Enzymatic oxalate decarboxylation in Aspergillus niger

ARCHIVES

OF

BIOCHEMISTRY

Enzymatic

AND

BIOPHYSICS

Oxalate

106,

488-493

Decarboxylation

E. EMILIAN12 From

the

Departamento

(1964)

de Microbiologia, Litoral,

niger’

AND P. BEKES

Facultad Santa

Received

in Aspergillus

de IngenierZa Fe, Argentina July

Quimica,

Universidad

National

de1

1, 1963

Common fungi of the Aspergillus niger group produce an oxygen-dependent oxalate decarboxylase which is especially abundant in those strains that yield greater amounts of citric acid. Oxygen is not consumed during the reaction and causes a denaturation that is proportional to its partial pressure. Certain reducing compounds (e.g., o-phenylenediamine) and proteins (e.g., gelatin) protect and stimulate the purified enzyme when it acts in the presence of air.

quires the presence of a certain amount of oxygen, although this element is not consumed during the reaction. The present communication deals with the cultural conditions under which the common strains of Aspergillus niger produce (rapidly and without a period of induction) a decarboxylase that, although resembling that from Collybia, exhibits somewhat different characteristics. Some interesting properties of this enzyme are also described.

Several enzymes attacking oxalic acid have been identified in microorganisms. In certain bacteria (1, 2) there is an enzymatic system that decarboxylates oxalate to formate, under anaerobic conditions, in the presence of Mg, ATP,3 CoA, ThPP, and acetate (or succinate). With respect to fungi, Vaisey et al. (3) obtained an extract of Tilletia controversa (a pathogenic fungus from wheat) that oxidizes oxalic acid under aerobic conditions and produces carbon dioxide and, probably, hydrogen peroxide. This reaction is markedly stimulated by riboflavin and FMN (but not FAD). Shimazono (4) and Shimazono and Hayaishi (5, 6) have described an enzyme obtained from a wooddestroying fungus, Collybia velutipes, that decarboxylates oxalate yielding stoichiometric quantities of carbon dioxide and formate. However, this enzyme is different from that obtained from bacteria, since it does not need any of the above mentioned cofactors. It re-

EXPERIMENTAL

ORGANISMUSEDANDCULTURALCONDITIONS The strain of A. niger [A. phoenicis: Thorn and Raper (7)J used was a good producer of citric acid; its characteristics have been described in an earlier paper (8). For liquid surface cultures of the fungus the following medium was used (grams per liter): sucrose (practical), 100; ammonium chloride, 2.8; monopotassium phosphate, 2; crystallized magnesium sulfate, 1; and zinc acetate, 0.02. Aspergillus was grown for 3 days at 30°C. in 256ml. Erlenmeyer flasks containing 50 ml. of medium. Under these conditions each flask yielded 8-10 g. of wet mycelium (about 1 g. dry wt.).

1 Presented in part at the 8th Latin-American Congress of Chemistry, Buenos Aires, September 16-22, 1962. 2 Fellow of the Consejo National de Investigaciones Cientfficas y Tecnicas, Buenos Aires, Argentina. 3 Abbreviations used: ATP, adenosine triphosphate; CoA, coenzyme A; ThPP, thiamine pyrophosphate; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; FDA, o-phenylenediamine.

MANOMETRIC METHODS Carbon dioxide was determined by the usual manometric methods (9). The reaction was normally carried out in an atmosphere of air. Each flask contained 0.3 ml. of 0.1 M oxalate solution, pH 4.7, 0.1 ml. of enzyme solution, and 0.2 M 488

ENZYMATIC

OXALATE

acetate buffer, pH 4.7, up to a total volume of 3.5 ml. Bath temperature: 30°C. Time of analysis, 5 minutes. The reaction was started by tipping the enzyme from the side arm. Decarboxylase activity of the intact mycelium was determined on 0.1 g. of wet mycelium (weighed after storage at -20°C. for 1-7 days) and 2.9 ml. of the buffer. In this case, the reaction was started by the addition of oxalate. Dry weight determinations were made on aliquots of frozen mycelium. To carry out reactions under anaerobic conditions, oxygen-free nitrogen was used (6, 10). Different mixtures of nitrogen and oxygen (9) were also tested. A unit of decarboxylase activity is defined as the amount of enzyme that catalyzes the decomposition of 1 pmole of oxalate per minute. Specific activity is expressed in units per milligram of protein (11).

CHEMICAL DETERMINATIONS Formic acid was determined methods of Warner and Raptis Samuelson (13), and proteins Lowry et ~2. (14).

by the combined (12) and Ahlen and by the procedure of

DECARBOXYLATION

489 TABLE

OX.ILK

ACID

II

DECBRBOXYLhSE

STR.UNS

OF

IN

SOME

ASPERGILLUS

CO?dg. dry wt. x 11)

AF;;tiV;i

Yield citric acid $& of sucrose

12.8

47

12.3

48

8.5 8.4

40 43

niger (M-l-81) niger (M-l-154) niger (M-1-192) jlavus-oryzae

6.8 5.1 2.3 traces

38 30 20 0

jlavus-oryzae

traces

0

lerreus fumigatus

traces traces

0 0

Strain

Aspergillus (A. niger Aspergillus (avellaneus Aspergillus Aspergillus (NRCA-l-253) Aspergillus Aspergillus ilspergdlus dspergillus (M-l-72) Aspergillus (M-l-89) Aspergillus Aspergillus

phoenicis group) phoenicis variety) niger Tc. niger

n Conditions Table I.

of analysis:

the

same

as those

of

RESULTS

INFLUENCE OF PH ON THE DECARBOXYLATING ACTIVITY OF THE IM~~ELIIJM When A. phoenicis was cultivated under the above-mentioned conditions, the starting TABLE INFLUENCE THE

OF

Ammonium Ammonium Potassium Potassium

I OF

DECARBOXYLSTING THE

Nitrogen

pH

THE

MEDIUM ACTIVITY

MYcELIUM Activity of oxalic aad decarboxylasea (IllOkS

source

chloride nitrate nitrate nitrate

ON OF

1.1 1.5 2.0 2.5”

0 traces 0.15 0.52

12.8 4.0 0.3 0

a Conditions of analysis : main compartment of Warburg flask: 2.8 ml. of 0.2 M acetate buffer, pH 4.7, and 0.1 g. of wet mycelium previously frozen for 7 days. Side arm: 0.3 ml. of 0.1 M oxalate, pH 4.7. Temp: 30°C. b pH obtained by adding sodium hydroxide solution 2 or 3 times a day.

pH of the medium (4.5) dropped to 1.14 at the third day. Acidity was due to the citric acid produced from sugar and to the small amounts of hydrochloric acid formed from the ammonium chloride used as the only nitrogen source. To obtain pH 1.5 and 2.0, ammonium chloride was substituted for ammonium and potassium nitrate, respectively, in equivalent quantities with respect to nitrogen. To maintain the pH at 2.5 sodium hydroxide was periodically added to the cultures. Three days after inoculation, the mycelia were washed with running tap water for 1 hour and stored at -20°C. for l-7 days. Freezing practically destroyed respiratory processes, thus facilitating the determination of decarboxylase activity. Values obtained are shown in Table I. It is evident that the pH of the medium had a great influence on t.he presence of decarboxylase in the mycelium and of oxalic acid in the medium. At pH 1.1 no oxalic acid was found in the medium, but small amounts were identified 4 pH (approx.

inside 5).

the

cells

is,

of

course,

higher

490

EMILIANI

AND

BEKES

in the mycelium by a technique already described in an earlier paper (15). A confirmation test with purified decarboxylase was also made. The amount, of oxalic acid in the mycelium developed with ammonium chloride as nitrogen source (pH 1.1) was about 4 mg./lOO g. of dry wt.

Partial Purification of the Enzyme. All operations were performed at 2”-3°C. First, 500 ml. of cold methanol (-30°C.) were slowly added to 500 ml. of crude extract with continuous stirring. The mixture was allowed to stand for about, 4 hours and the precipitate was then separated by centrifugation (3000 g, 10 minutes) and finally DECARBOXYLATING ACTIVITY OF SOME suspendedin 40 ml. of 0.1 M acetate buffer, STRAINS OF Aspergillus pH 5.6. After 4-8 hours the suspensionwas The activity of oxalic acid decarboxylase clarified by centrifugation at 13,000 g for 20 in some strains of A. niger and of other minutes and the solid was discarded. This speciesis shown in Table II. precipitation (50% MeOH) and the consequent clarification were repeated twice EXTRACTION AND PROPERTIES OF OXALIC more under the same conditions, but with ACID DECARBOXYLASE reducing of the volume of buffer and methanol each time (see Table III). After a fractional Preparation of Crude Extract. Three-dayold mycelia (maximal decarboxylating ac- precipitation, most of the enzyme was in the fraction precipitating between 15 and 30 % tivity) were washed with running tap water for 14-16 hours to eliminate the strong of methanol (v/v). This precipitate was disacidity. They were then squeezed slightly to solved in 2 ml. of acetate buffer, and the reduce the water retained in the mycelial solution was dialyzed against 500 ml. of 0.1 M acetate, pH 5.6, for 8 hours, replacing mat, and were stored at -20°C. for l-7 fresh solution twice in the meantime. The days. Twenty g. of frozen mycelium (about 3.5 results of a typical purification are shown in g. dry wt.) were ground in a mortar; when Table III. Purification by means of acetone and amthe material began to thaw, 10 ml. of 0.1 M monium sulfate was also tested in several acetate buffer, pH 5.6, were added, and grinding was continued until 80% of the assays,but the results were not satisfactory. Effect of Some Compounds.At a concentracells were destroyed. Finally 40 ml. more of buffer solution were added; the suspension tion of 5 X low4 M certain reducing substances such as o- and p-phenols, o- and was shaken for 15 minutes and then centrifuged at 18,000 g for 15 minutes. The super- p-aromatic amines, ferrocyanide, and asnatant was then filtered at normal pressure corbic acid had a favorable action on the through a fritted-glass filter to eliminate decarboxylation. On the contrary, sodium some floating lipids. The filtrate was stored sulfide, sodium azide, thioglycolic acid, at 0°C. for several weeks without loss of ac- phenylhydrazine, hydroxylamine, mercuric tivity. chloride, and periodic acid were very harmful TABLE PURIFICATION Fraction

Crude extract 1st precipitation 2nd precipitation 3rd precipitation 4th precipitation after dialysis

(50% MeOH) (507, MeOH) (50y0 MeOH) (1430y0 MeOH)

III

OF OXALATE Total

volume (ml.)

500 40 10 4 2.2

DECABOXYLASE Total

units

215 193 159 102 85

Protein h./ml.)

2.5 3.1 3.0 2.5 1.4

Specific

activity

0.17 1.5 5.3 10.2 28.3a

a As will be seen later, this value would be higher if gelatin and certain reducing compounds added, or if gas phase was Nz with 1% of O2 (partial pressure of 02 = 0.01) instead of air.

were

ENZYMATIC

0

OXALATE

491

DECARBOXYLATION

40

20

60

MINUTES Main compartment: 0.6 ml. FIG. 1. Eject of o-phenylenediamine (FDA) and gelatin. (120 pmoles) of oxalate, pH 5.2; 2 pmoles of FDA and/or 1 mg. of gelatin; 0.2 IV acetate buffer, pH 5.2, up to 3.0 ml. Side arm: 0.1 ml. of diluted enzyme and 0.4 ml. of acetate buffer. Gas phase: air. -

at the same concentration. The most potent poisons were sulfite and dithionite, with which the enzyme was instantly and completely inhibited, at a concentration of 1 X 1O-4 &I. Formate had no inhibitory action. The addition of gelatin, albumin, or protamine to the Warburg flasks (1 mg. per flask) favorably influenced the activity of the enzyme. Figure 1 shows the effect of gelatin and o-phenylenediamine. Ascorbic acid and the other reducing substances already mentioned had the sameeffect when the decarboxylase acted in an atmosphere of air. Some Properties of the Oxalate Decarboxylase. The enzyme solution in 0.1 M acetate buffer, pH 5.6, did not show any loss of activity by dialysis. Addition of boiled juice (“kochsaft”) had no effect on the decarboxylase activity. Manometric and chemical determinations confirmed the production of 1 mole of formate and 1 mole of carbon dioxide per mole of oxalate. The enzyme dissolved in acetate buffer, pH 5.6, did not lose its activity for several weeks at O”C., nor at 30°C. for several hours,

nor at 60°C. for 10 minutes, but at 65°C. for 10 minutes its activity was reduced 50 % and at 70°C. for 10 minutes it was destroyed completely. At values of pH lower than 5.2, the enzyme lost activity rapidly, but with the addition of gelatin it lost activity more slowly. With gelatin the maximal activity of the enzyme (time of analysis 5 minutes) was obtained at pH 4.7 (Michaelis constant, 2 X 10e3 M; concentration of saturation, 1OV M). Without gelatin the maximal activity was at pH 5.2 (Michaelis constant, 4 X 1OP M; concentration of saturation, 3 x 1OP M). Injluence of Composition of Gas Atmosphere. In a pure nitrogen atmosphere, the enzyme becameinactive in about 30 minutes, but this inactivation was reversible, since if air was introduced into the Warburg flask, the reaction was re-established after 10 minutes. Figure 2 showshow enzymatic activity decreasedin the presenceof air. The loss of activity depended, partially at least, on oxygen. In fact, as the partial pressure of this gas

492

EMILIANI

AND

BEKES

80 c( 8 Y 40

50

100 MINUTES

FIG. 2. Injhence of the partial pressure of oxygen. Main compartment 0.6 ml. of oxalate 0.2 M, pH 5.2; 0.1 ml. of gelatin 1%; 0.2 M acetate buffer pH 5.2 up to 3.0 ml. Side arm: 0.1 ml. of diluted enzyme, 0.4 ml. of acetate buffer. Gas phase: pure oxygen or mixtures of Nz and 02. The numbers above the curves indicate the partial pressure of oxygen.

was increased, the irreversible denaturation of the enzyme took place more rapidly and vice versa. It is interesting to note that on bubbling 02 for 1 hour through the enzyme solution in the absence of substrate, no inactivation was observed. SpeciJicity. The purified Aspergillus oxalate decarboxylase had no action on the following acids : formic, acetic, propionic, glyoxilic, glycolic , mesoxalic , pyruvic, oxalacetic, oxalsuccinic, succinic, fumaric, L( -)malic, L(+)tartaric, itaconic, citric, cisaconitic, a-ketoglutaric, gluconic, and 2-ketoglutaric, nor on any of the common amino acids.

Aspergillus niger produces the oxalate decarboxylase at pH 1.1. Under these conditions the medium contains no traces of oxalic acid. On the contrary, if the pH of the medium is increased, the decarboxylase is not produced and considerable quantities of oxalic acid accumulate in the medium. When speaking about citric fermentation, it is commonly assumed that Aspergillus does not produce oxalic acid at a low pH. It appears that this assumption is not quite correct since traces of oxalic acid are present in the cells. This suggeststhat at a low pH this acid is destroyed by the decarboxylase as soon as it is formed. The oxalate decarboxylase extracted from DISCUSSION A. niger appears to have similar properties to The strains of the A. niger group that that found by Shimazono (4) in Collybia yield great amounts of citric acid are es- velutipes, but they differ with respect to pecially good sources of oxalate decarboxylsolubility, optimal pH, resistance to high ase. A 3-day-old mycelium, grown on a temperature, etc. synthetic medium composed of sugar and The enzyme seemsto be highly specific ; mineral salts, exhibits a high decarboxylatit might thus be used either for the identifiing activity. It is not necessary to add oxalic cation or for the quantitative determination acid to the medium to induce the formation of this enzyme. The presence of oxalate de- of oxalic acid. Nothing can be said, for the present. recarboxylase could not be detected in other garding the mechanism of the oxalate despeciesof Aspergillus, such as A. terreus, A. jlavus orizae, and A. fumigatus. carboxylation.

ENZYMATIC

OXALATE

ACKNOWLEDGMENT The authors are indebted from the National Research for the supplying of a strain

to Dr. I). S. Clark, Council of Canada, of A. niger.

REFERENCES 1. J~K~BY, W. B., OHMURA, E., AND HAYAISHI, 0. J. Bid. Chem. 233, 435 (1956). 2. KEECH, 1). B., AND TAYLOR, G. A., Biochem. J. 76, 3P (1960). 3. VAISEY, E. B., CHEDELIN, 1.. H., IND NEWBITRG, R. W., ilrch. Biochem. Biophys. 96, F6 (1961). 4. SHIMAZONO, H., J. Biochem. Japan 42, 321 (1955). 5. SHIM.\ZONO, H., .~ND HAYAISHI, O., Abstracts American Chemical Society, 130th meeting, Atlantic City, 46C (1956). 6. SHIM.IZONO, H., AND H.ZYAISHI, O., J. Bid. Chem. 227, 151 (1957). 7. TIIOM, C., AND RAPER, K. B., “A Manual of the &pergilZi,” p. 215, 222. Williams and Wilkins, Baltimore, Maryland, 1945.

DECARBOXYLATIOX 8. EMILIANI, E., F.4~~0, F., .~ND RICCURDI, A. I., Rev. Fat. Ing. Quim. Univ. Nacl. Litoral, Santa Fe, Argentina 24, 27 (1955). 9. UMBREIT, W. W., BURRIS, R. H., AND STAUFFER, J. F., “Manometric Techniques.” Burgess, Minneapolis, Minnesota, 1959. 10. STONE, H. W., AND SKAVINSKY, E. H., Ind. Eng. Chem. (Anal. Ed.) 17, 495 (1945). 11. “International Union of Biochemistry, Symposium Series,” Vol. 20. Pergamon Press, 1961; in THOMPSON, R. H., Xature 193, 1227 (1962). 12. W>IRNER, B. R., AND R.UWS, L. Z., Anal. Chem. 27, 1783 (1955). 13. AHLEN, I,., AND SAMUELSON, O., Anal. Chem. 26, 1263 (1953). 14. LOWRY, 0. H., ROSEBROUGH, N. J., F~RR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193, 265 (1951). 15. EMILIANI, E., AND GOLDRING, B., IZev. Fat. Zng. Quim.. Univ. Nacl. Litoral, Santa Fe, Argentina

26,19

(1956).