Purification and some properties of the extracellular acid proteases from Mucor renninus

Purification and some properties of the extracellular acid proteases from Mucor renninus I. P. Belyauskaite, V. J. Palubinskas, O. E. Anchenko,* V. S. Vesa and A. A. Glemzha All-Union Research Institute for Appfied Enzymology, Vilnius 232028, USSR

(Received 26 January 1979; revised 7 August 1979) A complex ofproteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid o f the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I - I I I were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, H and III, respectively. Milk-clotting activity was exhibited only by protease IL A ll three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases H and 111. Trypsinogen was activated only by protease L All three enzymes have a molecular weight ~35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis, lsoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca 2+, Mg 2+, Cu 2+, Co 2+) on proteolytic activities o f these enzymes studied.

Introduction

Materials

The ever increasing requirement for calf rennet in the cheese industry and its limited availability makes the search for microbial rennet substitutes increasingly important. Strains of Mucor have been reported to be potent sources of milk-clotting enzymes. 1-11 Milk-clotting protease with molecular weight 29 0 0 0 - 3 0 600 and pH optimum at 4.0 (haemoglobin) has been isolated from Mucor pusillus.l-3 Other authors 4-9 have isolated protease having the properties of pepsin and rennin, with molecular weight 39 000 and isoelectric point at 4.2 from Mucor miehei CBS 370.65. Sternberg lo, 11 purified milk-clotting enzyme (molecular weight 27 000 and pH optimum at 3.5) from M. miehei NNRL 3420, using haemoglobin as substrate. It was determined that the enzyme hydrolyses casein and bovine serum albumin at a slower rate than haemoglobin. Some authors 12,13 have reported that strains of Mucor produce not only milk-clotting (rennin-type) enzymes, but also proteases without any milk-clotting activity. These proteases destroy the clumps of clotted milk and impart the bitterness to cheese during its maturation. So far, rennin-accompanying proteases, with their detrimental effect on cheese, have been poorly studied. The limited data available on these proteases make it difficult to remove them from the enzyme preparations. Regarding the practical and theoretical importance of this question, the aim of this paper was to study the complex of proteases from M. renninus, ~4 to isolate individual proteases and to investigate the properties of the enzymes. Biospecific adsorbent is on which proteases of Bacillus subtilis have been successfully resolved 16,17 was used for fractionation and purification of individual proteases.

The following chemicals were used: Sephadex G-75 (Pharmacia, Sweden), haemoglobin, Na caseinate, trypsinogen (Chemical Plant, Olaine, USSR), trypsin (SPOFA, Czechoslovakia), chymotrypsinogen, cytochrome c, horse myoglobin, ovalbumin, bovine serum albumin (SERVA, West Germany), human serum albumin (Koch-Light Laboratories, England) ampholines, pH 3 - 1 0 , 3 - 6 (LKB, Sweden). Other chemicals were analytical grade. The strain ofM. renninus used was obtained from the Moscow Technological Institute of Food Industry.

* Experimental-lndustrial Plant of Enzyme Preparations. 0141 --0229/80/010037--08 $02.00 © 1980 IPC BusinessPress

Methods G r o w t h o f f u n g a l culture a n d recovery o f crude e n z y m e

Cultures were grown in a 1.5 m 3 fermenter on a medium containing 3% maize starch, 1.5% casein, 0.2% CaCI2 and 0.05% KH2PO a. The medium was inoculated with 3% of a 24 h culture ofM. renninus. Fermentation was carried out at 30°C, aeration 0.8-0.9 m3/m 3 medium.rain and stirring at 200 rev/min. During growth the pH, initially 5.5, of the medium was not regulated. By the end of fermentation the culture fluid was filtered through filter-perlite. The obtained filtrate was freeze-dried. Preparation o f biospecific a d s o r b e n t Biospecific adsorbent was obtained by coupling N-(paminobenzyl)-L-tyrosine to chlorosulphonated polystyrene crosslinked with 0.5% divinylbenzene. 15 Preparation o f s u l p h o c a t i o n e x c h a n g e r Sulphocation exchanger was obtained by chlorosulphonation of polystyrene crosslinked with 0.5% divinylbenzene

Enzyme Microb. Technol., 1980, Vol. 2, January

37

Papers and by the subsequent hydrolysis of sulphonated groups to sulpho-groups, l 8

Determination o f proteolytic activity The proteolytic activity was measured by the modified Anson's method 19 using 2% haemoglobin, denatured by heating for 15 min at 50°C, in 0.1M universal buffer (0.1 Macetic acid, 0.1 M-boric acid and 0.1 u-phosphoric acid mixed in equal volume proportions; 1 N-NaOH for adjusting pH) as the substrate. The reaction was allowed to proceed for 10 min at 30°C. One protease unit was defined as the amount of enzyme to liberate 1 #tool tyrosine from haemoglobin in one minute at 30°C.

Amino acid analys& The amino acid composition of the proteases was determined by the method of Moore and Stein 23 using an LKB-3201 amino acid analyser. Protein hydrolysis was carried out in 6 N-HC1 at 110°C for 24h. Cystine and methionine were determined as cystic acid and methionine sulphone, respectively, after performic acid oxidation according to Moore's method. 24

Carbohydrate determination The total carbohydrate content was determined directly using the phenol sulphuric acid procedure of Dubois et al. 2s

Determination o f trypsinogen activation Determination o f milk-clotting activity Milk-clotting activity was determined from the time taken for the enzyme to coagulate milk. 2° One milkdotting unit was defined as the amount of enzyme which clots 1 ml substrate (fresh milk) in 40 rnin at 35°C.

The method of Sodek and Hofman 26 was used for the activation of trypsinogen. Trypsin activity was determined as the rate of hydrolysis of a-N-benzoyt-DL-arginine-pnitroanilide.27

Isoelectric focusing Protein determination Protein was determined by the method of Lowry et al. 21 with bovine serum albumin as the standard.

Enzyme purification Freeze-dried crude enzyme preparation (2 g) was dissolved in 50 ml of 0.01 M universal buffer, pH 4.4, containing 0.002 M-calcium acetate. The solution was clarified by centrifugation at 4000 rev/min for 20 min at 4°C and applied to a 2.4 x 30 cm column packed with 50 ml of biospecific adsorbent which had been washed with 150 ml of 1 M-CaC12 and equilibrated with the buffer described above. Some proteolytically inactive proteins and pigments were eluted by washing the column with the starting buffer. Subsequently the fraction with proteolytic activity (fraction I) was eluted. However, the majority of proteolytically active proteins were retarded by the column. These proteases were eluted as two peaks (fractions II and III) by increasing the concentration of NaCI in the eluant. The fractions of all three proteases were collected separately, concentrated by ultrat~dtration with membrane YAM-100 (All-Union Research Institute of Sorbent Synthesis, Vladimir, USSR) and once again applied to a column of biospecific adsorbent. Fraction I was eluted by washing with the starting buffer. Fractions I1 and III were released by a linear gradient of NaCI. After rechromatography the enzymes were concentrated by the same ultrafdter and applied to a 3 x 100 cm column of Sephadex G-75. After gel chromatography, electrophoretically homogeneous proteases were obtained.

Study o f protease fractions by gel ehromatograJahy on Sephadex G-75 and by sodium clodecylsulphate disc electrophoresis Protease fractions were studied by gel chromatography on a calibrated Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis 22 to determine the molecular weight, homogeneity and the number of polypeptide chains. Mercaptoethanol (5%) and sodium dodecylsulphate (2%) were used for the destruction of disulphide bonds. Cytochrome c, MW 12 400; horse myoglobin, MW 17 800; trypsin, MW 23 800; chymotrypsinogen, MW 27 000; ovalbumin,Ml¢ 45 000 and human serum albumin, MW 69 000 were applied as standard proteins.

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Enzyme Microb. Technol., 1980, Vol. 2, January

The method of Westerberg and Swenson was used. Isoelectric focusing was carried out on a 110 ml column with ampholines 3 - 1 0 in a 0 - 5 0 % sucrose gradient. 28

Determination o f optimum pH The optimum pH for enzyme activity was determined by assaying proteolytic activity with 2% haemoglobin, dissolved in buffer of the corresponding pH, as the substrate.

Determination o f pH stability range The solutions of each protease in 0.01 M universal buffer of various pH values were incubated at room temperature for 1 and 24 h. Proteolytic activity was measured after the incubation.

Influence o f metal ions on proteolytic activity Solutions of CaC12, MgC12, CuCIz and CoC1z were prepared in the concentrations 2 x 10 -2, 2 x 10 -3 and 2 x 1 0 - 4 M in 0.01 M universal buffer, pH 4.4. Enzyme solutions were mixed with metal ion solutions in the ratio 1 : 1 and incubated for 30 min at 30°C. Proteolytic activity was determined after the incubation.

Results

Isolation and purification o f the proteases jrom M. r e n n i n u s Data on the chromatographic behaviour of crude enzyme preparation from M. renninus on biospecific adsorbent are given in Figure 1. Considerable amounts of inactive protein and pigments were removed from the column by eluting a column with starting buffer. At the end of the elution the protein fraction having proteolytic activity appeared. The fraction thus obtained was designated protease I. By increasing the ionic strength of the eluate by a linear gradient of NaCl, ~60% of the initial proteolytic activity was released from the column; this was distributed between two protein fractions, termed proteases II and II1, respectively. The Figure indicates that proteases II and I|I were not completely resolved. Milk-clotting activity was found in protease !I alone, while proteases ! and Ill did not exhibit this activity.

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Proteolytically active protein fractions (proteases I - I l l ) were collected separately and concentrated by ultrafiltration. The data given in Table i show that by using ultrafiltration with YAM-100 membrane, the enzyme solutions were not only concentrated and desalted but the specific activity of the proteases increased considerably. This is explained by the observation that a certain portion of apparently low molecular weight proteins, were removed by the membrane, while the proteases were completely retarded. This purification stage resulted in the specific activity of individual proteases increasing 2 - 1 0 times. To demonstrate the availability of low molecular weight proteins, protease prepara-

tions were chromatographed on Sephadex G-75. Figure 2 shows that enzyme preparations obtained by chromatography on biospecific adsorbent consist of proteases and contain a considerable amount of low molecular weight proteins. The latter were effectively separated from proteases by gel chromatography. As a result the specific activity was increased by a factor of 35, 84 and 87 for proteases I, II and III, respectively. It follows, therefore, that only a part of low molecular weight proteins was removed by ultrafiltration. Sodium dodecylsulphate disc electrophoresis data show that all three protease preparations contain a major protein band and a minor one (Figure 3). The major protein in proteases I and II has a molecular weight of 36 000, and the minor protein 34 000. In contrast, for protease III the molecular weight of the major protein is 34 000, and the minor one 36 000. Thus, it was assumed that the preparations of proteases I and I1 contained traces of protease III, and that protease III contained traces of protease I and/or II. To obtain individual, homogeneous proteases, the preparations were concentrated by ultrafiltration and then rechromatographed on the biospecific adsorbent. The data given in Figure 4 show that traces of protease III were present in the preparations of proteases I and II, and successfully separated; traces of protease II were isolated from the protease III preparations. Protease preparations, free of contaminants, were chromatographed on a Sephadex G-75 column. The chromatographic data are given in Table 1, and show that a definite increase in the specific activity was obtained in the final stages of purification. However, a considerable loss of activity was observed. The resultant total activity yield was ~21% of the overall proteolytic activity of the crude enzyme preparation. Protease I was purified 75 times, protease II 120 times, and protease III 125 times. Thus, it is clear that proteases obtained from culture fluid ofM. renninus represent only

Table 1 Purification of M. renninus proteases Step

Protein (mg)

Proteolytic activity, PA (units)

Specific proteolytic activity (U/mg)

Crude enzyme preparation

12 860

720.0

0.056

1.0

100.0

1 051 514 278

90.4 296.2 1~.4

0.086 0.576 0.376

1.5 10.3 6.6

12.5 41.1 14.5

103 139 148

90.0 296.1 103.9

0.870 2.130 0.700

15.5 38.0 12.5

12.5 41.1 14.4

R e c h r o m a t o g r a p h y on biospecific adsorbent Fraction I F r a c t i o n II F r a c t i o n II I

33 52 11

53.6 270.8 46.2

1.640 5.2~ 4.1~

29.3 92.9 73.2

7.5 37.6 6.4

C o n c e n t r a t i o n by u l t r a f i l t r a t i o n Fraction I F r a c t i o n II Fraction III

20 47 9

52.0 265.5 45.0

2.6~ 5.700 5.2~

46.4 101.8 92.9

7.2 36.9 6.3

Gel chromatography on Sephadex G-75 Fraction I Fraction II Fraction III

5 15 4

20.4 100.4 30.0

4.200 6.800 7.000

75.0 121.4 125.0

2.8 13.9 4.2

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Purification (fold)

Yield (%)

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a small portion of the total protein in the crude enzyme preparation.

Molecular weight and amino acid composition o f proteases from M. renninus ÷

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40

Enzyme

Microb. Technol.,

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Following gel chromatography on Sephadex G-75, the homogeneity of proteases was checked by sodium dodecylsulphate disc electrophoresis. Electrophoretic patterns (Figure 5) show that fairly high quality protease preparations were obtained. Using standard proteins it was determined that proteases I, II and III have molecular weights of 36 500, 36 000 and 34 000 daltons, respectively. Molecular weight was also determined by gel chromatography on Sephadex G-75 and gave values of 36 700, 34 300 and 34 300 for proteases I, II and Ill, respectively. Taking into account the accuracy of the method, all three proteases can be considered to have the same or approximately equal molecular weight, ~35 000 daltons, which is close to that reported for microbial acid proteases. The fact that the molecular weight values determined by sodium dodecylsulphate disc electrophoresis and by gel chromatography on Sephadex G-75 are similar suggests that all three proteases consist of one polypeptide chain and that their structures are similar. To establish the existence of different proteins, amino acid composition was determined. The results in Table 2 indicate that all three proteases have a high content of acidic, hydrophobic and hydroxy amino acids, i.e. aspartJc acid, glutamic acid, serine, threonine, glycine, alanine, leucine and valine, although the composition is different for each of the three enzymes. The most noticeable difference is in the content of sulphur-c,mtaining amino acids. Neither methionine nor cystine is R)und in protease I while both are present in protease 11, with 8 cystine and 9 methionine per molecule. Protease Ill contains 11 cystine per molecule but no methionine. Considerable differences in the compositions of the isolated proteases were confirmed by the results of carbohydrate determination, which show that proteases I and 111 are devoid of carbohydrates, while carbohydrates make up ~8% of protease ll.

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Fraction number Figure 4 Rechromatography of individual proteases I - - I I I on biospecific adsorbent. 33 mg, 52 mg and 11 mg ( L o w r y ) of proteases I, II and I i i , obtained f r o m chromatography of crude enzyme preparation on biospecific adsorbent were applied to a 2.4 x 30 cm column at a f l o w rate of 14 ml/h. - - , Protein concentration; . . . . , proteolytic activity; - - . - - , NaCI gradient. (a) Protease I; (b) protease II; (c) protease III

Hydrolysis o f natural substrates by proteases 1-111 The action of isolated proteases on different natural substrates was studied for differences between the enzymes. Haemoglobin, Na caseinate, bovine serum albumin and trypsinogen were used as substrates. The results given in Table 3 indicate that protease I possesses the most wideranging activity, which differs slightly for different sub-

Table

strates. Protease I hydrolyses Na caseinate more rapidly than haemoglobin and albumin, and it activates trypsinogen. Proteases II and 1II do not activate trypsinogen and they hydrolyse haemoglobin more intensively than Na caseinate

÷

2 A m i n o acid composition of proteases I - - I I I

A m i n o acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan A m i n o acid residues Carbohydrate, %

Protease I 42 23 47 35--36 20 56 23 0 19 0 16 22 9 13 15 2 4-5 -346--348 0

Protease II

Protease III

42 21 26 33--34 14 35 24 8 16 9 11 20 8--9 12 14 1 6 -300--302 8

45 22 36 24 13 59 26 11 19 0 9 25 7 11-12 16--17 2--3 4--5 -329--333 0

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II

4.2 5.3 2.5 +

6.8 2.3 1.6 --

•

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7.0 3.2 1.5 --

A

B

C

Figure 5 Polyacrylamide disc electrophoresis of finally purified proteases I--I II. A, Protease I; B, protease II ; C, protease I I I

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and albumin. Proteases II and III are similar in their action towards different natural substrates, but protease II possesses milk-clotting activity and, in this respect, differs from protease III.

Study o f pH-dependence for proteases I - I I I The results of electrofi3cusing (Figure 6a) indicate that protease I produces a narrow and relatively symmetrical peak corresponding to a pI of 3.6. Protease II is eluted from the column in a symmetrical and broader peak corresponding to a pI of 5.1 (Figure 6b). The peak of electrofocused protease III is less symmetrical and corresponds to a pI of 5.4 (Figure 6e). The differences in pH-dependence of the proteolytic activities of proteases I - I I I indicate differences in regulation. The data in Figure 7 indicate that the pH optima for proteases I, II and III occur at pH 4.25, 3.5 and 2.5, respectively, when haemoglobin is the substrate. Determination of the pH-stability range (Figure 8) shows that all three proteases are stable for 24 h at 20°C in the pH range 3 . 0 6.5, and in this respect there are no essential differences in the enzymes.

the practical application of such preparations is limited because of the wide specificity of these proteases towards milk proteins. In this study we have shown that a crude enzyme preparation from M. renninus consists of at least three different proteases having about the same molecular weight, ~35 000, but that only one of them possesses milkclotting activity. This can be called rennin-type protease Mucor rennin - and is of great practical interest. This protease was shown to have its isoelectric point at pH 5.1 and pH optimum at 3.5, but its most distinguishing feature is that it contains a considerable amount of cystine and methionine, and ~8% carbohydrates. A comparison of rennin-type enzyme properties with the literature indicates

A

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Influence o f metal ions on activity o f proteases I - I I I The results in Figure 9 indicate that the influence of metal ion is identical in the case of proteases I and II, but that protease III behaves differently. All three proteases are strongly activated by Cu 2+, while other metal ions have only a slight effect. Calcium ions are without effect on all three proteases.

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Discussion

Acid proteases possessing milk-clotting activity have been found and isolated from various microorganisms. Crude preparations of such microbial proteases are used as substitutes for calf rennin in cheese manufacture. However,

42

Enzyme Microb. Technol., 1980, Vol. 2, January

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3

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5

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pH Figure 7 O p t i m u m pH f o r proteases I - - I I I . (), Protease I; e, protease

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that although they possess many similar properties, some differences are apparent. Molecular weights for rennin-type proteases from M. pusillus, M. miehei 8 are reported to be 30 000, 27 000 and 39 000, which are close to that of the rennin-type enzyme isolated in this work, taking into account the accuracy of the determination methods. The isoelectric points 3.5-3.8 and 4.2 reported in the literature for rennin-type proteases 4 differ considerably from the value of 5.1 obtained by us. On the other hand, a pH optimum of 3.5 for the protease isolated in the present work agrees closely

with that reported (pH 4.0) in the literature for rennintype proteases from Mucor. This agreement also holds for the pH-stability range. We believe that we have identified the most important differences in amino acid content. The cystine and methionine content is two to three times higher than the cited literature data. 8 Also the carbohydrate content is higher by 1.5-fold. Despite the similarity between the rennin-type enzyme isolated by us and previously reported enzymes from Mucor, essential differences exist. We have successfully obtained a considerable amount of two proteases which do not show any milk-clotting activity

Enzyme Microb. Technol., 1980, Vol. 2, January 43

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hydrate content and isoelectric points of rennin-type proteases from M u c o r w i t h that obtained by us for rennintype protease, we may conclude that the authors 2,4,8 have studied mixtures of enzymes rather than individual enzymes. There is the possibility that differences between rennin-type enzymes so far reported, and that characterized by us, are caused by different strain producers.

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The authors wish to thank Dr K. Janulaitene for isoelectrofocusing and sodium dodecylsulphate disc electrophoresis and D. Machernite for the amino acid analyses.

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50

Fraction number

Figure 10 Gel chromatography of crude enzyme preparation on Sephadex G-75. 580 mg ( L o w r y ) of crude enzyme preparation in 0.01 M universal buffer in 0.002M-Ca acetate, pH 4.4, was applied to a 3 x 100cm column. - - , Protein concentration; - - - - - - , proteolytie activity

References 1 2 3

0.09

Iwasaki,S., Tamura, G. and Arima, K. Agric. BioL Chem. 1967, 31,546 Yu, J., Tamura, G. and Arima, K. Biochim. Biophys. Acta 1969, 171,138-144 Yu, J., Tamura, G. and Arima, K. Agric. BioL Chem. 1971, 35, 1194

4

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Fraction number Figure 11 Chromatography of crude enzyme preparation on sulphocation exchanger. 68 mg (Lowry) of crude enzyme preparation in 0.01 M universal buffer, pH 4.4, was applied to a 1.2 x 13 cm column. - - , Protein concentration; . . . . , proteolytic activity

in the crude enzyme preparation from M. renninus. Protease I with its properties (molecular weight, isoelectric point, pH optimum, activation of trypsinogen, lack of methionine and carbohydrates) resembles acid pepsin-type proteases isolated from Aspergillus saitoi,29penicillium roqueforti 3° and Penicillium janthinellum. 31 Thus, the protease I described in this work may be called M u c o r pepsin. This is the first time that such a protease has been isolated, purified and characterized from the fungal strain Mucor. Protease III has also been isolated for the first time, but we could not find an analogue for it in the literature. The data reported by Ottesen and Rickert 4 indicate that there occurs a partial resolution of proteolytic activity into some peaks during ion-exchange chromatography. This phenomenon is explained by the authors rather strangely: the separation of the enzymatic activity in some peaks occurs because of the high load on the columns. In previous reports, the homogeneity of obtained rennin-type proteases from M u c o r is insufficiently argumented. Considering the almost identical molecular weights and similar isoelectric points of proteases, the enzymes may not be separated by gel chromatography or ion exchange chromatography. Parallel experiments carried out by us show that only one peak of proteolytic activity is obtained by chromatography of a crude enzyme preparation on Sephadex G-75 column and sulphocation exchange columns (Figures 10 and 11). Comparison of the reported data of amino acid pattern, carbo-

44

E n z y m e M i c r o b . T e c h n o l . , 1 9 8 0 , V o l . 2, J a n u a r y

5 6 7

9 10 11 12 13 14 15

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

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