Alkaline proteinases of the genus aspergillus

BIOCHIMICA ET BIOPHYSICA ACTA BBA

257

36007

ALKALINE PROTEINASES OF THE GENUS ASPERGILLUS ~

J. T U R K O V ~ a, O. M I K E ~ a, K. H A Y A S H I b, G. D A N N O c

AND

L. P O L G A R a

aDepartment of Protein Chemistry, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) and bCentral Research Laboratories of Kikkoman Shoyu Company, Noda-shi, Chiba-ken, Noda (Japan) and cDepartment of Agricultural Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe (Japan) and alnstitute of Biochemistry, Hungarian Academy of Sciences, Budapest (Hungary) (Received A u g u s t 9th, 1971)

SUMMARY

I. Extracellular alkaline proteinases from the molds Aspergillus flavus, Aspergillus oryzae, Aspergillus sojae and Aspergillus sulphureus, isolated and characterized by individual authors in four different laboratories, have been compared using uniform methods under identical conditions in one of our laboratories. 2. The enzymes studied were compared by gel filtration, disc electrophoresis, amino acid analysis, determination of the pH optima of their proteolytic activity, estimation of their specificity by cleavage of the B-chain of oxidized insulin, and by peptide maps of the tryptic, chymotryptic, and peptic digests of the inactivated proteinases. 3. The results of this comparison led to the following conclusion: Alkaline proteinases from A.flavus and A. oryzae are probably indentical (or very closely related), alkaline proteinase from A. sojae is closely related but not identical, alkaline proteinase from A. sulphureus is homologous yet less related. 4. The combination of methods used in this study requires only small quantities of enzymes. It is therefore suitable for preliminary comparison of proteinases and for studies on the degree of their homology.

INTRODUCTION

During the past few years, a number of papers have appeared describing the isolation and characterization of alkaline proteinases from various molds of the genus Aspergillus 1-1°. The reported data were only partly in agreement. It is thus logical that the similarities existing between some of these proteinases have been discussed.L6. None of the studies dealing with this subject, however, has provided enough * A p r e l i m i n a r y r e p o r t on this s t u d y h a s b e e n included in t h e C o m m u n i c a t i o n s b y O. MIKEg AND J. TURKOVA p r e s e n t e d a t t h e A n n u a l Meetings of t h e Czechoslovak Biochemical Society 14,16 a n d a t t h e 7 t h F E B S Meeting 16.

Biochim. Biophys. Acta, 257 (1972) 257-263

258

J. TURKOV2{et al.

experimental material to permit the problem of the possible degree of similarity of these enzymes to be elucidated. I t was therefore necessary to undertake comparative experiments carried out by the same standard methods. The alkaline proteinases from Aspergillusflavus of different originS, it have been compared by Tb'RKOVAet a112, while BRETSCHNEIDER el al. la compared aspergillopeptidase C from Aspergillus oryzae 4 with the alkaline proteinase from A.flavus 5. These comparative experiments proved very useful and it was appropriate to extend them to involve also other alkaline proteinases, above all from A. oryzae of a different origin (L. POLGAR8), from Aspergillus sojae (K. HAYASHI et al.8,9), and from Aspergillus sulphureus (G DANNO7). Preliminary reports on these studies have appeared 14-16. This paper presents experimental data which have been compiled by the participating workers and conclusions emerging from the discussion of the comparative experiments with the alkaline proteinase from A.flavus~, 1°, A. oryzae s, A. sojae3, 9, and A. sulphureus 7, carried out by identical methods in the same laboratory. The results obtained permit us to conclude that there exists a significant degree of homology between these enzymes. MATERIALS The alkaline proteinases from A. flavus 5, A. oryzae s, A. sojae 3, and A. sulphureus ~ were prepared by the authors cited and by the procedures described in their studies. Subtilopeptidase B was a product of Novo Industry. Sephadex G-ioo and blue dextran were products of Pharmacia, Uppsala, Sweden. Hemoglobin was from Zdravotnick~ potreby, Prague. METHODS The molecular weight determinations by gel filtration were carried out on a column of Sephadex G-Ioo, 4 o - I 2 o # m (2.5 c m - 64 c m ) i n 0.02 M ammonium acetate buffer at p H 6. Fractions, 4.8 ml in volume, were collected at 15 min intervals. The samples applied on the column contained 4 mg of the enzyme dissolved in 2 ml of the buffer, always with the admixture of blue dextran. Disc electrophoresis was carried out in fl-alanine buffer at p H 4-5 by the procedure used for basic proteinslL The amino acid analyses were carried out by the method of SPACKMAN et al. TM. The hydrolysis of the enzyme was performed always in 6 M HC1, 20 h at r i o ° in evacuated sealed tubes. No corrections for zero-time hydrolysis or for complete hydrolysis were made. The determination of the optimum p H of proteolytic activity was effected by a modification s of the method of ANsoN1% The specificities were compared b y the investigation of peptide maps of the B-chain of oxidized insulin digested with the individual enzymes. The digests were prepared by I h incubation of 200 #I of a solution containing 0.5 mg of the B-chain of oxidized insulin in o.I M (NH4)2CO 3 (pH 8.5) at 37 ° with the proteinase at an enzyme to substrate molar ratio of i :ioo. The whole amount of the dried sample was used for the peptide map prepared by a combination of (I) high-voltage electrophoresis ~° at p H 1. 9 and (II) chromatography in the system ~1 butanol-acetic acid-pyridine-water (45:9:30:36 by vol.) on a sheet of W h a t m a n No. 3 paper. Biochim. Biophys. Acta, 257 (1972) 257-263

ALKALINEPROTEINASESOF ASPERGILLUS

259

The enzymatic digestions were carried out after inhibition of the alkaline proteinase. For tryptic or chymotryptic hydrolysis, 6 mg of the alkaline proteinase was dissolved in 300 #1 of ice-cold water and immediately precipitated by the addition of 12o F1 of 20% trichloroacetic acid. The precipitate was washed three times with 200 #1 of water, twice with 200 F1 of acetone, and twice with 300 #1 of ether in conical centrifugation cuvettes and dried i n vacuo in a desiccator. Finally, the precipitate was suspended in I ml of water and 15o #1 of the enzyme solution was added to give an enzyme to substrate molar ratio of approximately 1:75. The pH of the solution was adjusted to 8.3-8.5 by using o.I M (NH4)2CO3 and I drop of the indicator solution (a mixture of i vol. of o.I °,/o aqueous solution of cresol red and 3 vol. of o. I °/o aqueous solution of thymol blue). The closed cuvette was thermostatically controlled at 37 ° for 4 h with occasional stirring and pH measurement. The digestion was discontinued by cooling, acidification by dilute formic acid, and evaporation to dryness i n vacuo in a desiccator. The peptic digestion was carried out with 3 mg of the alkaline proteinase dissolved in 0.5 ml of ice-cold water. The solution was immediately acidified to pH 2 by the addition of o.I M HC1 using I drop of the indicator solution (o.1% aqueous solution of methyl violet). The acidified solution was allowed to stand for 15 min at room temperature in order to inactivate the proteinase. The pepsin solution (15o #1) was added afterwards (enzyme to substrate molar ratio approximately 1:75) and the solution was incubated 4 h at 37 °. The digestion was discontinued by cooling and rapid evaporation to dryness i n vacuo. Two thirds of the total amount of the digest were dissolved in each case in 20-30 #1 of water and applied to the origin of peptide maps prepared by the combination of (I) electrophoresis and (II) chromatography as described above. The remaining one third was used for mixed peptide maps. RESULTS AND DISCUSSION We considered it appropriate to carry out all comparison experiments with the proteinase by uniform procedures and under identical conditions. We therefore accepted the offer of the Department of Protein Chemistry of the Institute of Organic 10

I

MW x 10"*

_

I

I

I

I

6

I

~

I

_

Volume (ml)

Fig. I. Comparison of elution volumes of alkaline proteinases from the molds Aspergillus by gel chromatography, expressed on a molecular weight scale. Standards used: I, human serum albumin; 2, ovalbumin; 3, bovine chymotrypsinogen; 9, subtilopeptidase B; IO, bovine pancreatic ribonuclease. Alkaline proteinases: 4, from A. flavus inhibited by DFP; 5, from A. oryzae; 6, from A. flavus of French originzl (not inhibited by DFP) ; 7, from A. sojae; 8, from A. sulphureus. Biochim. Biophys. Acta, 257 (1972) 257-263

260

j. TURKOV2~et al.

Chemistry and Biochemistry of the Czechoslovak Academy of Sciences and the experiments were carried out in the Prague laboratory. The molecular weights of the investigated proteinases were compared in parallel experiments by gel filtration on Sephadex G-Ioo. As is obvious from Fig. I, tile elution volumes of all proteinases were very similar. The mixed sample of all proteinases gave one symmetrical peak. An elution volume similar to the alkaline proteinases from the mold Aspergillus was, however, found also for subtilopeptidase B which had been added as a standard. With respect to its elution volume the molecular weight of subtilo peptidase B read from the graph should be 22 ooo, a value which is in disagreement with its actual molecular weight of 27 600 calculated from its amino acid composition 22. Similar low value of molecular weights obtained KEAY et al. 23 who examined also other alkaline proteinases of the genus Bacillus. The data in the graph in Fig. I can serve merely as a proof of the identity of molecular size of the compared enzymes, their molecular weight, however, cannot be reliably deduced (cf. ref. IO). Similar to the gel filtration experiments, all the investigated proteinases from the mold Aspergillus showed practically the same mobility also when subjected to disc electrophoresis. In order to compare the amino acid composition of the proteinases we performed the amino acid analysis under identical conditions (Table I). We obtained similar results with the alkaline proteinases from A.flavus, A. oryzae and A. sojae, while the amino acid analysis of the alkaline proteinase from A. sulphureus was markedly different. The pH profile of proteolytic activity of all the proteinases studied showed a

TABLE

I

COMPARISON

OF AMINO

ACID ANALYSES

OF ALKALINE

PROTEINASES

FROM

MOLDS OF GENOS

ASPERGILLUS

This Table contains uncorrected numbers of residues per mole (mol. wt. -- 2 3 ooo for all enzymes; cf. refs. 7-1o) calculated only from the data obtained on 2o-h hydrolysates, prepared under identical conditions and analyzed on the same analyzer. The content of tryptophan was not analyzed. The table serves only for comparison purposes and does not represent the correct amino acid comp o s i t i o n of these enzymes.

A m i n o acid

A. flavus

A. oryzae

A. sq~'ae

A. sulphureus

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine

13. 7 5.o 2.9 24.8 13.3 23.4 15. 3 6. i 24. 3 29.2 o 15. 7 1.5 12.o 12. 5 6.4 6.0

13. 4 4.7 2.7 25.2 14.0 25.2 15.8 5.6 24. 9 29.2 o 15.1 t.6 I I .o i 1.7 6.7 5.8

13.I 5.2 3.0 25. 7 14.4 23 .1 16.5 5.7 24. 3 27.8 o 14.2 1.6 11.8 12-5 6.7 6.2

8.5 4-8 4.o 23.6 18.9 25.4 12.1 5.o 28.9 30.9 o 18.4 o.8 8.2 13.7 9.7 4-7

Valine

Methionine Isoleucine Leucine Tyrosine Phenylalanine

Biochim. Biophys. Acta, 2 5 7 (1972) 2 5 7 - 2 6 3

261

ALKALINE PROTEINASES OF ASPERGILLUS

11

~e

o

oo@0

o@

(mo~

~©o

@

A

B

I

0

o

@

@00

I

e

o c::~ c : ~ 0

O

0

@

G

~0 D

Fig. 2. P e p t i d e m a p s of B - c h a i n of oxidized insulin, digested b y alkaline p r o t e i n a s e s f r o m m o l d s Aspergillus. I, Horizontally, electrophoresis; II, vertically, c h r o m a t o g r a p h y . T h e m a i n s p o t s are h a t c h e d . T h e p r o t e i n a s e s were p r o d u c t s of t h e s e species: A, A. flavus ; B, A. oryzae; C, A. sojae ;

D, A. sulphureus.

rf

q ~

o

O,

I

e

II

X

x

X

~

0

x

o

~

~

0 Fig. 3. P e p t i d e m a p s o f t r y p t i c digest of d e n a t u r e d alkaline p r o t e i n a s e s f r o m m o l d s Aspergillus. See legend to Fig. 2. Main s p o t s differing in position f r o m m a p of t h e digest of A. flavus p r o t e i n a s e are m a r k e d Q; a b s e n t m a i n s p o t s are m a r k e d X.

Biochim. Biophys. Acta, 257 (1972) 257-263

262

j. TURKOV2{et a l

~I

A

c

B

D

Fig. 4. Peptide m a p s of c h y m o t r y p t i c digest of d e n a t u r e d alkaline proteinases from the molds Aspergillus. See legend to Figs. 2 and 3-

broad optimum in the pH-range 7-I o, very similar to the optimum of subtilopeptidase B. Fig. 2 shows that the specificity of each individual proteinase is practically the same when tested by the method of peptide maps, using the B-chain of oxidized insulin as substrate (cf. ref. 24). We would like to emphasize that the comparison is

merely qualitative. TABLE II COMPARISON

OF

RESULTS

FROM

PEPTIDE

MAPS

OF

PARTIAL

HYDROLYSATES

OF

ALKALINE

PROTEI-

NASES

Source o[ proleinase

Tryptic hydrolysate

Chymotryptic hydrolysate

Number of main spots

Number of main spots

Number of spots identical with A. flavus proteinase

Number of spots identical wtih A. flavus proteinase

A. flavus A. oryzae

16 16

standard 16

33 33

standard 33

A. sojae

15

14

34

3°

A. sulphureus

12

IO

25

19

Biochim. Biophys. Acta, 257 (1972) 257-263

Degree o[ homology to A. ftavus proteinase

standard identical (or very closely related) closely related, b u t not identical homologous yet less related

ALKALINE PROTEINASES OF ASPERGILLUS

263

V e r y c o n v i n c i n g e v i d e n c e of t h e d e g r e e o f h o m o l o g y b e t w e e n t h e a l k a l i n e p r o t e i n a s e s w a s a f f o r d e d b y p e p t i d e m a p s of t h e i r e n z y m i c digests as s h o w n in Figs. 3 a n d 4. E s p e c i a l l y t h e r e s u l t s o b t a i n e d w i t h t h e r e p e a t e d t r y p t i c a n d c h y m o t r y p t i c digest, s u m m a r i z e d in T a b l e I I , i n d i c a t e t h e i d e n t i t y o f a l k a l i n e p r o t e i n a s e s i s o l a t e d f r o m t h e m o l d s A . f l a v u s a n d A . oryzae a n d t h e e x i s t e n c e o f h o m o l o g y b e t w e e n t h e s e p r o t e i n a s e s a n d t h e a l k a l i n e p r o t e i n a s e s f r o m A . sojae a n d A . sulphureus. T h e p r i m a r y s t r u c t u r e o f t h e a l k a l i n e p r o t e i n a s e f r o m A . sulphureus differs m o s t f r o m t h e p r i m a r y s t r u c t u r e o f t h e p r e c e d i n g p r o t e i n a s e s . T h e p e p t i c digests w e r e f o u n d to be less c o n v e n i e n t b e c a u s e o f t h e g r e a t n u m b e r of s p o t s w h i c h r e n d e r e d t h e e v a l u a t i o n o f t h e m a p difficult. T h e o b t a i n e d r e s u l t s s h o w t h e m e r i t o f a s i m i l a r c o m p a r i s o n o f e n z y m e s of d i f f e r e n t origin p r e p a r e d a n d c h a r a c t e r i z e d b y d i f f e r e n t a u t h o r s . T h e s e e x p e r i m e n t s w e r e s t i m u l a t e d b y t h e d i s c u s s i o n a t t h e S y m p o s i u m on M i c r o b i a l P r o t e i n a s e s , o r g a n i z e d b y Prof. B e n g t v o n H o f s t e n in U p p s a l a in 1967. A c o n s i s t e n t c o m p a r i s o n of this type permits a better orientation among the many reported enzymes.

ACKNOWLEDGEMENT T h e a u t h o r s are i n d e b t e d to Mr. J . Z b r o 2 e k for a m i n o a c i d a n a l y s e s a n d to Mrs. J . L u k e g o v A a n d Mrs. J . K o n e ~ n ~ for t e c h n i c a l assistance. REFERENCES i A. R. SUBRAMANIANAND G. KALNITSKY, Biochemistry, 3 (1964) 1861. 2 A. R. SUBRAMANIANAND G. KALNITSKY, Biochemistry, 3 (1964) 1868. 3 K. HAYASHI, D. FUKUSHIMAAND K. MOGI, Agric. Biol. Chem. (Tokyo), 31 (1967) 1171. 4 A. NORDWlG AND F. W. JAHN, Eur. J. Biochem., 3 (1968) 519. 5 J. TURKOVJ~,O. MIKEg, K. GAN~EV AND M. BOUBLfK, Biochim. Biophys. Acta, 178 (1969) IOO. 6 0 . MIKEg, J. TURKOV~., NGUYEN BAO TOAN AND F. ~ORM, Biochim. Biophys. Acta, 178 (1969) 112. 7 G. DANNO, Agric. Biol. Chem. (Tokyo), 34 (197 o) 264. 8 L. POLGAR, Acta Biochim. Biophys. Acad. Sci. Hung., 5 (197 o) 53. 9 K. HAYASHI, M. TERADA, D. FUKUSHIMAAND K. MOGI, Seas. Sci. (Tokyo), 17 (197 o) 17o. IO J. TURKOVJ, AND O. MIKE~, Collect. Czech. Chem. Commun., in the press. i i P. LALLOUETTEAND G. BOURDERON, Bull. Soc. Chim. Biol., 4 ° (1958) 1479. 12 J. TURKOVA, O. MIKEg, R. RICHOU AND P. LALLOUETTE,Revue Immunol. Ther. Antimicrob. in the press. 13 G. BRETSCHNEIDER,A. NORDWlG, O. MIKEg AND J. TURKOVA,Z. Physiol. Chem., 352 (1971) 1372. 14 O. MIKEg AND J. TURKOVA, Abstr. National Meetings Czech. Biochem. Soc., Martin, I97 o, Abstr. No. IV-6. 15 O. MIKEg AND J. TURKOV~,, Abstr. National Meetings Czech. Biochem. Soc., Praha, 1971 Abstr. No. ]II-5. 16 O. MIKEg AND J. TURKOVX, Abstr. 7th F E B S Meeting Varna, 1971, Abstr. No. 153. 17 R. A. REISFELD, U. J. LEwis AND I). E. WILLIAMS, Nature, 195 (1962) 281. 18 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 3° (1958) 119o. 19 M. L. ANSON, J. Sen. Physiol., 22 (1938) 79. 20 Z. PRUSIK AND t3. KEIL, Collect. Czech. Chem. Commun., 24 (196o) 2049. 21 S. G. WALEY AND J. WATSON, Biochem. J., 55 (1953) 328. 22 H. MATSUBARA,C. B. KASPER AND E. L. SMITH, J. Biol. Chem., 240 (1965) 1125. 23 L. KEAY, P. W. MOSER AND B. S. WILDI, Biotechnol. Bioeng., 12 (197o) 213. 24 J. TURKOV~. AND O. MIKEg, Biochim. Biophys. Acta, 198 (197 o) 386. Biochim. Biophys. Acta, 257 (1972) 257-263