Multiple proteolytic enzymes of Bacillus licheniformis

ARCHIVES

OF

BIOCHEMISTRY

Multiple FRANK ~~partnlent

AND

Proteolytic F. HALL?

of Biochernistr~

114, 145153 (19%)

BIOPHYSICS

Enzymes

of 5acillus

H. 0. KUNKEL,

and Ndrition,

AND

licheniformis’

J. %‘I. PRESCOTT

Texas A d%M University,a

Cdlege Station,

Texas

Received October 19,1965 By the use of synthetic substrates, organofluoride inhibitors, and heat denaturation, three types of proteolytic enzymes were identified in culture filtrates of Bacillus ~ic~en~~o~~~s. The proteinases detected were (i) an enzyme with high activity toward M-acetyl-L-tyrosine ethyl ester (ATEE) and toward proteins, highly susceptible to organofluoride inhibition, and rapidly denatured at G5”C; (ii) a proteinase with low activity toward ATEE, slowly inhibited by organofluorides, and inactivated relatively slowly at 65°C; and (iii) an aminopeptid~e, unaffected by organofluorides, and stable at 65°C for 60 minutes. In the unfractionated enzyme system, esterolytic activity was greatest toward N-substituted esters of aromatic L-amino acids, but substituted esters of the n-enantiomorphs were not hydrolyzed. Unsubstituted esters of L-amino acids were not susceptible, and N-substituted esters of basic amino acids were hydrolyzed only slowly, as were the amides of N-blocked aromatic amino acids. Gel-.infiltration on Sephadex G-209 separated two fractions which varied greatly in their relative activities toward ATEE and N-benzoyl-t-arginine ethyl ester (BAEE), thus furnishing further evidence for the presence of two endopeptidases. Gross proteolysis of the unfractionated system was enhanced by Ca++ ions, and was inhibited by EDTA. The aminopeptidase was stimulat,ed by Co* ions.

A decade ago, Damodaran and his coworkers (1) reported the existence and some properties of a proteolytic system in culture filtrates of Bacillus licheniformis. These workers observed rapid hydrolysis of protein suggestive of endopeptidase substrates, activity, but a number of synthetic substrates for trypsin, chymotrypsin, and pepsin were not susceptible. Several di-, tri-, and tetrapeptides were cleaved from the ~-ter~nus, and leu~inamide was hydrolyzed, thus indicating the presence of an aminopeptidase, which was shown by inhibition experiments to be different from the main protein-hydrolyzing enzyme. Subsequently, Bernlohr and Novelli (2) observed proteolytic activity in another strain of I?. 1This work was supported in part by grant A-003 of the Rabert A, Welch Foundation and by grant AI-00965 of the U.S. Public Health Service. SPredoetoral Fellow of the Graduate College, and of the Robert A. Welch Foundation. 3 Contribution of the Texas Agricultural Experiment Station.

licheniformis, and Bernlohr (3) reported some properties of an enzyme preparation made by autolyzing and dialyzing lyophitized crude culture filtrates. Damodaran et al. (1) made no attempt to fractionate the enzyme system, and Bernlohr (3) stated that attempts at purification by convenlional methods gave poor results. In view of the evidence of Damodaran et aE. (1) for at Ieast two proteolytic enzymes, it appeared relevant (i) to investigate the number and types of proteinases present in the crude B. lichf?niformis proteolytic system, as judged by their action on various types of synthetic substrate and by their susceptibilities to inhibitors, and (ii) to establish procedures for the fractionation of the crude system and the purification of individua1 p~~in~~. MATERIALS

AND

METHODS

Culture and Microbiological

Procedures

The organism used was a subculture of the original isolate of Damodaran et al. (1) obtained 145

146

HALL,

KUNKEL,

from American Type Culture Collection (No. 11560). When the subculture was received in the laboratory, its characteristics were determined by the procedures of Smith et al. (4) and were found to be identical to those listed for B. tichen& formis by these workers and by Breed et al. (5). Stock cultures were grown at 37°C on agar slants containing the compot~er~ts shown in Table I. Cultures were stored under a thin layer of sterile mineral oil at 5”C, and required transfer only at 6-8 month intervals.* The inoculum medium used for enzyme production contained twice the concentration of nutrients shown in Table I, with the agar omitted. Forty ml of the inoculum medium in a 125-ml Erlenmeyer flask were inoculated by loop, and the organism was grown on a rotary shaker for 12-18 hours at 37°C; this quantity of inoeulum was used for 5 liters of growth medium.

Enzyme Pv-o&&on The medium used for enzyme production was identical t,o that of Damodaran et al. (1) and consisted of 2% peanut meal, 170 glucose, 0.1570 CaC12, 0.1% K&PO+ and 0.05y0 MgSOa. A New Brunswick Scientific Co. fermentor (Model FS307), oomplet,e with drive assembly, water bath, and automatic antifoam units, was used for growing t,he cultures, which were aerated at a rate of 1.4 liters of air per minute per liter of medium and stirred at 400 rpm. Antifoam(General Electric Co.) was added from the antifoam units to retard foaming. The large cultures were grown at a temperature of 30”-32°C. At the end of the desired growth period, cells and any remaining insol~~bie prot,ein were removed by passage through a Sharplos Super Centrifuge. The supernatant fluid was filtered first through a oneinch pad of Hyffo Supereel, then successively through Millipore filters of 0.65 g and 0.45 s pore size. The dark brown, cell-free, culture filtrate was either frozen or stored at 5°C under a layer of toluene until needed for the enzyme experiments. On some occasions, the culture filtrate was concentrated approximately IO-fold by perevapora*Initially, cultures were maintained at room temperature on agar slants having twice the concentration of nutrients shown in Table I. This procedure resulted in a progressive decline in the abiIit,y of B. Eicheniformis to produce proteolytic enzymes. A fresh subculture, obtained from the American Type Cult,ure Collection and maintained as described in the text above, was highly stable and uniform in its proteinase production. Most of the experiments reported herein were performed with cultures maintained by t,he latter procedure.

AND

PRESCOTT TABLE MEDIU~X

FOR STOCK

Racillua

I CULTURES

Am~~nt/lit~r

Component

Agar Enzymically Glucose NaCl CaClz KHsPOa

digested

OF

licheniformia

caseina

~...

(pm)

20.0 10.0 5.0 1.0 0.75 0.50 _____..__ h-a)

MgSOs.7HzO FeSOc. 7H,O MnS04.Hz0

80.0 4.0 1.6

a Vit,amin-free cesein (Nutritioylal cals Corp.) was digested as described et al. (6).

Biochemiby Williams

tion from dialysis tubing and stored in t,he frozen state. Inasmuch as one of the major objectives of this investigation was to determine the number and types of proteinases elaborat,ed by B. Eichenijormis, all experiments unless ot,herwise stated, were performed with the crude enzyme solution prepared in this manner. Before assay, the enzyme sol&ion was dialyzed at least 12 hours at 6°C against buffers selected on the basis of the experimental objective.

Enzymic Assays Proteinase activity. Proteolytic activity was determined at 37°C with urea-denat‘ured hemoglobin substrate (pH 7.5) by the procedure of Anson (7), or by form01 titration with 1% casein substrate, as performed by Damoderan et al. (1). When the method of Anson was used, the extent of proteolysis was determined by reading the absorbancy of the TCA5-soluble degradation products at 280 rnr in a Beckman model DU or a Hitachi Perkin-Elmer model 139 spectrophotometer. One unit of proteolytio activity was 5 Abbreviations used: TCA, trichloroacetic acid; ATEE, N-acetyl-L-tyrosine ethyl ester; LNA, L-leucyl-@-naphthylamide; CGP, N-carbobenzoxygly~yl-L-phenylalanine; EDTA, d&odium ethylenediaminetetraacetate; tris, tris(hydroxymethyl)aminomethane; PMSF, phenylmethanesulfonyl fluoride; DFP, diisopropylphosphorofluoridate; APEE, N-acetyl-L-phenylalanine ethyl ester; TAME, p-tosyl-L-arginine methyl ester; BAEX, N-benzoyl-L-arginine ethyl ester; ATA, ~-acetyl-L-tyroaine amide; APA, N-acetyl+ phenylalanine amide; Hb, hemoglobin.

PROTEOLYTlC

ENZYMES

arbitrarily defined as the amount of enzyme that caused an inc Pease in absorbancy of 1 .O (280 mp) in 5 minutes. E&erase ~~~ui~~. Activity toward ester substrates was determined by t,he potent&metric method of Schwert et aE. (8), using 10 ml of lo-% M esters in deionized water. The reaction was performed at 25”C, and the pH was maintained at 8.0 by the addition of standardized, carbonate-free NaOH. These assays were done manually, using a Beckman Zeromat,ic pH meter, or automatically with a pH-stat (Radiometer TTTlc/SBR2c/ SBUla). One unit, of e&erase aet~ivity was defined as that, quantity of enzyme catalyzing the hydrolysis of ester substrate at a rate of one pmole per minute. Amidase activity. hssays for the hydrolysis of amides were performed ati 37°C and pH 7.5 by incubating 0.5 ml of enzyme, dialyzed in lO-% M Veronal-HCl buffer, with 0.5 ml of 2 X We M amide substrate solution dissolved in 2 X 10-* M Veronal-HCl buffer. The reaction was terminated by the addition of 2 ml of 0.1 N HCl, and the ammonia liberat,ed was detected by direct Nesslerization (9). Aminopeptidase activity. Aminopeptidase activity was determi~~ed at 37°C by the ~olorimetric method of Goldbarg and Rutenburg (10) with 3.4 X 1W4 M LNA in 10-Z M Veronal-HCl buffer (pII 7.5), or with leucinamide by t#he method described above for amidase activity. Unless otherwise indicat,ed, enzyme samples for aminopeptidase determinations were routinely rendered 5 X W4 A4 with respect to Co++ and incubated at 37°C for at least 2 hours prior to assay. One unit of amir~opeptid~e activity was defined as the amount of enzyme that catalyzed the hydrolysis of LNA at a rat,e of one rmole per minute under the conditions specified. CaTbo~y~e~t~dase activity. The subst,rate for carbox~~pept~dase activity was 2 X lo-% M CGP in 5 X lO+ M Tieronal-HCl buffer, pH 7.5, rendered 0.8 M with respect to NaCl. The reaction was performed by incubating one ml of substrate with one ml of enzyme solution at 37”C, and was terminated by adding one ml of ninhydrin reagent and heating in a boiling water bath for 15 minutes. Any liberated phenylalanine was detected by the quantitative ninhydrin method of Moore and Stein (11). Protein assays. Protein concentrations were estimated by the method of Lowry el al. (12); crystalline bovine plasma albumin (Armour Laboratories) was used as a standard.

Fractionation

Procedures

Fractionation of the proteolytic enzymes was accomplished by chilling a solution of culture

OF Budllus

Eicheniformis

147

filtrate, concentrated lo-fold, until it just commenced t’o freeze. Two volumes of acetone (-20°C) were added, and the resulting precipitate was eollect.ed by celltrif~lgation at 1560g. The precipitate was suspended in a minimum amount of tap water (pH ?: 8.0), and dialyzed overnight against running tap water to remove any residual acetone. Sephadex G-200 (Pharmaeia Fine Chemicals) was prepared for use by sLlspe~~di]lg approximately 40 gm of the material in dist,iiled water and allowing it t,o swell for 24 hours. Fine particles were removed by repeated decantation. The material was poured into a 4.2 X 56-cm ehromatographic tube and allowed to settle to a height of 50 cm. A filter paper disc was placed on the surface of the bed. The void volume of the Sephadex was determined with Blue Dextran-2000 (Pharmacia Fine Chemicals) and the column was eq~~ilibrated by allowing a quantity of elut,ing buffer (5 X 1W3 M Veronal-HCl, pH 7.5) equal to the bed volume of the Sephadex gel (700 ml) to pass through the column. Fifteen-ml samples of the dialyzed acetone precipitate were subjected to gel-filtration on the column. Protein in the effluent was detected by a Gilson Medical Electronics Ultraviolet Absorpt,ion Meter (280 met) and recorded on a Texas Instuments “Reetiriter” st.rip recorder.

Other Procedures Activator and inhibitor studies. The effects of a number of ions and compounds known to activate or inhibit other proteolytic enzymes were t.ested by incubating 10-d M, 5 X low3 M, and 10-* M concentrations of each with samples of R. lichenijormis enzymes. The ammonium sulfate salts of Al+++ and Fe+++ were nsed, the chlorides of Cu++, Hg++, Zn++, Co*+, Ni-+*, Ca++, Raft, and Sr++ were tested, and Mg++ and Mn++ were supplied as the sulfates. The compounds to be tested were made up in 5 X 10ea M tris-HCl buffer, pH 7.5, and the culture filtrate was dialyzed extensively against the same buffer. The enzyme sample and the test compound were incubated for 2 hours at 37”C, and an aliquot from each sample was assayed with hemoglobin substrate and compared t,o a control incubated with buffer. Organojluoride inhibition. The sensitivity of the enzyme syatem and of various enzyme fractions to organofluorides was tested using PMSF and DFP. PMSF inhibitiol~ was determined by mixing equai volumes of enzyme solut,ion and 2 X 1c3 M PMSF dissolved in 30% (v/v) 2-propanol. After one hour of incubation at 37”C, samples were assayed for remaining henloglobi~~ and ATEE activities; control samples were incubated in 15% (v/v) Z-propanol. To study DFP inhibition, samples rendered 5 X 1OW M with respect to the inhibitor were incubated for eit,her 2 or 24 hours at 25°C.

HALL,

148

KUNKEL,

Prior to incubation with DFP, samples which were to be assayed for hemoglobin and ATEE activities were dialyzed for 12 hours against 3 liters of 5 x 10-a M tris-HCl buffer, pH 7.5. Enzyme samples for LNA assays were dialyzed against 5 X 10-Z M Veronal-HCl, as tris buffer inhibited the aminopeptidase reaction. Thermal denaturation experiments. Studies of the effects of temperature were performed by assaying the enzyme system against hemoglobin for 5 minutes at temperatures from 6” to 65”C, and also by heating the enzyme in a 65°C water bath for varying intervals of time. The enzyme samples which were heated at 65°C were withdrawn at the desired time and immediately chilled in an iceNaCl bath, then were assayed as soon as possible against the various types of substrates at either 25°C or 37°C by the assay methods described above. Efects of pH. The optimal pH for the hydrolysis of hemoglobin by B. licheniformis culture filtrate was determined by adjusting hemoglobin substrate, prepared by the usual procedure, to various pH values from 5.0 to 10.0. To determine the effects of pH on est,erolytic activity, the hydrolysis of ATEE was carried out in the pHstat at values ranging from pH 5.6 to 10.1. The effects of hydrogen ion concentration on aminopeptidase activity were investigated by preparing LNA substrate in a composite buffer, 10-2 M with respect to acetic, diethylbarbituric, and boric acids. The pH values tested ranged from 5.1 to 9.5, and were obtained by titration of the stock solution of mixed acids with 6 N KOH; at each pH value, the ionic strength was adjusted to 5 X 10-z with KCl.

Reagents The hemoglobin and vitamin-free casein used for protein~e substrates were purchased from Worthington Biochemical Corp., and from Nutritional Biochemical Corp., respectively. Synthetic substrates were obtained from Cycle Chemical Corp., Nutritional Biochemical Corp., and Mann Research Laboratories. PMSF was purchased from Cycle Chemical Corp., and DFP from Aldrich Chemical Co. RESULTS

AN’D DISCUSSION

Proteolytic activity, as judged by the hydrolysis of hemoglobin or casein, was maximal after 2.5-3 days of growth, and was equivalent to that observed by Damodaran et al. (1) in 4 days with shake cultures. The shorter time required for maximum enzyme production can probably be ascribed to the

AND PRESCOTT TABLE EFFECTS

OF METAL F~EMOGLOBIN

II

IONS AND EDTA HYDROLYSIS

Percentage of Metal ion

CLP”

the controlactivity

Concentration of ionsa 10-s M

Fe”+ APf Hg++ Zn++ Co++ NP Ni++ Mn++ Ca++ SF Ba++ EDTA

ON

1

13 14 18 28 74 74 79 98 120 102 97 28

5

x 10-s M 12

13 17 22 59 83 96 84 85 114 104 96 27

lo-%+f

7s 85 60 95 92 100 94 99 85 105 95 97 19

1?The concentrations indicated are the final concentration in the enzyme solution before addition of the substrate. Enzyme concentrations during incubation were 0.160 mg/ml in the tests involving St+++ and Ba++ ions and 0.383 mg of protein/ml for all other ions. Appropriate blanks and controls were included in each experiment.

more vigorous aeration afforded by the fermentors. Ejfects of metal ions, EDTA, and reducing agents 012proteolysis. Damodaran et al. (1) showed the inhibition of casein hydrolysis by metal ions, in the following order: Cu++ > Hg++ > Co* > Mn*. Our experiments, using hemoglobin substrate, confirmed and extended these results (Table II). All of the ions tested except Gaff, Sr++, Ba* and &In++ adversely affected proteolysis at concentrations of 10-a M; the lower concentrations of ions were less inhibitory. The addition of Ca++ ions (W2 JQ consistently enhanced hemoglobin hydrolysis by 20-25%. The presence of a metal requirement for proteolysis is suggested by the inhibition produced by EDTA (Table 11). The omission of Ca++ ions from the growth medium resulted in a considerably lower yield of gross proteolytic activity. Calcium ions have been found by other investigator: (i) to prevent denaturation and au~digestion of certain proteolytic enzymes (13-15), (ii) to be required as a part of a metallo-proteolytic enzyme (15), and

PROTEOLYTIC

ENZYMES

(iii) to be necessary for the production of a bacterial proteinase (16). The present data do not reveal the specific role of Ca* ions in the 3. l~~~n~~orrn~ proteolytic system, but the effects of Ca* ions appear to be unique, as no other divalent cations tested were capable of stimulating proteolysis appreciably. It is noteworthy, however, that Sr++ and Ba* ions were non-inhibitory, and Sr++ ions apparently produced a slight stimulation at the two higher concentrations. Tests with reducing agents confirmed the observation of Damodaran et al. (1) that cyanide and cysteine at KF M do not affect proteolysis si~ificantly. In our tests, Na2S at 10e2 M also failed to enhance or diminish enzymic activity appreciably. Hydrolysis of synthetic substrates. Damodaran et al. (1) showed that their enzyme preparations degraded certain peptides from the N-te~inus, and hydrolyzed L-leueinamide, and thus estab~shed the presence of an aminopeptidase. Although they observed a rapid hydrolysis of protein substrates, their tests with some N-substituted peptides and amides failed to disclose endopeptidase substrates susceptible to hydrolysis. However, they did not test ester substrates. In order to determine the types of proteolytic activity present in the crude enzyme preparations, we tested the susceptibilities of a number of esters, amides, and synthetic substrates for aminopeptidases and car~xypeptid~es. The results of these experiments are summarized in Table III. hydrolyzed The preparations readily ATEE and APEE, which are classical substrates for chymotrypsin. Two trypsin substrates, TAME and BAEE, were hydrolyzed at much slower rates. In this respect, the B. ~ic~n~~o~~ system is similar t,o the endopeptidase from the fungus, Phymatotrichum Qmnivorum (17) and to the NOVO proteinase from B. s~btiZ& (18), both of which show a preference for esters of aromatic amino acids. Although ATEE and APEE were rapidly hy~olyzed, the analogous amides, ATA and APA, were attacked at barely discernible rates. This behavior also is reminiscent of that displayed by the fungal endopeptidase of Burgum and Prescott (17) and by the protozoan proteinases described by Dickie and Liener (19). The

OF Badlus

Eicheniformis TABLE

149 III

Acmvrm OF BaeilEte ~~che~~~~~rn~~ CRUDE ENZYME Towart~ SYNTHETIC SUBSTRATES Substrate

A’-Aeetyl-L-tyrosine ethyl ester (ATEE) iv-Acetyl-n-tyrosine ethyl ester ~-Acetyl-L-phenylalanine ethyl ester (APEE) L-Tyrosine ethyl ester (TEE) L-Phenylalanine ethyl ester (PEE) N-Carbobenzoxyglycyl-~-phenylalanine ethyl ester& p-Tosyl-L-arginine met,hyl ester (TAME 1 N-Bensoyl-L-arginine ethyl ester (BAEE) L-Arginine methyl ester L-Lysine methyl ester N-Acetyl-L-tyrosine amide (ATA) ~-Aeetyl-L-phenylalanine amide (APA) N-Carbobenzoxyglycyl-L-phenylalanine (CGP) n-Leucinamide L-Leucinamide + 5 X IO-* M Co++ L-Leucine-~-naphth~lamide (LNA) L-Leucine-~-naphthylamide + 5X 10-4 M co++

Relativea activity

100.0 0.0 27.9 0.0 0.0 6.4 7.7 4.3 0.0 0.0 trace trace trace 0.08 0.24 0.03 0.17

a Activities were determined from kinetic slopes under initial velocity conditions; relative activities are expressed as per cent of ATEE activity. Rate of ATEE hydrolysis was 1.7 amoles/ minfmg protein. Zero relative activity implies no measurable hydrolysis using a lo-fold concentrated enzyme solution. All substrates reported as “trace” were determined after at least 4 hours at 37°C. Assay procedures are described in the text. 5 Tested as a suspension in 2570 (v/v) ethanol.

low level of activity toward these amides would explain the failure of Damodaran et al. (1) to observe a chymotryptic type of as they used N-carbobenzoxyactivity, glycyl-L-phenylalaninamide as a substrate. Our results further indicate that only Nsubstitute esters of the L-~onfi~ration are susceptible. Although it is similar to chymotrypsin in its preference for aromatic amino acid residues, the esterolytic activity of B. &icheniformis is clearly different from ehymotrypsin, which attacks unsubst,ituted, as well as ~7-substitut~, esters. With respect

150

HALL,

5

6

7

8

9

KUNKEL,

IO

PH FIG. 1. Effects of pH on proteolyt8ic (A----A), esterolytic (O---O), and aminopeptidase (a---0) activities. Assays were performed as described in Materials and Methods. The values shown on the ordinate were obtained by multiplying the hemoglobiu and LNA activities by 100, and the ATEE activity by 2.

to exopeptidase activities, the data in Table III confirm the observations of Damodaran et al. (1) that, cob~t-activated aminopeptidase is present, and that carboxypeptidase A-like activity is absent. The abilit,y of Co* ions to stimulate aminopeptidase activity and to reduce endopeptidase activity indicated that different enzymes were responsible for these two types of activity, as suggested by Damodaran et aE. (1). The results in Table III, however, did not reveal whether the esterolytic and proteolytic act)ivities resided in one or several individual enzymes. Knowledge of t.he number of proteinases present was obviously desirable prior to attempting their fractionation. Consequently, a series of experiments was performed in order to resolve this qu&ion. ~~ec~~ oj pH on the e~~~e~. Figure 1 shows the effects of pH on the various enzymic activities identified in the crude enzyme from B. Eichenijormis. Maximum hydrolysis of both ATEE and LNA occurred at pH 1.5-9.0. The similarities in the curves for these two activities must be considered coincidental, however, as the experiments reported below unequivocally showed the enzymes to be separate entities. Damodaran et al. (1) found that L-leucinamide and DLat leucylglycine were cleaved optimally

AND PRESCOTT

pH 7.2 and 7.6, respectively. The more alkaline pH optimum for the hydrolysis of LNA is not surprising, considering the dissimilarity between LNA and the substrates used by Damodaran et al. (1). The pH optimum for the hydrolysis of hemoglobin is fairly broad, ranging from pH 7 to 8. Bernlohr (3) also found a broad, indistinct pH optimum for the hydrolysis of casein and azocasein by a proteinase from another strain of B. lichenijormis. Damodaran et al. (1) reported a somewhat, sharper optimum at pH 7.4 for casein hydrolysis. Although a broad pH optimum does not necessarily indicate the presence of multiple enzymes, this concept@is compatible with our other findings (see below). Effects of organo;lluorides. The esterolytic action of crude B. licheniformis enzyme, and the well-known ability of some organofluorides to inhibit certain esterolytic proteinases (e.g., subtilisin, trypsin, chymotrypsin) prompted us to test the effects of DFP TABLE

IV

EFFECTS

OF ORGANOFLUORIDES licheniformis PROTEOLYTIC

Inhibitor

PMSF DFP

FinaiGy’

Length of Incu$XILI~ b;z;

ON BaciEus ENZYMES ‘?&Inhibition activities

(hours)

(“C)’

Hb

lo-”

1

5 x lfl-*

2

37 25

92 85

of

ATEE

LNA

100 100

0

MINUTES FIG. 2. Inhibition of ATEE hydrolysis by PMSF. The arrow indicates the time at which sufficient PMSF was added to yield a final conoentration of 2 X 1V M.

PROTEOLYTIC

ENZYMES

AI

/I

~o.ll/ IO

20

30

40

TEMPERATURE,

50

\

60

OC

FIG. 3. Effect of reaction temperature on hemoglobin hydrolysis. For each temperature indicated, 5 ml of hemoglobin substrate (pH 7.5; EL= 0.22) were equilibrated before the addition of one ml of enzyme solution containing 0.386 mg of protein/ml in Veronal-HCl buffer (1.8 X 1P M, pH 7.5; p = 2.3 X IO-*). The reactions were allowed to proceed for 5 minutes.

and PMSF on the hydrolysis of different substrate. Both of these conlpounds completely inhibited e&eroIysis, but the inhibition of proteolysis was only partial, and aminopeptidase activity was unaffected (Table IV and Fig. 2). The residual proteolytic activity was consistently observed in t,he presence of concen~ations of DFP and PMSF sufficient to abolish hydrolysis of ATEE. The aminopeptidase, whieh was not inhibited by DFP, would hardly be expected to release from hemoglobin substrate significant amounts of material with absorbancy at 280 my. This is confirmed by fractionation experiments reported below, which yielded a fraction possessing aminopeptidase activity, but no measurable hemoglobin activity. The different extents of inhibition of esterolytic and prot,eolytic activities thus suggested that proteolysis was due to at least two enzymes, one of which rapidly hydrolyzed ATEE and was highly susceptible to inhibition by or~anofluorides, and one of which hydrolyzed ATEE slowly if at all, and was unaffected--or was inhibited slowly-by these reagents. I’hermal stabilities of the enzymes. A fur-

151

OF Baci&~s licheniformis

ther evaluation of the types and number of individual proteinases in the crude system was made by determining the effects of heat on the enzymes responsible for hydrolysis of the different substrates. Figure 3 shows the effect of assaying the crude enzyme with hemoglobin substrate at different temperatures. The greatly reduced prot.eolysis at 65°C prompted the use of this temperature t.o investigate the time-course of heat inactivation, the results of which are shown in Fig. 4. The loss in activity toward ATEE was logarithmic, and approached completion in 40 minutes. Although prot~lysis was substantially reduced by heating at 65”C, it was never diminished as drastically as esterolysis. The aminopept,i~se act,ivity was stable at this temperature, and in fact, increased slightly during the first 10 minutes of exposure. This increase was real, as demonstrated in repeated experiments, and may have resulted from the destruction of an inhibitor present in the crude enzyme. In the light of these resultas, it appeared that the crude proteolytic system of B. (i) an aminopep-

IO

30

30

40

30

MINUTES Fro. 4. Stabilities of the proteolytic (A-A), esterolytic (O--O), and aminopeptidase (@---a) activities of Bacillus l~chen~fo~~s to heating at 65’C. The enzyme solution contained 0.772 mg of protein/ml in 1.6 X 1P M VeronalHCl buffer, pH 7.5; ionic strengt,h of the prot,ein solution was 2.6 X lo-$. Samples were withdrawn at the times indicated, cooled quickly, and assayed against the appropriate substrates at eit,her 37” or 25°C. Esterofysis was assayed with ATEE substrate.

152

HALL,

KUNKEL,

AND PRESCOTT

40 =t

E

50

0 60 Q) N 70 < 00 $?

FRACTI~

NOR

5. Fractionation of the enzymes by gel-filtration on Sephadex G-200. Fractions 11 ml were collected from the column eluted with 5 X 10msM Veronal-HCl buffer, pH 7.5. Other conditions are described in the text. For simplification of t.he graph, have been multiplied by 2, the LPJA units (a----0) by 3, the hemoglobin units (A-A) and the ATEE units (O-O) by 0.01. The dashed line represents the percentage transmittance at 280 rnp. FIG. containing

TABLE V RELATIVE ACTIVITIES OF Bacillus lkheniformis PROTEINASE FRACTIONS TOWARD ATEE AND BAEE

TABLE EFFECT

Fraction

Unfractionated Fraction I6 Fraction IW

enzyme

activitiesa

toward:

ATEE

BAEE

1.4 1.4 5.9

0.06 1.0 0.1

ATEE/ - BAEE

23.3 1.4 59.0

DFP

VI

ON FRACTIONS

FBOM

SEPHADEX

G-200 Fractiona

Specific

OF

I I II III III

Substrate

Hb Hb LNA Hb ATEE

Incubation time (hounf

2 24 2 2 2

% Inhibition

68 100 0 100 100

Q Units/mg of protein. The assays and units are described in the text. * The fractions were obtained by gel-~ltration on Sephadex G-200 (Fig. 5).

a Each fraction was dialyzed as described in the text, incubated with 5 X lo-* M DFP for the times indic&ed, assayed for the various activities, and compared to a suitable control.

tidase, stable at 65°C for 60 min, which was unaffected by organofluoride inhibitors, (ii) a proteinase moderately resistant to thermal denaturation and which reacted slowly if at all with organofluorides, and (iii) a proteinase capable of cleaving ATEE, and which was highly susceptible to organofluorides and rapidly inactivated by heating at 65°C. Consequently, the separation of the co~tituent proteinases was unde~aken. Fract~~~~~on 0s enzymes. Acetone precipitates of the crude enzyme, prepared in the manner described in Materials and ~e~~o~~, were subjected to gel-filtration on Sephadex G-200; the results of a typical experiment are shown in Fig. 5 and Table V. Fraction I hydrolyzed hemoglobin substrate, and possessed low but measurable

activity toward ATEE, BAIZE and TAME; Fraction II hydrolyzed a~nopeptid~e substrates, and Fraction III readily attacked hemoglobin and ATEE, but hydrolyzed BAEE and TAME at barely discernible rates; it was devoid of a~nopeptid~e activity. Fraction I was obviously of high molecular weight, as it emerged in the void volume of Sephadex G-200 as determined by Blue Dextran-2000. These results confirmed the existence of at least two endopeptidases, having widely different ratios of activity toward N-substituted esters of tyrosine and arginine. Samples of each fraction were tested for ~sceptibi~ty to inhibition by DFP, with the results shown in Table VI. Fraction II, which contained the aminopeptidase activity, was unaffected by DFP,

PROTEOLYTIC

ENZYMES

but both the proteolytic and esterolytic activities of Fraction III were tot&y inhibited after 2 hours exposure to DFP. Fraction I was more slowly inhibited by DFP, the inhibition being only partial at 2 hours, but complete in 24 hours. The slow reaction of this enzyme with DFP explains the residual proteolytic activity observed in samples of crude enzyme incubated with DFP or PMSF for 2 hours or less (see above). The use of synthetic substrates, inhibitors, and heatmg experiments to differentiate various types of proteolytic activity thus revealed that B. Z~c~e~~jor~~~elaborates a rather complex array of these hy~ola~es. In the light* of our evidence for at least two endopeptidases (proteinases), the postulation of Darnodaran et al. (1) that this organism possesses a proteinase and one or more peptidases must be broadened. The possibility must be considered t,hat further fractionation e~:periments may reveal still others. Our observations concerning the cobaltactivated aminopeptidase agree closely with those of Damodaran et al. (l), but it appears appropriate not to refer to this enzyme as “leucine aminopeptidase,” as did these earlier workers, Recent work on amino peptidases has indicated that the name ‘{leucine aminopeptidase” should be reserved for the well-characterized enzyme from animal tissues (20). No evidence was obtained in our investigation for the existence of more than one exopeptida~, but this also is a possibility not to be excluded. Bernlohr (3) reported insignificant effects from the addition of divalent cations or EDTA to proteinase preparations. The lack of inhibition of gross proteolysis by Cu++, Hgi+, and Zn++ ions reported by Bernlohr (3) contrasts with our results and with those of Damodaran el al. (1). As Bernlohr did not use synthetic substrates, no comparison can be drawn concerning the number and types of enzymes present in his preparations. It seems likely that the differences in results may be attributable to the strains of 2% Zicheniformis used. ACKNOWLEDGMENTS We are indebted to Dr. Ruth E. Gordon for advice concerning the classification of the orga-

OF Bacillus

Eichenijormis

153

nism, and to Dr. W. A. Taber for suggesting the method used for preserving the cultures. The peanut meal used in the growth medium was generously supplied by Brady Mills, Inc., Brady, Texas.

REFERENCES 1. DAMODARAN, M., GOVINDARAJAN, V. S., AND SUB~~MANIAN, S. S., B~och~m. ~~ophy~. Aeta

17,99 (1955). 2. BERNLOER, R. W., AND NOVELLI, G. D., Arch. Biochem. Biophys. 103, 94 (1963). 3. BERNLOHR, R. W., J. Biol. Chem. 239, 538 (1964).

4. SMITH, N. R., GORDON, R. E., AND CLARK, F. E., “Aerobic Sporeformin~ Bacteria,” Agriculture Monograph No. 16, p. 40. United States Department of Agriculture, Washington, D.C. (1952). 5. BREED, R. S., MURRAY, E. G. D., AND SMITH, N. R., “Bergey’s Manual of Determinative Bacteriology,” 7th edition, p. 619. Williams and Wilkins, Baltimore, Maryland (1957). 0. WILLIAMS, W. L., HOFF-JORGENSEN, E., AND SNELL, E. E., J. Bid. Chem. 17’7,933 (1949). 7. ANSON, M. L., J. Gen. Physiol. as,79 (1938). 8. SCHWERT, G. W., NEURATH, H., KAUFMAN, S., AND SNOBE, J. E., J. Biol. Chem. 172, 221 (1948)f 9. JOHNSON, M. J., J. Biot. Chem. 137,575 (1941)’ 10. GOLDBARG, J. A., AND RUTENBURG, A. M., Cuncer 11, 283 (1958). 11. MOORE, S., AND STEIN, W. H., J. Biol. Chem. 211,907 (1954). 12. LOWRY, 0. H., RO$EBROU~XX, N. J., FARR, A. L., AND RANDALL, R. J., J. BioE. Chem. 193,265 (1951). 13. MATSUBARA, EL, HAGIHARA, B., NAKAI, M., KOMAKI, T., YONETANI, T., AND OKUNUKI, K., J. Biochem. 46,251 (1958j. 14. DESNUELLE, P., in “The Eneymes” (P. D. Boyer, H. Lardy, and K. Myrb&k, eds.), 2nd edition, Vol. 4, Part A, p. 119. Academic Press, New York (1960). 15. MORIHARA, K., AND TSUZUXI, H., Biochim. Biophys. Acta 92, (1964). 16. MORIHARA, K., J. Bacterial. 88, 745 (1964). 17. BURGTJM, A. A., AND PRESCOTT, J. M., Arch. B~och~. &o&s. 111,391 (1965). 18. OTTESON, M., AND SPECTOR, A., Compt. Rend. Trav. Lab. Car&berg 32, 63 (1960). 19. DICKIE, N., AND LIENER, I. E., Biochim. Biophys. Acta 64, 52 (1962). 20, PATTERSON, E. K., HSIAO, S., AND KEPPEL, A., J. Biol. Chm. 238,361l (1963).