydroxyacetone was oxi Activation by alkali. The enzyme was dized at a rate of 59 % to that for glycerol. activated about two-fold by dialysis of the 1, 3-Propanediol, 1. 3-butanediol, and 1, 4- enzyme against 0.05 M borate buffer, pH 10.0, butanediol were oxidized at low rates, while for 24 hr (Fig. 9). But in more alkaline pH glycerol 3-phosphate, dihydroxyacetone pho than 11.0, the enzyme was rapidly inactivated. sphate, methanol and ethanol were not at 2044 T. UWAIIMA and 0. TERADA

TABLE III. EFFECT OF METAL IONS

Enzyme activity was assayed by measuring hydro

gen peroxide formation as described in the text. Glycerol oxidase (2 ƒÊg of protein, specific activity 142.2) was incubated with glycerol in the presence of

variable concentrations of metal ions under the standard assay conditions.

FIG. 8. Effect of Glycerol Concentration on Reac

tion Velocity.

Enzyme activity was assayed by measuring the forma tion of hydrogen peroxide. The reaction velocity, V was expressed as ƒÊmol hydrogen peroxide • min-1.

mg protein-1.

•@ assigned to cytochrome B. Potent inhibitions of the enzyme by potassium cyanide, hydroxyl amine, and sodium azide also confirmed the

participation of iron porphyrin in the oxida tion process. On the other hand, non-heme iron was probably not involved in the enzyme

FIG. 9. Activation of Enzyme by Alkali. reaction, since the addition of iron-chelating

After the enzyme was dialyzed for 24 hr against 0.02 M agents such as ƒ¿,ƒ¿•Œ-dipyridyl and o-phenan-

buffer of pH as indicated, the enzyme activity was throline resulted in no inhibition of enzyme assayed by measuring hydrogen peroxide formation activity. Diethyldithiocarbamate was some under the standard assay conditions. The buffer what inhibitory to glycerol oxidase activity. used were TES-NH4OH for pH 6.0•`9.0, boric acid- However, after inhibition of the enzyme by KCl-Na2CO3 for pH 10.0•`11.0, and NaOH-Na2HPO4 for 12.0. diethyldithiocarbamate, the enzyme activity was fully restored by dialysis or dilution with a Effect of metal ions. The enzyme activity buffer solution. Whether or not copper is was increased by several divalent metal ions directly involved in the enzyme activity cannot such as Zn2+, Mgz+, Ni2+, Co2+ and Mn2+ be ascertained from the data presented here. (Table III). Among them, Zn2+ was the most The stoichiometry of the enzyme reaction effective, showing 2.3-fold activation at a indicated that glycerol oxidase might belong concentration of 1.0 mM. to a group of oxidases which catalyze two electron reductions of oxygen, one molecule- DISCUSSION of hydrogen peroxide being formed from one molecule of the oxygen consumed. It has been Glycerol oxidase, a novel glycerol-oxidizing demonstrated so far that the two-electron enzyme, was found to occur in some molds transfer enzymes are flavoproteins (e. g. and was purified to apparent homogeneity glucose oxidase9)), copper-proteins (e. g,

from the cell-free extract of Aspergillus japoni galactose oxidase10)), or non-heme iron-pro- cas AT 008. Spectrophotometric studies with teins (e. g. secondary alcohol oxidase1l,12)) the purified preparation indicated that glycerol Thus, glycerol oxidase is considered to be a oxidase was a hemoprotein which could be novel example of two-electron transfer enzyme Properties of New Enzyme Glycerol Oxidase 2045 containing heme as its prosthetic group. lipoprotein lipase, may be employed for the It is of interest that glycerol oxidase was specific determination of triglyceride in sera. activated by dialysis against a buffer of al kaline pH. Matsumura et al.13) have also Acknowledgments. The authors thank Dr. Y. reported that bovine cytochrome oxidase was Matsuda in their laboratory for his valuable suggestions activated on alkali or SDS. The cytochrome and Mrs. Masako Shigemasa and Miss Hiromi Iriuchi jima for thier excellent technical helps. - oxidase was shown to separate into two active monomers in a suitable alkaline pH, indicating that the increase of activity was due to an in REFERENCES crease in the number of active species. It 1) T. Uwajima, H. Akita, K. Ito, A. Mihara, K. was felt that consideration of dissociation Aisaka and O. Terada, Agric. Biol. Chem., 43, association of glycerol oxidase molecule might 2633 (1979). be necessary for the elucidation of activation 2) T. Uwajima, H. Akita, K. Ito, A. Mihara, K. Aisaka and O. Terada, Agric. Biol. Chem., 44, 399 mechanism by alkali. (1980). The oxidative degradations of hydroxyl 3) T. Uwajima, H. Yagi, S. Nakamura and O. Terada compounds by microorganisms have been Agric. Biol. Chem., 37, 2345 (1973). extensively investigated at the enzymatic level. 4) C. C. Allain, L. S. Poon, C. S. G. Chan, W. Gunsalus et al.14) demonstrated glycerol 3- Richmond and P. C. Fu, Clin. Chem., 20, 470 (1974). phosphate oxidase which was specific for gly- 5) O. H. Lowry, N. J. Rosebrough, A. L. Farr and cerol 3-phosphate in certain lactic acid . R. J. Randall, J. Biol. Chem., 193, 265 (1951). Tani et al.15) isolated a yeast primary alcohol 6) D. A. Yphantis, Ann. N. Y. Acad. Sci., 88, 586 oxidase which was equally active for methanol (1960). and ethanol. These enzymes and the secon 7) P. Andrews, Biochem. J., 91, 22 (1964). 8) K. G. Paul, H. Theorell and A. Akeson, Acta dary mentioned above,16,17) Chem. Scand., 7, 1248 (1953). however, did not act on glycerol at all. 9) W. Franke and M. Deffher, Ann., 541, 117 (1939). Glycerol was the best substrate for glycerol 10) D. Amaral, L. Bernstein, D. Morse and B. L. oxidase. Dihydroxyacetone was also oxi Horecker, J. Biol. Chem., 238, 2281 (1963). dized at a significant rate. But glycerol 3- 11) T. Suzuki, Agric. Biol. Chem., 42,1187 (1978). 12) M. Morita, N. Hamada, K. Sakai and Y. Wata phosphate, dihydroxyacetone phosphate, nabe, Agric. Biol. Chem., 43, 1225 (1979). methanol, ethanol, and glucose were not 13) Y. Matsumura, Y. Orii and K. Okunuki, J. Japan. attacked. Glycerol oxidase seems to require Biochem. Soc., 42, 646 (1970) (in Japanese). the structure of CH2OHCXCH2OH as its 14) I. C. Gunsalus and W. W. J. Umbreit, J. Bacteriol. substrate. It is assumed that a primary al 49, 347 (1945). cohol group in the structure is oxidized, and 15) Y. Tani, T. Miya and K. Ogata, Agric. Biol. Chem., 36, 76 (1972). another primary alcohol may be essential 16) T. Suzuki, Agric. Biol. Chem., 40, 497 (1976). when the enzyme binds with the substrate. 17) Y. Watanabe, N. Hamada, M. Morita and Y. Since glycerol oxidase is a stable enzyme Tsujisaka, Arch. Biochem. Biophys., 174, 575 and has the high structural specificity toward (1976). glycerol, the enzyme in a combined form with