Some observations on the aspergillin of Aspergillus niger

ARCHIVES

OF RIOCHEMISTRY

AND BIOPHYSICS

Some Observations

124, 418421

on the Aspergillin ARUN

Department

of Biochemistry,

Received

(1968)

August

KUMAR University

of Aspergillus

niger

RAWAT of Lucknow,

29, 1967; Accepted

Lucknow,

November

India

6, 1967

The presence of a general inhibitor of enzymes has been established from several strains of Aspergillus niger, although the amount present showed wide variations. The inhibitor is identical with or closely related to the black pigment aspergillin, and is absent from all but black-colored spores. The inhibitor appears to function, at least partially, by the proteolysis of enzymes. All the mold spores have been shown to posses enzymic proteolytic activity. It has been shown that, except for a few, fungal spores do not contain a full complement of enzymes (Gottlieb, 1964). Some of the missing enzymes only appear later during germination (Staples and Weinsten, 1959). In preliminary experiments aimed at demonstrating the glycolytic enzymes in the resting spores of a strain of Aspergillus niger, it was observed that broken cell preparations of spores did not have hexokinase and phosphoglucomutase activity, but actually inhibited externally added enzymes (Nandan et al., 1962; Khanna et al., 1963). It was subsequently recognized that inhibition brought about by the spores was not confined to the hexokinase and phosphoglucomutase but that a large number of enzymes tested were all inhibited in vitro. The active factor in the spores was therefore an inbibitor of enzymes in general (Tiwary et al., 1964). There was reason to believe that inhibitor was either identical with or closely related to the pigment aspergillin, although its relation to humic acid was not clear. The isolated inhibitor had powerful proteolytic activity as tested against casein or albumin (as well as inhibitory activity against enwas heat-stable, and was thus zw-4, different from the true protease activity. A preliminary account of this work has been published (Rawat, 1965).

MATERIALS

AND

METHODS

CULTURES

Before large scale inoculation, the organisms were subcultured from stock cultures on a potatosucrose-agar medium. Aspergillus giganteus was cultured on a medium consisting of yeast extract, glucose, potassium dihydrogen phosphate, and magnesium sulfate. For final inoculation of stock suspension of spores was prepared in sterile distilled water, and samples were spread aseptically on the surface of the medium solidified with agar in Petri dishes. The cultures were allowed to grow at 30” for 6 days, when profuse sporulation occurred. The spores were collected by suction as described by McCallan et al. (1936). In a few cases where such a technique was not possible, the spores were harvested with the flooding technique. After exhaustive washing in the cold, the spores were transferred to a flat dish and dried at room temprature in vacua over calcium chloride, and the drying was continued until constant weight was attained. With fresh desiccant it was found shat a 2-gm batch of spores could be dried completely in about 48 hours. The dried spores were bottled and stored at -13’ in a deepfreeze. PREPARATION

OF

SPORE

HOMOGENATES

One-hundred mg of dried spores was ground with purified sand in a chilled mortar with 0.02 M Tris buffer (pH 7.6) for 10 minutes. The volume was made up to 10 ml with Tris buffer to give a 1% (w/v) spore suspension. Dilute preparations were obtained by diluting 1 ml of stock homogenate to 10 ml with water to give a 0.1% spore suspension; by diluting a further 1 ml of the latter to 10 ml with water, a 0.01% spore suspension was ob-

1 Present address: Department of Biochemistry A, University of Copenhagen, Copenhagen, Denmark. 418

ASPERGILLIN tained. Suitable used to inhibit IHIBITORY

OF Aspergillus

aliquots of spore suspensions were the enzyme in the desired range. ACTIVITY

AGAINST HEXOKINASE

In the assay mixture for assaying the inhibitory activity of the broken cell preparations from spores, 0.5 ml of broken spore preparation was added. The assay mixture contained the following components: 0.2 M Tris buffer in 0.5 ml, 10 pmoles of glucose in 0.2 ml, 50 pmoles of NaF in 0.1 ml, and 0.2 mg hexokinase (Sigma Chemical Co., yeast). When the aliquot of spore preparation was less than 0.5 ml, the difference was made up by the addition of required amount of water. Hexokinase action. Five pmoles of ATP, pH 7.8, in 0.10 ml, and 5 @moles of MgC12 in 0.10 ml were used. At the end of 15 minutes of preincubation at 30”, the hexokinase reaction was started by adding ATP, followed by MgC12. After 30 minutes incubation at. 30”, the reaction was arrested by adding 1 ml each of 5% (w/v) ZnSOd and 0.3 N Ba(OH) 2 (Somogyi, 1945). The control tubes which contained added MgCll now received ATP. The volume was made up to 8 ml with water, and reducing sugar was estimated on aliquots of the centrifuged supernatant fraction by the arsenomolybdate method of Nelson (1944) as modified by Somogyi (1952). In control experiments, where the full activity of the enzyme was measured, the spore suspension was replaced by water. One unit of hexokinase activity was equivalent to the disappearance of 1 pmole of glucose under the conditions of assay, whereas one unit of inhibitor was the amount of inhibitor required to inhibit the activity of hexokinase by 50yo under the condition of assay. PROTEOLYTIC ACTIVITY When tested against casein or albumin, the inhibitor preparations caused the proteolysis of the proteins, which was evidenced by an increase in absorption at 280 rnp. Casein was used for testing the proteolytic activity in the present investigations. The spectrophotometric method used was essentially t,hat of Kunitz (1947). The assay system contained 300 pmoles phosphate buffer, pH 7.5, in 1.5 ml; 5 mg casein in 0.5 ml 1% spore suspension; and water, 0.5 ml. The total volume of the assay mixture was 3 ml. The experiments were run in duplicate. The reaction was started by adding the spore suspension to the experimental tubes, and was allowed to proceed at 37” for an hour. Two ml of 10% trichloroacetic acid was added to precipitate the protein and the inhibitor. Control tubes now received the spore suspension. The suspensions were immediately centrifuged at 6009 for 30 min-

419

niger

utes. The suspension was diluted a-fold with water, and an increase in absorption at 280 rnp of experimental tubes over the controls was measured. For estimating the heat-stable proteolytic activity, the spore suspension was heated in boiling water for 10 minutes. The difference between the heat-stable proteolytic activity and total proteolytic activity was taken as the measure of enzymatic proteolytic activity. The determinations were reproducible within a range of error of l%%. ESTIMATION

OF ASPERGILLIN

The determination of aspergillin was based on the method of Sharma et al. (1966). The phenol reagent was prepared according to the method of Folin and Ciocaltue (1927) and was diluted with water to make it 1 N in acid. Reagent C, consisting of cu++ and tartarate in alkaline Na&Ch, was the same as that of Lowry et al. (1951). ISOLATION OF ASPERGILLIN Six strains of Aspergillus niger with different levels of inhibitor were used. One gm of spore was ground in a mortar with 0.1 N NaOH at room temperature. The residua1 material was reextracted twice with alkali. A volume of about 50 ml was used for extraction, the period of contact being about 90 minutes. Further extraction did not yield more pigment. The pH of the intensely dark-colored solution was adjusted to 2.5 with 1 N HCl, and the bulky precipitate was allowed to settle at room temperature. The precipitate was collected by centrifugation at 600s for 10 minutes, washed with 10 ml of water, redissolved in 0.1 N NaOH and precipitated with 1 N HCI, sedimented on the centrifuge, and washed once with water. A second washing was not attempted as a rule, since trial experiments revealed that pigment tended to pass into solution on exhaustive washing. The pigment was dissolved in 20-30 ml of 0.1 N NaOH, and the pH was adjusted to 7 with 1 N HCl. Suitable aliquots of the aspergillin preparation were diluted with water and assayed for inhibitory activity against externally added hexokinase, heat-stable proteolytic activity, and aspergillin content calorimetrically (Table II). One-ml aliquots of the aspergillin solution were precipitated once again with acid, the precipitate was washed, dried to constant weight at 80”, and the dry weight was determined. RESULTS

Tested against externally added hexokinase, only the black spores of Aspergihs

420

RAWAT TABLE RELATION

OF INHIBITOR

Inhibitory activity (units/l00 mg spores)

Speciesand strains

A. niger,

NRRL

A. niger,

548

A. niger,

NRRL

8 (@ @I (a) (b) (a) (b) (a) (b)

337

599

A. niger, 612 A. A. A. A.

niger, 1015 oryzae, 15 oryzae, 456 giganteus a (a) and (b) represent

I

TO PROTEOLYTIC ACTIVITY

the spores of the same strain

A. A. A. A. A. A.

niger, niger, niger, niger, niger, niger,

NRRL 337 548 NRRL 599 612 1015 Perlman-1

0.60 0.50 2.07 1.65 3.31 2.07 4.14 2.27 0.62 Nil Nil Nil

grown on two different

TABLE

Strains

Heat-stable

2.3 2.4 22.8 18.3 33.7 22.1 25.6 27.5 7.6 0.2 0.3 0.3

125 50 4761 4761 2272 833 2631 2564 500

Yield of aspergillin (mg/gm spores) Calorimetrically “vh;sfsh’ determined

niger contained the inhibitor; the green spores of Aspergillus oryzae and the blue spores of Aspergillus giganteus do not contain the inhibitor (Table I). The pale brown spores of Aspergillus terreus and the salmon pink spores of Neurospora crassa also did not contain the inhibitor. Among the black spores of A. niger, 23 strains of which were analyzed, there was considerable variation in the inhibitory act,ivity. Strain 337 contained less than 200 unit,s of activity, whereas strain Perlman-1 had over 8000 units/100 mg of spore. Other strains had intermediate levels of activity. As was expected, the spores of all strains of A. miger contained aspergillin. The amount, however, showed wide variations. There was verv little difference between the values determined in the presence and absence of Cu+, which indicated that the present preparation was free from protein

Total 1.30 0.83 3.30 3.30 4.14 3.72 8.28 3.72 2.07 1.80 1.03 0.82

batches.

II

OFASPERQILLINISOLATEDFROM

50 129 158 320 150 240

CONTENT

Proteolytic activity (units/100 mg spores)

Aspergillin content (rn.g/loo mg spores)

Nil Nil Nil

YIELDANDACTIVITY

AND ASPERGILLIN

30 125 149 355 137 222

Aspergillusniger

STRAINS

Inhibitory activity against externally added Proteolytic activity enzyme (units/mg (unit&g aapergillin) aspergillin) 20 218 182 188 15 162

0.06 0.12 0.12 0.12 0.03 0.15

contamination. Spores of other organisms did not show inhibitory activity against the externally added hexokinase. Spores of organisms other than Aspergillus niger contained insignificant amounts of pigment calculated as aspergillin equivalent (Table I). All black spores were again characterized by possessing heat-stable proteolytic activity, which varied from strain to strain. Such activity was absent in other spores. The yield of aspergillin on the basis of the dry weight of the pigment was in close agreement to the calorimetrically estimated pigment (Table II). The isolated aspergillin, like the spore homogenates, showed inhibitory activity against the externally added hexokinase and proteolytic activity against Casein (Table II). However, unlike the spore homogenates, the heat-stable and total proteolytic activity of the pigment was the. It was clear from the results that, among

ASPERGILLIN

OF AspergiEZusniger

the strains of A. niger which showed inhibitory activity against externally added hexokinase, there was in general a direct proportionality among the inhibitory activity against hexokinase, the content of aspergillin, and the heat-stabIe proteolytic activity. Statistical evaluation of the data on the basis of the coordination coefficient and the application of the t test revealed the direct relationship between the inhibitory activity and aspergillin content on one hand, and between the inhibitory activity and heat-stable proteolytic activity on the other, was significant in 95 % of the population, with the exception of only 5%. The relation found among the spore of A. niger was further strengthened by the finding that the eleven other spores which did not, inhibit hexokinase, also did not, contain aspergillin nor have heat-stable proteolytic activity. Relation between the total and heat-stable proteolytic activity. The total proteolytic activity of the spores was always higher than the heat-stable component. Even the organisms which did not have inhibitory activity had demonstrable true proteolytic activity. The conclusion was drawn that all the mould spores contain the true proteolytic activity, but this enzymic activity did not lead to the inactivation of enzymes added in vitro. Reproducibility of the data. When spores of the same strain were grown on different batches, some variation in the analytical values were obtained. The variation was found to be more pronounced in strains having a lower inhibitory activity, but the degree of variation was never more than 50%. Endogenous hmokinase activity. Aspergillus niger strain NRRL 337 was characterized by a low content of inhibitor. Tests for hexokinase carried out on the homogenates of this sample gave negative results, indicating that the enzyme was either absent or present in too low an amount to escape the activity of the inhibitor. As was expected, strain 548 and Perlman-1, which

421

have a high inhibitor activity, did not show any hexokinase in their homogenates. Activity of isolated pigment prepardions. The yield of pigment aspergillin was high from strains which possessed high inhibitory activity and low from other strains which possessed low activity. It was anticipated that, if same aspergillin were to occur in different spores, the inhibitory activity and the heat-stable proteolytic activity would reasonably the same for all the preparations. Three out of the six samples had more or the less the same specific activity, calculated as inhibition per unit of calorimetrically estimated aspergillin content. However, preparations derived from strains with low inhibitory activity had an extremely low activity against, externally added hexokinase. ACKNOWLEDGMENT The author is grateful to The Council of Scientific and Industrial Research, New Delhi, for a research fellowship, and to Professor F. Lundquist for his valuable suggestions in preparing the manuscript. REFERENCES 1. GOTTLIEB, D., Endeavour 23, 85 (1964). 2. STAPLE, R. C., AND WEINSTEIN, L. H., Conk. Boyce. Thompson. Inst. 20, 71 (1951). 3. NANDAN, R., TIWARY, K. K., AND KRISHNAN,

P. S., J. Sci. Industr. Res. ale, 260 (1962). R., AND TIWARY, K. K., Arch. Microbial. 46, 398 (1963).,; TIWARY, K. K. SHARMA, 0. K., AND KRISHNAN, P. S. Indian. J. Exptl. Biol. 2, 56, (1964). RAWAT, A. K., Abstr. Joint Convention Indian Chem. Sot., Chem. Res. Comm. B 10 (1965). MCCALLON, S. E. A., AND WILCOXIN, D. D., Contrib. Boyce. Thompson. Inst. 8,151 (1936). SOMOGYI, M., J. Biol. Chem. 160, 69 (1945). NELSON, N., J. BioZ. Chem. 163, 375 (1944). SOMOGYI, M., J. Biol. Chem. 196, 19 (1952). KUNITZ, M., J. Gen. Physiol. 30, 291 (1947). FOLIN, O., AND CIOCALTUE, V. J. Biol. Chem.

4. KHANNA, 5. 6. 7. 8.

9. 10. 11. 12.

78, 627 (1927). 13. SHARMA, 0. K., .4ND KRISHNAN, P. S., Anal. Biochem. 14, 11 (1966). 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. L. J. Biol. Chem.

193, 265 (1951).