Isolation of aminopeptidase from Aspergillus flavus

Biochimiea et Biophysica Acta, 420 (1976) 309-315 © Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands

BBA 37250 ISOLATION OF A M I N O P E P T I D A S E F R O M A S P E R G I L L U S FLA VUS

J. TURKOVA a, O. VALENTOVAa and J. (~OUPEKb alnstitute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, 166 10 Prague 6 and blnstitute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, 162 06 Prague 6 (Czechoslovakia) (Received July 4th, 1975)

SUMMARY A mixture of aminopeptidase and neutral protease from the Aspergillusflavus mould obtained by chromatography on DEAE-Sephadex was fractionated by chromatography on the hydroxyalkyl methacrylate gel with chemically bonded 1,6 hexamethylene diamine and D-leucine. Aminopeptidase thus obtained was electrophoretically homogeneous. Conditions for chromatography were worked out allowing a one stage isolation of a highly active aminopeptidase sample directly from the alcoholic precipitate of the culture medium of the Aspergillus flavus mould.

INTRODUCTION The alkaline proteinase from the mold Aspergillusflavus has been studied for several years in our laboratory [1-6]. In a series of comparative studies we have shown the existence of a close relationship between the alkaline proteinase isolated by us and the alkaline proteinase from Asp. oryzae [7, 8]. We have proved that the culture medium of Asp. flavus contains proteinases analogous to those found in the culture medium of Asp. oryzae by the Japanese authors Nakadai, Nasuno and Iguchi [9]. In addition to the alkaline proteinase, an endopeptidase of the metalloenzyme type and of the carboxyl proteinase type, a carboxypeptidase, and an aminopeptidase have been found by us in the culture medium of Asp. flavus. Our aim has been the isolation and characterization of all these proteinases. This paper reports on the isolation of an aminopeptidase from the mould Asp. flavus. MATERIAL AND METHODS The culture medium of the Asp. flavus mould and the alcoholic precipitate of the culture liquid were obtained by courtesy of the Food Industry Research Institute of the Czechoslovak Academy of Agriculture, Prague. The hydroxyalkyl methacrylate gels of the Spheron type were prepared by the suspension copolymerization of monomers in presence of inert solvents [10]. Abbreviation : DAF, dialysed alcoholic precipitate of the culture liquid of Asp. flavus.

310 N-benzyloxycarbonyl-D-leucine was a product of Schuchardt, IMeucine-pnitroanilide was produced by Koch-Light Laboratories, and haemoglobin was a product of L6~iva, Prague.

Preparation of D-Leu-NH2-Spheron To the hydroxyalkyl methacrylate gel of the Spheron 300 type, particle size 120-300/zm, activated with cyanogen bromide [11], 1,6-hexamethylenediamine was bonded chemically according to a modified procedure suggested by Cuatrecasas [12]. Ten ml of activated gel was mixed with 10 ml of 10~o, 1,6-hexamethylenediamine, pH 10, and stirring was continued at room temperature for 24 h. On completion of the reaction the gel was washed subsequently with a 0.1 M acetate buffer solution containing a 1 M NaC1 solution, pH 4.1, with a 0.1 M borate buffer solution, 1 M NaC1 pH 8.5, and finally with distilled water. The procedure was repeated until the eluate contained no hexamethylenediamine. The gel thus modified was named NH2Spheron. 10 ml of swollen NH2-Spheron was added to a solution of 100 mg of Nbenzyloxycarbonyl-D-leucine in 10 ml dimethylformamide, and the pH of the suspension was adjusted to 4.7. One ml of a 15 ~o solution of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide methoiodide was added dropwise to the suspension during 5 min. The reaction mixture was stirred at room temperature 20 h, after which the gel was washed with a 50 ~o solution of dimethylformamide and water. The analysis of amino acids after hydrolysis [11, 13] showed that 3.6 micromoles of N-benzyloxycarbonylD-leucine were bonded to 1 ml of the swollen NHz-Spheron gel. The protective benzyloxycarbonyl group was split off by adding 15 ml of hydrogen bromide in acetic acid to the dry derivative of the gel (2.5 g) followed by stirring of the suspension at room temperature (30 rain). The gel was then washed with water, alcohol, acetone and ether.

Chromatography of the separation products on DEAE-Sephadex The culture medium (20 1) from the Asp. flavus mould was sorbed on Amberlite IRC-50 by techniques described earlier [6]. After desalting of the desorbate on Sephadex G-25 equilibrated with 0.01 M calcium acetate the protein fraction was directly chromatographed on DEAE-Sephadex A-50 (Fig. 1), and the joined fractions were lyophilized. The yield of fraction C was 3.8 g. Chromatographic separation was carried out at 4 °C. 1 g of the |yophilized material of fraction C was dialyzed twice against 10 1 water, which yielded 8 mg of the protein material DC.

Comparison of the adsorption of aminopeptidase on D-Leu-NHz-Spheron, NH2-Spheron, and Spheron 3 mg of the dialyzed material of fraction C (DC) was dissolved in 30 ml of 0.05 M Tris.HC1 buffer and 0.01 M CaCl2 pH 8.0. 7 ml of the swollen D-Leu-NHzSpheron was added to one third of the prepared solution, 7 ml of the swollen NHzSpheron was added to the second third and the same amount of the swollen Spheron was added to the remaining third. Prior to sorption, all gels were washed with distilled water in the same way until the conductivity of the eluted water equalled that of water applied at the inlet of the column. After this the gels were equilibrated with 0.05 M Tris-HC1 buffer and 0.01 M CaC12 pH 8.0. The course of sorption is shown in Fig. 2. All sorptions and desorptions on the hydroxyalkyl methacrylate gels were carried out at room temperature.

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Chromatography of the protein components of fraction C on D-Leu-NH2-Spheron 2.5 m l o f s w o l l e n D - L e u - N H 2 - S p h e r o n filtered b y s u c t i o n was a d d e d to a sol u t i o n o f 50 m g o f f r a c t i o n C in 5 m l o f 0.05 M T r i s - H C 1 buffer s o l u t i o n a n d 0.01 M CaC12 p H 8.0. S a m p l e s f o r t h e d e t e r m i n a t i o n o f t h e a m i n o p e p t i d a s e a c t i v i t y w e r e t a k e n in 5 m i n . i n t e r v a l s f r o m t h e s u s p e n s i o n w h i l e stirring. T h e gel c a p a c i t y was

312 s a t u r a t e d d u r i n g l 0 min. ( a b s o r b a n c y at 405 n m c o r r e s p o n d i n g to the a m i n o p e p t i d a s e activity fell f r o m 0.85 to 0.27) a n d no further a m i n o p e p t i d a s e was sorbed. The gel with the sorbed a m i n o p e p t i d a s e was transferred to the c o l u m n where after washing o u t the non-specifically sorbed m a t e r i a l a m i n o p e p t i d a s e was displaced by a change in the ionic strength as shown in Fig. 3.

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Fig. 3. Chromatography of protein components of fraction C on D-Leu-NH2-Spheron. 2.5 ml of ge with aminopeptidase sorbed by procedure described in text was transferred to column (diameter 0.8 cm), then washed with 0.05 M Tris.HC1 and 0.01 M CaCl2 buffer solution pH 8.0. 6 ml fractions were taken in 10 min intervals. Aminopeptidase was desorbed with buffer solution of the same composition containing also 2 N NaC1. , Absorbancy at 280 nm; -- -- --, proteolytic activity; . . . . . . , aminopeptidase activity; . . . . . , conductivity 1/.(2. Fig. 4. Chromatography of dialyzed alcoholic precipitate of culture medium (DAF) on o-Leu-NH2Spheron. 7.1 ml of gel with aminopeptidase sorbed by procedure described in text was transferred to a column 1 cm in diameter. Further course of chromatography is the same as described for Fig. 3.

Chromatography of the dialyzed alcoholic precipitate of the culture liquid of Asp. flavus (DAF) on D-Leu-NHz-Spheron 60 m g D A F was dissolved in 20 ml 0.05 M Tris. HC1 buffer solution a n d 0.01 M CaC12 p H 8.0.5 ml o f the solution was t a k e n for the d e t e r m i n a t i o n o f the p r o t e o l y t i c activity as a function o f p H . 7.1 ml o f the swollen sucked-off o - L e u - N H 2 S p h e r o n was a d d e d to the r e m a i n i n g 15 ml o f the solution. 5 ml o f the solution was t a k e n after stirring for 15 min. for the d e t e r m i n a t i o n o f the p r o t e o l y t i c activity as a function o f p H . The r e m a i n i n g solution with gel suspension was transferred to the c o l u m n a n d c h r o m a t o g r a p h e d as shown in Fig. 4. The specific activities o f the starting r a w p r o teolytic sample a n d o f the d e s o r b e d a m i n o p e p t i d a s e were 0.336 u n i t s / m g o f p r o t e i n a n d 2.19 u n i t s / m g o f protein, respectively. The dependence o f the p r o t e o l y t i c activity on p H o f the D A F solution before a n d after s o r p t i o n on o - L e u - N H 2 - S p h e r o n is illustrated in Fig. 5.

Aminopeptidase and proteolytic activities The a m i n o p e p t i d a s e activity was d e t e r m i n e d b y a modified m e t h o d after

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Pfleiderer [14] using 1.66 mM L-leucine-p-nitroanilide in 0.05 M Tris.HCl buffer pH 8.0. The amount of nitroanilide released after 10 min of incubation at 37 °C was measured on the basis of absorbancy at 405 nm and read off from the calibration curve. The unit of the aminopeptidase activity of the enzyme was defined as the amount of enzyme which releases 1 #mol ofp-nitroanilide in one minute under the described experimental conditions. The specific activity was calculated by relating these units to 1 mg of protein. The protein concentration in solutions was determined by means of Folin's agent after Lowry [15]. The proteolytic activity was determined by using a modified Anson's method [16] for use with hemoglobin. The polyacrylamide gel electrophoresis of aminopeptidase was carried out at pH 9.4 according to Davis [17]. RESULTS A N D DISCUSSION

Isolation of aminopeptidase from fraction obtained by chromatography on DEAESephadex The isolation of alkaline protease from Asp. flavus [1, 6] by chromatography on DEAE-Sephadex (Fig. 1) yielded a fraction containing a mixture of neutral endopeptidase (optimum of the proteolytic activity at pH 6) and aminopeptidase. Konoplych and coworkers [18] were the first ones to point to the difficulties involved in the separation of this mixture, Morihara and coworkers [19] had the same experience with an analogous mixture from Asp. oryzae. Vosbeck et al. used specific adsorption to 1,6-diaminohexane-agarose to isolate an aminopeptidase from a commercial preparation of pronase from K-I strain of Streptomyces griseus [20]. A comparative experiment with the adsorption of the aminopeptidase from Asp. flavus to Spheron, NH2-Spheron, and o-Leu-NH2-Spheron is shown in Fig. 2. Under the experimental conditions used there is no adsorption at all to the unmodified hydroxyalkylmethacrylate gel. Considerable adsorption, observed also elsewhere [20], takes place on NH2-

314 Spheron. The aminopeptidase was adsorbed at the highest rate to D-Leu-NH 2Spheron which was therefore used for its isolation. As can be seen in Fig. 3, at pH 8 and a low ionic strength aminopeptidase becomes specifically adsorbed on this gel, while neutral endopeptidase is eluted. Aminopeptidase was released by using a solution with increased ionic strength. For a successful course of chromatography it is absolutely necessary to apply a well washed gel and to use a sample with a low ionic strength. In the case of sorption of the protein material of the fraction C directly on the column of the modified gel it was necessary first to dialyze the material. The isolated aminopeptidase was homogeneous in disc electrophoresis at pH 9.4; its specific activity was 2.45 units per 1 mg of protein.

Isolation of aminopeptidase from ethanol precipitate of culture medium The conditions worked out for the isolation of aminopeptidase by chromatography on D-Leu-NHz-Spheron were used for its direct isolation from the dialyzed alcoholic precipitate of the culture medium DAF. As shown by Fig. 4, the fraction of the displaced aminopeptidase still exhibits a negligible proteolytic activity determined by means of hemoglobin at pH 6. After rechromatography under identical conditions we obtained an aminopeptidase which no longer showed proteolytic activity and was homogeneous on disc electrophoresis at pH 9.4. The finding that there is practically no adsorption of endopeptidases while the aminopeptidase is adsorbed from the DAF solution to D-Leu-NH2-Spheron, was confirmed by an examination of curves expressing the dependence of proteolytic activity on pH in the DAF solution before and after the adsorption procedure (Fig. 5). The curves obtained show an entirely analogous course; the differences observed can be ascribed exclusively to dilution caused by the addition of the gel slurry. The increase in the speicfic aminopeptidase activity from 0.336 units/rag in the case of the dialyzed raw proteolytic sample to 2.19 units/mg in the case of isolated aminopeptidose is in good agreement with the results of Nakadai et al. [21], who by a multistage purifying operation obtained from the raw sample with 0.482 units/mg aminopeptidase having a specific activity of 2.12 units/mg, determined by the splitting of leucinefl-naphthylamide. The results presented show the high efficiency of the isolation procedure developed. The problem to be solved is the nature of the specific interaction both with D-Leu-NHz-Spheron and with NHz-Spheron. We cannot exclude the possibility of simple ion-exchange chromatography on a support with free NH2-groups sterically accessible to the protein isolated; hydrophobic affinity chromatography on the alkylamine derivatives of solid supports, reported by Shaltiel and Er-el, may also play a role [22]. According to Vosbeck et al. the ability of selective binding which shows supports on the 1,6-hexamethylenediamine basis can be also ascribed to a similarity between the compound bound and the amino termini of polypeptide chains [20]. Even though the binding of D-leucine to NH2-Spheron increased its adsorption ability, this fact by itself does not provide evidence strong enough to support significantly this third possibility. Kettner et al., however, have shown that, e.g. L-leucine ketones are good competitive inhibitors of the aminopeptidase from Aeromonas [23]. Leucine methyl ketone for example has an inhibition constant Ki = 18 #M, for leucine chloromethyl ketone Ki = 0.67 #M, and for leucine bromomethyl ketone Ki = 0.20 #M.

315 We hope that the rapid isolation of the h o m o g e n e o u s proteinase by the procedure developed by us will facilitate n o t only a detailed characterization of the aminopeptidase from Asp. flavus b u t also an investigation of its specificity a n d inhibition. ACKNOWLEDGEMENTS We are indebted to Mrs. J. Luk~eovh for careful technical assistance, Mrs. M. K~iv~ikov~i CSc. for p r e p a r a t i o n of the basic carriers of the Spheron type, a n d Mr. J. Zbro~ek a n d Mrs. V. Himrov~i for the analyses of a m i n o acids. REFERENCES 1 Turkovfi, J., Mikeg, O., Gan~v, K. and Boublik, M. (1969) Biochim. Biophys. Acta 178, 100-111 2 Mikeg, O., Turkovfi, J., Nguyen bao Toan and ~orm, F. (1969) Biochim. Biophys. Acta 178, 112117 3 Turkov~i, J. and Mikeg, O. (1970) Biochim. Biophys. Acta 198, 386-388 4 Turkov~i, J. (1970) Biochim. Biophys. Acta 220, 624-627 5 Turkov~, J. and Mike,~, O. (1971) Collection Czech. Chem. Commun. 36, 2739-2743 6 Mike~,, O., Worowski, K. and Turkovfi, J. (1973) Collection Czech. Chem. Commun. 38, 33393351 7 Turkov~, J., Mike~,, O., Hayashi, K., Danno, G. and Polgfir, L. (1972) Biochim. Biophys. Acta 257, 257-263 8 Bretschneider, G., Nordwig, A., Mike~, O. and Turkov~i, J. (1971) Hoppe-Seyler's Z. Physiol. Chem. 352, 1372-1376 9 Nakadai, T., Nasuno, S. and Iguchi, N. (1972) Agr. Biol. Chem. 36, 261-268 10 ~oupek, J., K~ivfikovfi, M. and Pokorn~, S. (1973) J. Polym. Sci., Symp. 42, 182-190 11 Turkov~t, J., Hub~ilkov~.,O., K~ivfikov~i,M. and (~oupek, J. (1973) Biochim. Biophys. Acta 322, 1-9 12 Cuatrecasas, P. (1970) J. Biol. Chem. 245, 3059-3065 13 Spackman, D. H., Stein, W. R. and Moore, S. (1958) Anal. Chem. 30, 1190-1206 14 Pfleiderer, G. (1970) Methods in Enzymology 19, 514-521 15 Lowry, H. O., Rosebrough, N. J.- Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193,265275 16 Anson, M. L. (1939) J. Gen. Physiol. 22, 79-89 17 Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404--427 18 Konoplych, L. O., Tsyperovich, O. S. and Kolodzeiska, M. V. (1973) Ukr. Biokhim. Zh. 45, 161165 19 Morihara, K., Tsuzuki, H. and Oka, T. (1968) Arch. Biochem. Biophys. 123, 572-588 20 Vosbeck, K. D., Chow, K. F. and Awad, W. M., Jr. (1973) J. Biol. Chem. 248, 6029-6048 21 Nakadai, T., Nasuno, S. and Iguchi, N. (1973) Agr. Biol. Chem. 37, 757-765 22 Shaltiel, S. and Er-el, Z. (1973) Proc. Natl. Acad. Sci. U.S. 70, 778-781 23 Kettner, C., Glover, G. I. and Prescott, J. M. (1974) Arch. Biochem. Biophys. 165, 739-743