Multiple forms of Rhizopus oligosporus protease

ARCHIVES

OF

RIOCHEMISTRY

AND

Multiple

BIOPHYSICS

Forms HWA

Northern

of Rhizopus

L. WANG Regional

Received

459-463 (1970)

140,

oligosporus

c. W. HESSELTIKE

.4ND

Research Laboratory,’ May

Protease

15, 1970; accepted

Peoria,

Illinois

61604

Jul,v 9, 1970

Ext,racellular proteases of Rhizopus oligosporus have been purified and separat’ed into five active fractions (A-E) by ammonium sulfate fractionation, gel filtration, or diethylaminoethyl cellulose chromatography. All fractions showed single bands on acrylamide gel electrophoresis. Preliminary characterization studies showed t,hat the enzymes respond similarly to inhibitors and have t,he same PI-I stabilities. ITowever, consistent differences in pH optima, temperature opt,ima, and ratio of casein digestion to milk-clotting activit,y were found between two groups consisting of Fractions A, B, C and Fractions D, E. Crystalline enzymes nere obtained from Fractions A and B.

Acid protease that was found in the filtrates of Rhizopus oliqosporus cultures (1) exists in more than one form (2). In this communication, evidence will be presented to show that the multiple forms of the Rhizopus protease occur naturally. Biochemical differences of these multipIe forms will be described. MATERIALS

AND

METHODS

Extracellular enzyrrte preparation. R. oligosporus NRRL 3271 from the ARS Culture Collection at the Northern Laboratory was used throughout the course of this investigation. The culture medium was 547, skimmed milk powder. Cult,ivation procedures and ammonium sulfate fractionation of the culture filtrates were carried out as previously described (2). The precipitates collected from 30-75% saturation with ammonium sulfate were dissolved in a minimum amount of water. The protein solution was then dialyzed against 0.05 M phosphate buffer (pEI 5.8) at 4” and subjected to gel filtration on Sephadex G-l00.2 Gel-filtralion chronzatograph?J. Sephadex G-100 1 This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of .4griculture. 2 The mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned. 459

from Pharmacia Fine Chemicals was allowed to swell for 3 days in 0.05 M phosphate buffer (pII 5.8) at room temperature. The gel filt,ration of t,he extracellular enzyme was performed at 25” under the following condit,ions: bed dimensions, 2.5 X 36 cm; flow rate, 20 ml/hr; eluant, 0.05 M phosphate buffer of pH 5.8. The enzyme fractions were pooled, dialyzed against, water, and freeze-dried. sample was then dissolved in a The freeze-dried small volume of 0.1 M phosphate buffer and subsequently fractionated over a diethylaminoethy13 (DEAEj-cellulose column. L)E.gE-cellulose chromalography. This anionexchange material (Rio-Rad Laboratory) was equilibrated with the startiug buffer of 0.1 M phosphate at pH 5.8 and packed in a 2.5 X 42-cm colunm with a water jacket. The temperature of the jacket was maintained at 4”. After addition of the protein sample, the column was eluted with the starting buffer of 0.1 M phosphate at pH 5.8, and then p-ith a salt gradient of 0.1 to 1.0 Y NaCl prepared in the starting buffer. The gradient was established by connecting two identical vessels, one of which (the mixing vessel) contained 300 ml of 0.1 M KaCl in t,he buffer and the other, 300 ml of 1.0 M NaCl in the same buffer. Gel electrophoresis. Electrophoretic studies were made with an 12-C vertical gel electrophoretic cell. The electrophoresis was performed with a 7yti Cyanogum-ll (95% acrylamide and 5% bisacrylamide) gel at pH 9.0. The electrophoretic 3 Abbreviations used: DEAE, diethylaminoethyl.

4u.l

WANG

ANI)

separation W&S carried out for 2 hr at 300 V and about 60-120 Ma. The gel plate was stained with amid0 black; the stained background was removed with a mixture of methanol:H?O:acetic :tcid (5:5: 1). I*:nzyme assays. Proteolytic activity was meas[Irod by the casein digestion method described by Krlnitz (3). Unless otherwise indicated, the proteolytic assay was conducted at, pH 3.0 and a react ion temperature of 40” (2). The proteolytic activity was expressed in terms of pmoles of tyrosine formed per hour at. 40” or as A On at 280 rnp. Milk-clothing activity was det,ermined by measuring the time required for 1 ml of enzyme solut.ion to clot 10 ml of a reconstituted milk substrate as suggested by Berridge (1) and under conditions we determined (2). Twelve grams of nonfat dry milk was homogenized in 100 ml of 0.01 M calcium chloride solution with a PotterElvehjem homogenizer. The milk was then kept for an hour at 25”. The pH was adjusted to 6.1 if necessary. The experiment was ronducted at 25” in a test tube on a rotator set at 8 rpm. When a thiil film of milk on the glass surface broke int.o visible specks, that time was t.akeu as the end poiilt. The rennet unit of an enzyme solution is obtained by comparing its clotting t,ime with t.hat of a rennet, tmrmal solution used ill the dairy industry which arbitrarily contains 100 rennet nnit.s/ml. The amount of protein was estimated by the use of Folirl-Ciocaltenu reagent, according to Lowry el al. (5). RESULTS

Isolation of the en.zynze. Gel filtration on Sephadex G-100 of the crude enzyme preparation obtained from ammonium sulfate fractionation yielded two overlapping peaks absorbing at 280 mp. The first absorbing peak, which had the larger molecular size,

Effluent, ml

FIG. 1. Fractiollation of Rhizapus oligosporus proteases by gel filtration on Sephadex G-100.

HII;SHELTINE NaCl Gradient from 0.1 to 1.0 M

401

1I

n

30

40

50

60 90 100 Tube No 15 ml\

110

120

130

FIG.

2. Ion-exchange chromatography of 11. proteases on diethylaminoethyl (DEAE) cellulose. (a) Enzyme fraction from gel filtratiou; (1)) same as (a) after incubat,ing at 28” for 25 hr. ICnzyme fractions A-E.

oligosporus

displayed proteolytic activity (Fig. 1). After gel filtration further fractionation of the proteolytic enzyme 011 DEAE cellulose gave several active peaks. A typical elution pattern of the enzyme preparation from a DEAE-cellulose column is reproduced in Fig. 2a. Two peaks were noted when the column was eluted by the starting 0.1 AI phosphate buffer at pH 5.5. The first peak had no enzymic activity, while the second peak displayed strong casein digestion and milk clotting activities. This active fraction was designated as Fraction A. The materials eluted by the KaCl gradient were designated Fractions B, C, 11, and E. Although the quantities of Fractions C, D, and E were much less than for Icractions A and B, experimental results indicate that they all indeed exist naturally. Tests for autolysis awl artifacls. The possibility that these fractions might be the product of self-digestion during the periods of cultivation and purification was checked by the following experiment: The erbzyme fraction from gel filtration was dlvlded equally into t\w portions. One portsion was

Khizopus

oligosporus

immediately applied to a DEAE-cellulose column and eluted as usual; the other was incubated at 28” for 25 hr and then fractionated on the DEAE-cellulose column. The elution patterns of these two enzyme preparations are compared in Pig. 2. There is no annreciable difference after a 25-hr autolysis.’ Therefore, the components are probably not the degraded products of self-digestion. To eliminate the possibility that these multiple forms are not artifacts from column chromatography, an individual fraction was rechromatographed. Each fraction appeared as a single peak at the expected position. Furthermore, when R. oligosporus was cultured in different media, 2 % wheat flour, 2% wheat bran, or 5% skimmed milk, proteases were oroduced at different ratios (Fig. 3). The en&me oreuaration from wheat flour medium displayed only one major fraction corresponding to Fraction D (Fig. 3a), whereas that from either milk or wheat bran medium yielded the same five active fractions (Fig. 3b and c). The magnitude of each fraction. however, was characteristic of the growth medium. The major fractions of 20

t

[al Growth Medrum: Wheat Flour

I

IY

--NaCI Gradient from 0 to 1.0 M in pH 5.8 Buffer 40 [c) Growth Medium: Wheat Bran 60

n

t 80-

46 1

PROTEASE

FIG. i. Gel electrophoresis of crrlde enzymes and protease fractions of R. oligosporm. Left to right: crude ellzyme, Fractions A. IS, C, I>, and IC.

the enzymes from wheat bran \vere A, B, and E; whereas those from milk were A and B. In spite of the variations that occurred from one batch of enzyme preparation to another and in spite of the elution patterns that varied slightly from different DEhEcelluloae columns, enzyme preparations from wheat flour always yielded only one major peak, and those from wheat bran or skimmed milk invariably gave five peaks. I’roteolytic ~IIzymes which might originally occur in the substrates had, of course, been denatured during sterilization of the medium. When these fractions were subjected to gel each fraction displayed electrophoresis, different electrophoretic mobility (Fig. 1). The bands were homogenous. Corresponding bands were noted in a gel-elrctrophoretic pattern of crude enzyme preparation.

100 10

20

30

40 50 60 Tube No. (5 ml)

70

80

90

FIG. 3. Iowexchange chromatography 011 DEAF, cellulose of R. oligosporus proteases produced on three culture media. Enzyme fractions A-E.

Comparatil~e

Studies of T’arious Protease F?-actions

Solubility. The solubility of Fractions LI and B in water was low. Fractions C, B, and E, however, were easily soluble in water. All

4G?

WANG AND HESSELTINE 0.7

& c

2 '\

0.2-

.\

0

o,‘L

3

PH

4

r

I I 1 40 50 6?+Temperature, “C

I 30

FIG. 6. Effect of rcact.iau temperatlue 011 the rate of proteolysis for each fraction (A-E).

5

FIG. 5. The pTT activity curves of protense Fractiolls A-K of R. oliyo.spo~~~s.

TABLE

I

EFFECTS OF VARIOUS COMPOUNDS ON THE ACTIVITY OF PROTEASE FJJACTIONS FROM Rhizopus 0ligo.sporu.s

fractions were soluble in dilute salt solutions. Optimal pi7 ad pH stab&y. The pH activity curves of the five fractions (Fig. 5) did not differ greatly. They exhibited their maximum activity nt pH 2.5-3.0. All fractions were stable between pH 2.2-6.0, but they were rapidly denatured above pH 7.0. ReactiorL temperature ad ih~emostability. The effect, of reaction temperatures on the enzyme activities of all fractions is shown in Fig. 6. Optimum activity was displayed by Fractions D and I? at SO”, and their activities decreased rapidly thereafter. Vractions A, B, and C reached their maximum activity at CO”, which indicated that their stability was greater than lcractions D and IX. Above 60” their activities began to decline. However, preincubation of Fractions A, B, and C at 70” for more than 30 min inactivated the erkzymcs completely. Inhibitom and aclivatom We have already shown that soybean trypsin inhibitor does not affect the activity of R. oligosporus protease (1). The effects of other inhibitors and activators on each protease fraction are given in Table I. The data indicate that all protease fractions were inactivated completely by sodium lauryl sulfate. No other significant inhibition or activation was noted except that the activity of Fraction D was inhibited considerably by CUSOA .

Compoundsadded 5 x 10-sM

A

c

H

n

I?

Activity remaining (‘5)

Xa-lauryl sldfate L-Cysteine EDTA CaCl% cuso4

0 98 85 97 92

0 99 84 102 80

TABLE

II

0 103 93 112 85

0 71 99 81 4i

0 88 111 93 72

ACTIVIT~~ OF EACH ENzYA~E F~~ACTI~N FROM 12. &gosporus TOWAIW CASEIN DIGESTION AND MILK CLOTTING EIl2ylIle

fractions

Activity (unit/m6 protein) Ratio of casein digestion to --Casem -. digestion Milk clotting milk clotting

.- __

-

A B C 1) E

330 248 274 114 155 -___--.

5 .:3 5.9 5.9 0.8 0.4 .__----- -.

-..~-_

62 42 47 136 380

Ratio of proteolytic to milk-clottirq activity. All fractions clotted milk and digested casein, but their degree of activity varied. The relationship between milk-clotting a~tivity and casein-digesting activity is shown in Table II. Fractions A, B, and C had similar relative activities, whereas Fractions D and E behaved alike with comparatively

FIN;. 7. Crystalline

highrr activity clotting. Cryslalli2atiou

proteases

for casein dig&on oj

obhined

from Fraction

than milk

proleases J’ronl R.

dip

sporus. Fractions from DEXII:~cellulose chromatography lvere dialyzed against water and then freeze-dried. Tile freeze-dried ew zvmw were dissolved in a minimum amount ok distilled water and enough acetone \~as added t,o make the solution cloudy. The solut.ions I\-ere then kept at 0” for 1 week. Crystxllized enzymes rrsultcd from Fractions A and B (Fig. 7). l)IsCuSsIOu

i

Earlier we demonstrated (2) the separaprotease into four action of R. ok~ospoms tive fractions, which correspond to Fractions B, C, D, and E here. Previously, we chromatographed the enzyme preparation from ammonium sulfate fractionation directly on a DINE-cellulose column without gel filtmtion. Obviously, Fraction A is masked by the bulk of inactive materials, x&h behave similarly on DEAE cellulose as the enzyme. Sow we know that gel filtration removes almost all the inactive material. Fraction A, therefore, is distinctly separated from the innctive material. A better separation can be

A (a) and Fraction

B (b).

obtained \vhrn the chromatogram is devcloped I\-ith a buffer gradient of 0.01 to 0.1 11 at pH 53 instead of 0.1 11 phosphate buffer. Although substantial evidence has been presented that the multiple forms of R. oli~osporus are real and reproducible entities, there is not enough evidence to consider them :w either isoenzymes or different PI+ zymeh. 1,‘ractions A, IS, and C act alike as :I group in terms of pH optima, temperature optima, and ratio of casein digestion to mill TIESSI.:I.TIm, 1. W.ING,

c. w., Can. J. !~~icrobioz. 15, 99 (Km). 3. KUNITZ, 11.. J. Gen. t'hgsiol. 30, 291 (194i). 4. BICRRIDG~:, N. J., Biochem. J. 39, 179 (1!)45). 5. LowKY,O.H., IIOREBKOUGH,N. J., FAIW~ X. C., Chem. 193, .\xD R.\NI).\LL, R. J., J. EM.

x5

(1951).