JOURNAL OF BACTERIOLOGY, May 1971, p. 394-402Copyright © 1971 American Society for Microbiology

Vol. 106, No. 2Printed in U.S.A.

Purification of Bacteroides amylophilus ProteaseE. M. LESK AND T. H. BLACKBURN

Department of Microbiology, The University of British Columbia, Vancouver 8, British Columbia, Canada

Received for publication 19 October 1970

A protease was released by Bacteroides amylophilus cells in late stationaryphase, approximately 12 hr after maximum cell density was reached. The proteasewas concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and waspurified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel fil-tration, and by isoelectric focusing. The purified protease was active between pH4.5 and 12.0 with optima at pH 6.0 and 1 1.5. Evidence against there being a singleprotease was given by the differential inhibition of esterase and protease activitiesby some inhibitors. There was some evidence that only a single protease waspresent as the ratio of protease activity at various pH values did not alter signifi-cantly during purification or when the purified protease was partially heat-in-activated or treated with two specific trypsin-type protease inhibitors: N-a-tosyl-L-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of thesame protease were found by acrylamide gel electrophoresis. Gel filtration con-firmed the presence of protease in 30,000 and 60,000 molecular-weight forms.Treatment with 1 mM ethylenediaminetetraacetic acid or with 4 M urea failed toconvert the 60,000-molecular-weight to the 30,000-molecular-weight species.

Bacteroides amylophilus H 18, a gram-negativeproteolytic organism, was isolated from therumen of a sheep by using strict anaerobic condi-tions (4). Isolation of such an organism was con-sidered unusual, since, with the exception ofmembers of the genus Pseudomonas and Vibrio,most bacteria which produce exoenzymes aregram-positive (17). Protease production by B.amylophilus H18 was not subject to either re-pression or induction (2). The presence of 0.1%(w/v) Tryptose in the basal medium decreased thelag period. It was assumed that this resultedfrom a stabilization of the Eh since additionalnutrients did not affect either growth or proteaseproduction (2).The cell-bound protease, liberated by sonic

disruption of B. amylophilus H18 cells harvestedduring exponential phase, was particle bound andcould not be easily purified (3). In this respect itresembled the penicillinase of Bacillus licheni-formis which, when liberated by lysozyme treat-ment, appeared to be bound to membrane frag-ments (12). Therefore, it was necessary to deter-mine whether the protease which was liberated inthe stationary phase was of small molecularweight, indicating preferential liberation, orwhether it was of large molecular weight andpresumably membrane or particle bound. If thelatter were true, there would be no advantage inattempting its purification as similar materialcould more conveniently be prepared from expo-nential-phase cells. There was evidence, based on

the complex pH activity spectrum and on dif-ferent ratios of tosyl-L-arginine methyl ester-es-terase to protease in different samples and ondifferential inhibition of these activities by di-isopropyl-phosphofluoridate that more than oneprotease was produced by B. amylophilus H18(3). The purpose of this investigation was topurify the protease active at pH 6.7; this pH wasclose to the pH optimum of the unpurified pro-tease.

MATERIALS AND METHODS

Chemicals. Sephadex G-200, diethylaminoethyl(DEAE)-Sephadex A-50 and sulfoethyl (SE)-SephadexC-50 were purchased from Pharmacia, Uppsala, Swe-den. The media constituents and other chemicals were ofreagent grade and obtained from Fisher Scientific Co.,Fairlawn, N.J.; soluble starch was from The British DrugHouses, Poole, England. Bovine serum albumin, N-a-tosyl-L-lysylchloromethyl ketone (TLCK)-hydro-chloride and N-a-benzoyl-L-arginine methyl ester(BAME) were from Calbiochem, Los Angeles, Calif.Trypsin (X2 crystallized, lyophilized) and phenyl-methane-sulfonyl fluoride (PMSF) were from SigmaChemical Co., St. Louis, Mo.

Protease assay. The assay which measured trichloro-acetic acid soluble-peptides was a modification of themethod of McDonald and Chen (14). The reactionmixture contained 0.2 ml of enzyme solution and 1.8ml of a 2.0% (w/v) solution of casein in 0.01% Mer-thiolate-0.1 M phosphate buffer (pH 6.7). Where neces-sary, reactions were run at other pH values by dis-solving the casein in water and adjusting the pH witheither NaOH or HCI. The mixture was incubated at 38

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C for various lengths of time, depending on the amountof proteolytic activity within a given sample. At theend of the incubation period, 2.0 ml of 0.72 N trichlo-roacetic acid was added and mixed well to precipitateall of the protein. The precipitated digest was allowedto stand at room temperature for 10 min. After centri-fuging at 1,000 x g for 10 min, 1.0 ml of the trichloro-acetic acid-soluble supernatant was removed with a

pipette and added to 5.0 ml of copper reagent. Thecopper reagent was prepared by adding 1.0 ml of 0.5%CuSO4 in 1.0% sodium citrate to 50 ml of 0.2 N NaOHin 2.0% Na2CO3. After 15 min of incubation at 38 C,0.5 ml of 1.0 N Folin reagent was added with immediatemixing. The tubes were then incubated at 38 C for 10min, and the absorbancies were measured at 700 nm on

a Spectronic 20 spectrophotometer (Bausch & Lomb,Inc., Rochester, N.Y.). Controls were 0.2 ml of water

plus 1.8 ml of casein for zero time and for incubation.These controls showed that there was little or no in-crease in absorbancy from nonspecific hydrolysis of thecasein even at high pH values except after prolongedincubation. The absorbancy at zero time (where tri-chloroacetic acid was added before incubation) was

subtracted from the absorbancy of the test, and thecorrected value was compared with a standard bovineserum albumin (BSA) curve (200 ug of BSA giving an

absorbancy of 0.32 at 700 nm). A unit of proteolyticactivity was defined as the amount of enzyme whichunder standard conditions would solubilize the equiva-lent of 1.0 Ag of BSA per min. There was a linear rela-tionship between the time of incubation and theamount of casein solubilized. Specific activity was de-fined as units of protease activity per milligram of pro-

tein (13).Determination of amylase activity. Amylase activity

determination was essentially the method of Fischerand Stein (10), using dinitrosalicylic acid.

Determination of esterase activity. Esterase activitywas measured by titration (18) by using a Radiometertype PHM 28, titrator (Radiometer Ltd., Copenhagen,Denmark). The reaction mixture contained 0.4 ml of0.1 M ester substrate (e.g., BAME), 0.5 ml of water,and 0.1 ml of enzyme preparation and was incubated at25 C under N2. The pH of the mixture was maintainedby titration with 0.1 N NaOH from a 0.5 ml of mi-crometer syringe.

Culture. B. amylophilus H 18 was cultured and main-tained as described by Blackburn (2). The basal me-

dium contained (g/liter): K2HPO4, 0.45; KH2PO4, 0.45;(NH4)2SO4, 0.9; NaCI, 0.9; MgSO4 -7H20, 0.09; CaC12,0.09; resazurin, 0.001; L-cysteine-hydrochloride, 0.5;NaHCO3, 5.0; maltose, 3.0; and Tryptose, 3.0.

Isolation of proteases from B. amylophilus HIt. A32-liter stainless-steel milk can containing 29 liters ofgrowth medium was inoculated with I liter from an

exponential-phase culture of B. amylophilus H 18 andwas incubated anaerobically under CO2 for 23 hr at 38C. The can and its contents were cooled rapidly with a

water hose, and the cells were removed by centrifuga-tion at 8,700 x g at 4 C, using a GSA centrifuge withKSB: R continuous flow adaptor (Sorvall, Norwalk,Conn.).

DEAE-Sephadex A-50 (0.2 g, dry weight/100 ml)was added batchwise to the culture fluid, and CO2 was

bubbled through the suspension for 12 hr at 4 C toensure good mixing. The optimal pH for the attach-ment of the protease to the ion exchanger was found tobe pH 5.5, which was the pH of the 23-hr culture of B.amylophilus H 18. After allowing the suspension to set-tle, the supernatant fluid was poured off, and the gelwas collected on a sintered-glass filter. The gel wassuspended in 500 ml of 1.0 M NaCl; the suspension wasmixed well and then centrifuged. The supernatant fluidwas poured off and stored. The DEAE-Sephadex wasextracted in this way a total of six times. The first fiveextractions (total volume, 2,500 ml) were pooled anddialyzed against 0.05 M phosphate buffer (pH 7.0).A DEAE-Sephadex A-50 column (3.0 by 60 cm)

was equilibrated with 0.05 M phosphate buffer (pH 7.0),and the dialyzed protease was passed through it. Theenzyme was eluted with a linear gradient of 0.2 to 1.0M NaCl in the same buffer, and the fractions weretested for proteolytic activity. The active fractions werepooled and dialyzed against 0.05 M phosphate buffer(pH 7.0). The dialyzed enzyme was concentrated to 70ml in a Diaflo ultrafiltration cell with a UM10 filter(Amicon Co., Lexington, Mass.; exclusion limits,10,000 daltons) at 20 psi of nitrogen. Some of the con-centrated protease (31 ml) was filtered by upward flowthrough a Sephadex G-200 column (5.0 by 90 cm),which was equilibrated with 0.05 M phosphate buffer(pH 7.0). The protease was eluted with the same buffer;fractions were collected, and the distribution of proteinwas established by absorbancy readings at 280 nm. Theprotease was again concentrated by using an AmiconUM10 filter.The further purification of the protease was at-

tempted by using 8100 Ampholine ElectrofocusingEquipment (LKB Produkter, A.B., Stockholm,Sweden). Low-molecular-weight ampholines were usedin the range pH 3.0 to 6.0 and pH 3.0 to 5.0. Both theampholines and the proteases were randomly distrib-uted throughout the column before application of thepotential gradient. The methods were as recommendedin the Ampholine Instruction Manual.

Disc gel electrophoresis. Disc gel electrophoresis wascarried out in a standard acrylamide gel (7%), whichwas stacked at pH 8.9 and run at pH 9.5 by themethod of Davis (7) by using the Canalco model 6system (Canal Industrial Co., Bethesda, Md.). Theenzyme samples were run in duplicate. After electro-phoresis, one gel was immediately stained for proteinwith aniline black, whereas the duplicate gel was usedto assay for various activities. Protease activity andamylase activity were detected by laying the sliced gelson the flat surface of an agar medium containing anappropriate substrate; the enzymes diffused from thebands in the gel into the agar layer.

Protease activity at pH 6.0. Glass slides were layeredwith 1.0% casein made up in 0.2 M phosphate buffer(pH 6.0) plus 1.0% agar.

Protease activity at pH 11.0. Glass slides were lay-ered with 1.0% casein made up in 0.8 M sodium car-bonate (pH 11.0) plus 1.0% agar.

Protease activity at pH 4.0. An underlay of 0.8 Macetate buffer (pH 4.0) with 2.0% agar was made in apetri dish. Three ml of 2.0% casein was mixed with 3.0ml of 1.0% agar and was added as an overlay. This

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method was adopted as even suspensions of casein atpH 4.0 could not be produced on slides by simple pHadjustment.

Amylase activity. Glass slides were layered with1.0% starch made up with 0.2 M phosphate buffer (pH6.0) plus 1.0% agar.

The sliced gels were placed on the various slides andplates at 37 C for 30 min. The gels were then removedand a 12% HgCI2-16.5% HCI solution was applied tothe casein slides and plate to precipitate nonhydrolysedcasein. The starch slides were flooded with an aqueoussolution of 0.3% iodine, 0.6% potassium iodide. Theresults of the casein and starch were compared to theprotein-stained acrylamide gels.

Cystein content. A protease' sample was hydrolyzedunder vacuum with 6 N HCI for 18 hr at 110 C. Ap-proximately 0.3 mg of protein hydrolysate was ana-lyzed (20) by using the amino acid analyzer (model120; Beckman Instruments, Palo Alto, Calif.). Cysteinewas titrated in the native protein with p-chloro-mercuribenzoate (5).

Determination of molecular weight. The molecularweight was determined by the method of Andrews (1).Blue dextran (Pharmacia) and the protein markers,chymotrypsinogen, ovalbumin, and bovine serum al-bumin (Mann Research Laboratories, Orangeburg,N.Y.), were used to calibrate the large Sephadex G-200column (5 by 90 cm).

RESULTSPreliminary experiments indicated that the

major portion of the cell-free protease was re-tarded by G-200 Sephadex and was thus not at-tached to membrane fragments. This proteasehad casein hydrolytic activity between pH 3.0and 11.5 with a broad plateau of activity betweenpH 5.5 and 7.5 and a second sharper peak at pH11.0 (Fig. 1). It seemed probable that more than

16

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FIG. 1. Protease activity as a function of pH. Thedigests contained 0.2 ml of culture fluid and 1.8 ml of2.0% casein at different pH values. The points are themean of duplicate determinations.

one enzyme was responsible for this proteolyticactivity, and throughout this work the proteaseactivity was assayed at pH 5.0, 6.7, 8.0, and11.0. Protease(s) which operated at these pHvalues were liberated from cells simultaneously.The liberation of the pH 6.7 protease is illus-trated in Fig. 2, but the curves for the other pHactivities paralleled this plot and reached peaksat the same time. This time did not coincide withpeak cell density nor with the subsequent charac-teristic fall in cell density. The maximum concen-tration of extracellular protease was found 10 hrafter maximum cell density was reached. Themaximum concentration of amylase, another ex-tracellular enzyme produced by B. amylophilusH 18, was found only 5 hr after maximum celldensity. There were unexplained fluctuations inthe extracellular protease and amylase activitieswhich were not due to inaccuracies of the assayprocedures.

Protease purification. The procedure was de-scribed above, and the results are summarized inTable 1.

Culture supernatant. A good yield of proteasewas obtained in the 23-hr culture supernatant.This represented 80% of the total protease (cell-bound and cell-free) which had reached a max-imum at 13 hr. A sample of the supernatant wasdialyzed thoroughly against 0.05 M phosphatebuffer (pH 7.0) to remove cysteine and Tryptosepeptides before performing the protein assay.

Concentrate from DEAE-Sephadex. The con-centration step was accomplished by binding theprotease to DEAE-Sephadex. The eluted, di-alyzed protease represented 74% of the startingmaterial. Although this step gave only a four-fold purification, it reduced the volume from 29to 2.6 liters.

AGE OF CULTURE(HOUR)

FIG. 2. Liberation of protease. The optical densityat 660 nm (OD.) (-) and the cell-free protease (0)were plotted against time of incubation.

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TABLE 1. Summary ofprotease purification

Ratio of proteaseTroteale Seii

Purifi- Per cent activityFraction Vol (ml) Pr(1ot Spctivi cation recov- at pH(l~ activity (fold) erya

units) 5.0 6.7 8.0 11.0

1. Culture supernatant ........... 29,000 272 29.7 0.78 1.00 0.85 0.912. Concentrate from DEAE-

Sephadex .................... 2,600 202 126.2 4 743. DEAE-Sephadex chromatography. 71 332 1165 39 122 1.03 1.00 0.70 0.924. G-200 Sephadex gel filtration .. A 14 75 15,800 532 63 1.05 1.00 0.86 1.00

B 11.5 30 7,830 .263 25 1.00 1.00 0.84 0.97

a Calculated on the basis that all the protease from one step was processed in the next step.

DEAE-Sephadex chromatography. Furtherpurification and concentration (2,600 to 500 ml)were achieved by adsorption on a DEAE-Se-phadex column with subsequent elution by a so-dium chloride gradient (Fig. 3). The pooled, Di-aflo-concentrated protease was purified 10-foldin this step, with an apparent increase in proteo-lytic activity.

G-200 Sephadex. In the experiment shown inTable 1, filtration of no. 3 through a G-200 Se-phadex gel resulted in the elution of the proteasein two fractions, A and B, the approximate mo-lecular weights of which were 60,000 and 27,000daltons, respectively (Fig. 4). For conveniencethese are referred to as 60,000- and 30,000-mo-lecular-weight proteins, although it is appreciatedthat these are not accurate figures. The proteasewas purified 532-fold in A, 263-fold in B, with acombined recovery of 88%. Except for the rela-tive increase in the pH 5.0 activity, the ratios ofprotease activity at various pH values did notvary significantly throughout the purification.This would indicate that a single protease wasresponsible for proteolytic activity over the broadpH range examined. Since the proteases in thetwo fractions, A and B, from gel filtration dem-onstrated activity over the broad pH range, itmust be assumed that they were at least closelyrelated and might possibly represent a monomer-dimer relationship.

In other purifications, in which exactly thesame procedures were followed, all of the pro-tease was located in the 60,000 dalton peak.

Isoelectric focusing. The protease was found inone main fraction, the isoelectric point of whichwas pH 4.3 (A, Fig. 5). Some of the protease (B)was found at the alkaline electrode boundary.The protease activity was measured at pH 5.0,6.7, 8.0, and 11.0, and the distributions werefound to be coincident. This again substantiatedthe argument for a single protease. The tubescorresponding to A and B were pooled, dialyzed,concentrated, and assayed for protease activity.In general, the recoveries were poor, and little

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FIG. 3. DEAE-Sephadex chromatography. The di-alyzed protease was adsorbed to a column (3.0 by 60cm) of DEAE-Sephadex A-50 which had been equili-brated with 0.05 M phosphate buffer (pH 7.0). Theprotease was eluted by a linear gradient of NaCl, 0.2to 1.0 M, in 0.05 M phosphate buffer (pH 7.0) and wascollected in 20-ml fractions. The protease activity atpH 6.7 (0) and the absorbancy at 280 nm (OD280)(solid line) were measured.

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FIG. 4. Gel filtration through a Sephadex G-200 (5by 90 cm) column of 31.0 ml of protease no. 3 (Table1). The protease was eluted in 0.05 M phosphate buffer(pH 7.0) and collected in 12.5-ml fractions. The voidvolume (Vo) of the column was determined with BlueDextran, and the protease activity at pH 6.7 (0) andthe OD280 (solid line) were measured.

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FRACTION NUMBER

FIG. 5. Isoelectric focusing. A portion of purifiedprotease (5.8 ml, specific activity 11,800) was added toan LKB-8100 electrofocusing column with LKB Am-pholine carrier ampholytes to give a gradient from pH3.0 to pH 6.0 after 1,000 v for 48 hr. One-milliliterfractions were collected; the pH (solid line) and proteo-lytic activity at pH 5.0 (0), pH 6.7 (-), pH 8.0 (A),andpH 11.0 (0) were measured.

purification was achieved. The protease in B wasthought to be a denatured product, as a proteinwith these characteristics would not be likely tobind to DEAE-Sephadex at pH 5.5 in purifica-tion step 2. The protein in fraction A gave a vis-ible opacity in the column after isoelectric fo-cusing for 48 hr. A second experiment was there-fore run for 24 hr in a gradient from pH 3.0 to5.0. The main protease activity was located in amore diffuse fraction, the isoelectric point ofwhich was pH 4.25.

Activity. The purified protease had approxi-mately the same proteolytic activity as crystal-line trypsin, the specific activity of which was14,600 at pH 8.0, when assayed as describedabove. The specific activity of the B. amylophilusH18 protease 4A was 15,800 at pH 6.7. The es-terase activities of trypsin and the protease at pH8.0, expressed as micromoles of N-a-benzoyl-L-arginine methyl ester hydrolyzed per minute permilligram of protein were 250 and 230, respec-tively. N-a-benzoyl-L-argininamide was not hy-drolyzed by the protease.

Purity and size. Acrylamide gel electrophoresisof the 4A and 4B fractions demonstrated twomajor protein bands in each, both bands wereproteases which were active at pH 4.0, 6.0, and11.5. It would be unlikely that two distinctly dif-ferent proteases would have this same very broadpH spectrum, and a possible explanation wouldbe that the two bands represented polymericforms of the same enzyme. If this were true andthat an equilibrium existed between the twoforms, it would then be anticipated that protease4B (30,000 molecular weight) should polymerize.When 4B was rerun through Sephadex G-200, itwas found to be exclusively in a 60,000-molecu-

lar-weight form. The 60,000-molecular-weightform appeared to be the more stable as it pre-dominated in purification step 4.

Treatment of the 60,000-molecular-weight pro-tease with 1.0 mM ethylenediaminetetraaceticacid (EDTA) or with 4 M urea, followed by fil-tration through G-200 Sephadex columns equili-brated with the same reagents in 0.05 M phos-phate buffer (pH 7.0), failed to convert the pro-tease to a 30,000-molecular-weight form. Morerigorous treatment with 0.01 mM sodium dodecylsulfate resulted in a loss of protease activity, andthere was insufficient protein present to be de-tected by chemical methods.The acrylamide gels of both 4A and 4B also

contained a small band of fast-moving unidenti-fied protein. In addition, 4A, but not 4B, con-tained three other minor protein bands, twoabove the upper protease band and one below it;these bands were identified as being amylaseswhich are also secreted by B. amylophilus H 18.

Further purification of the protease 4A wasattempted by using the cation exchangers SE-Sephadex C-50 and phosphocellulose at pH 4.1(isoelectric point of protease was pH 4.25), butno purification was achieved.pH optimum. The pH spectrum of the purified

protease (Fig. 6) was not very different from thestarting material except for a sharpening of thelower pH optimum to pH 6.0 rather than exhib-

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FIG. 6. Protease activity as a function of pH. Thereaction mixture contained 0.2 ml of diluted enzyme(specific activity, 11,900) and 1.8 ml of 2.0% casein atdifferent pH values.

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iting a plateau. The upper optimum was at pH11.5. N-a-benzoyl-L-arginine methyl ester washydrolyzed most rapidly at pH 8.0 and exhibitedonly one pH optimum.Temperature. The optimal temperature for the

purified protease was between 60 and 65 C (Fig.7). An Arrhenius plot of the same data showedthat there was probably some inactivation of theprotease, at temperatures above 50 C, under theassay conditions.Temperature inactivation was used to deter-

mine whether proteases with different thermalsensitivities might be responsible for the proteaseactivity at different pH values. The results of thisexperiment (Table 2), in which protease washeated at 60 C for 30 min, showed the same de-gree of inactivation for protein hydrolytic ac-tivity at pH 5.0, 6.0, 8.0, and 11.0.The thermal stability of the purified protease

(specific activity, 11,800) was not due to disulfidebridges, since no half-cystine was detected inamino acid hydrolysates and no cysteine wasdetectable by titration in the native enzyme. Thepossibility that divalent ions conferred a rigid

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FIG. 7. Effect of assay temperature on protease ac-

tivity. Samples of diluted purified protease (specificactivity, 11,800) were added to 1.8-mI volumes of 2.0%casein in 0.1 M phosphate buffer (pH 6.7), which were

preheated in a thermal gradient. The tubes were incu-bated for 45 min.

TABLE 2. Thermal inactivation ofproteasea

Incubation Percentage of activity remainingtime at 60 C

(min) pH 5.0 pH 6.0 pH 8.0 pH 11.0

0 100 100 100 10010 100 91 82 8420 82 82 79 78

a A 10.0-mi amount of 1/50 dilution of purified pro-tease (specific activity, 11,800) in water was incubatedat 60 C. Samples (2.0 ml) were removed at 0, 10, and20 min, cooled immediately, and assayed in duplicatefor protease activity at pH 5.0, 6.0, 8.0, and 11.0.

tertiary structure to the protease was investigated(Fig. 8). Dialysis against 10 mM EDTA increasedprotease activity, possibly by chelating inhibitoryheavy metal ions; the addition of 1.0 mM Ca2+gave a further 10% increase but did not result ina significant increase in thermal stability. Theprotease in 4A and in 4B were affected to a sim-ilar extent by 10 mM EDTA and 1.0 mM Ca2+treatment. This was additional evidence thatboth preparations contained the same protease.

Protease inhibitors. PMSF and TLCK at 1.0mM were effective inhibitors of protease activity(Table 3). There was no significant difference inthe degree of inhibition of protease activity atdifferent pH values, except perhaps the greaterinhibition of pH 11.0 activity. Incubation witheither inhibitor for 3 hr resulted in almost com-plete inhibition. Results from different experi-ments showed some variability, but the evidenceindicated the presence of only one protease or ofproteases which were susceptible to these inhibi-tors and presumably had a dibasic amino acidspecificity. Contradictory evidence for the pres-ence of more than one proteolytic enzyme wasgiven by the fact that the BAME activity wascompletely inhibited by exposure to I mM TLCKunder the conditions outlined in Table 3.

DISCUSSIONThe protease which was liberated in cultures

containing late stationary-phase cells of B. amy-lophilus H 18 was shown to be of relatively lowmolecular weight. Its purification was under-taken in anticipation that it could be morereadily purified than the particle-bound proteasewhich Blackburn (3) liberated from disintegratedexponential-phase cells of this microorganism.The relatively weak proteolytic activity found inthe culture supernatant made it necessary toprocess large volumes. Fortunately, the proteo-lytic activity could be removed from the superna-tant by adsorbing it to DEAE-Sephadex. Theprotease could then be eluted in a concentratedand purified form.

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FIG. 8. Thermal stability of protease. Protease no. 4A and protease no. 4B were dialyzed against 10 mMEDTA, and then a portion was dialyzed against 1.0 mm calcium. The enzymes were diluted to 1/S0 of their orig-inal concentration with 10 mm EDTA or 1.0 mm Ca2+, and 0.2-mi samples were placed in tubes in a thermal gra-dient for 15 min, after which they were removed, cooled, and assayed for proteolytic activity at pH 6.7. The activi-ties of untreated (A), 10 mm EDTA (-)-, and 1.0 mM Ca2+ (@)-treated protease in no. 4A and no. 4B were

plotted against temperature in A and B, respectively.

TABLE 3. Protease inhibitorsa

Per cent inhibition of protease by

Assay pH m I mm

PMSF TLCK

5.0 37 206.7 22 298.0 37 3011.0 67 48

a The test contained 1.0 ml of purified protease (spe-cific activity, 11,800), 0.2 ml of 10 mM N-a-tosyl-L-ly-sylchloromethane (TLCK) or 0.2 ml of 10 mM phenyl-methane-sulfonyl fluoride (PMSF) in n-propanol, 0.5 mlof 0.1 M phosphate buffer (pH 6.7), and 0.3 ml water.A control tube contained 0.2 ml of n-propanol insteadof inhibitor. The tubes were incubated at 25 C for I hr,and 0.2-ml portions were immediately added to 1.8-mlvolumes of 2.0% casein at pH 5.0, 6.7, 8.0, and 11.0and incubated at 38 C for 15 min for protease assay.The results are mean of two experiments.

Blackburn (3) demonstrated that B. amylo-philus H 18 protease was active between pH 5.5and 9.5 (higher pH values were not examined)and suggested that two or more proteases mightbe present. In this present work, it was estab-lished that the culture fluid possessed proteolyticactivity over a wide pH range (pH 3.0 to 11.5)with optima at pH 6.0 and 11.0. Again, this sug-gested the presence of more than one protease.Other bacterial proteases have broad pH optima,e.g., Arthrobacter sp., pH 4.0 to I 1.0 (referenceI 1), and Bacillus subtilis N-, pH 4.5 to 1 1.5 (ref-erence 15). These proteases did not demonstratethe multiple optima observed for that of B. amy-lophilus H18. It was found at each step in the

purification, by DEAE-Sephadex chromatogra-phy, gel filtration, and isoelectric focusing, thatthe protease activities at the different pH values,neither were separated from each other nor didthe ratio of their activities change significantly.These results favored the hypothesis that all theproteolytic activity, over the pH range examined,was due to a single protease and this was sub-stantiated by the uniform heat inactivation ofprotease active at all pH values. There was some

evidence against this hypothesis in that the prote-olytic activities at the more neutral pH values(pH 5.0, 6.7, 8.0) and at pH 11.0 were inhibitedto slightly different extends by treatment of thepurified enzyme with TLCK or PMSF. In addi-tion, the BAME-esterase activity at pH 8.0 was

completely inhibited by TLCK under conditionswhich did not completely inhibit the protease atpH 8.0 or at the other pH values tested. Thismight be taken to mean that the BAME-esteraseactivity was due to a distinct and different pro-tease. However, the almost complete inhibition,by continued incubation with TLCK of thecasein hydrolytic activity over the whole pHrange would indicate that the same active sitewas operative at all pH values. Work is currentlyin progress to determine the exact peptide bondspecificity at pH 6.0 and 11.5 by using polypep-tides of known linear sequence. The two pH op-tima might be a result of pH-dependent changesin the protease or of different ionizations in thesubstrate. Trypsin exhibits two pH optima forhydrolysis of substrates: pH 8.0 for benzoyl-L-arginine p-nitroanilide and pH 9.25 for lysine p-nitroanilide (9).Although it is true that the esterase and pro-

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tease activities of trypsin are apparently due toone and the same active site as determined bysimultaneous activation or inhibition, it was re-cently shown that formylation of tryptophan intrypsin abolished the protease activity but didnot destroy the esterase (6), and protease activitymay be increased by conditions which greatlyreduced the esterase activity (8). In addition, anonproteolytic, arginine ester-hydrolyzing en-zyme was isolated from snake venom (21). Dif-ferential inhibition or activation of one functionwithout an effect on the other has thus been es-tablished so it is possible that TLCK may inhibitthe esterase activity at a more rapid rate than itinhibits the protease activity.TLCK is, however, rather different from the

other modifying agents (6, 8) in that it reactswith histidine at the active site (19). It is possi-ble, however, since the serine protease chymo-trypsin probably hydrolyses esters and peptidesby different mechanisms (8) that the mechanismused by B. amylophilus H18 esterase may be in-hibited by TLCK more rapidly than the protease.

Further evidence for the presence of only asingle protease was found when gel electropho-resis failed to resolve the protease into bands ac-tive at different pH values. In other words, aband showing the pH 6.0 proteolytic activity wasalso active at pH 4.0 and 1 1.0.The interesting observation was made that gel

filtration could resolve the proteolytic activityinto two fractions having molecular weights ofapproximately 30,000 and 60,000. Since the pHrange of both proteolytic fractions was the same,it seemed possible that the 60,000-molecular-weight species might be a dimer of the 30,000-molecular-weight species. This possibility wassubstantiated by a demonstration that the30,000-molecular-weight fraction, on rerunningthrough the gel, gave a peak characteristic of the60,000-molecular-weight protease. Further evi-dence that the two molecular-weight speciesmight exist in equilibrium was given by the factthat gel electrophoresis always demonstrated twocharacteristic protease bands with the same pHactivity spectrum. It is proposed that the dimermight be more stable than the monomer, as the60,000-molecular-weight species predominatedon gel filtration and the upper band on acryla-mide gel electrophoresis was always greater inprotein content and protease activity.

Neither EDTA nor 4.0 M urea treatment of thepurified protease resulted in the disappearance ofthe 60,000-molecular-weight species. The condi-tions for splitting the dimer, if indeed that iswhat the 60,000-molecular-weight material repre-sented, remain unknown. More drastic methodsof effecting a separation could not be used as the

protease was inactivated.Amino acid analysis of the protease revealed

an absence of half-cystine. It has been shown forseveral other extracellular proteases purifiedfrom bacteria that disulphide bridges are notinvolved in maintaining tertiary structures (17).The temperature optimum for protease activity

was between 60 and 65 C in spite of the tempera-ture of inactivation of the enzyme being only 50C.

Microbial proteases may be divided into twogroups, metalloenzymes and enzymes with aserine at the active site (16). The protease of B.amylophilus H 18 does not appear to belong tothe first category, since it was not inhibited byEDTA nor was it activated by the divalent ionstested. It is presumably a serine protease since itis inhibited by diisopropylphosphofluoridate (3)and by PMSF, but it also appears to be similarto trypsin in that it is inhibited by TLCK andthat it hydrolyses BAME at pH 8.0.

ACKNOWLEDGMENTS

This investigation was supported by grant A 4286 from theNational Research Council of Canada.

LITERATURE CITED

1. Andrews, P. 1964. The gel-filtration behaviour of protcinsrelated to their molecular weights over a wide range.Biochem. J. 96:595-606.

2. Blackburn, T. H. 1968. Protease production by Bacteroidesamylophilus strain H 18. J. Gen. Microbiol. 53:27-36.

3. Blackburn, T. H. 1968. The protease liberated from Bac-teroides amylophilus strain H18 by mechanical disinte-gration. J. Gen. Microbiol. 53:37-51.

4. Blackburn, T. H., and P. N. Hobson. 1962. Further studieson the isolation of proteolytic bacteria from the sheeprumen. J. Gen. Microbiol. 25:69-81.

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6. Coletti-Previero, M.-A., A. Previero, and E. Zuckerkandl.1969. Separation of proteolytic and esterasic activities oftrypsin by reversible structural modifications. J. Mol.Biol. 39:493-501.

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8. Epand, R. M. 1969. Evidence against the obligatory for-mation of an acyl enzyme intermediate in the a-chymo-trypsin catalysed reactions of amides. Biochem. Biophys.Res. Comrmun. 37:313-318.

9. Erlanger, B. F., N. Kokowsky, and W. Cohen. 1961. Thepreparation and properties of two new chromogenic sub-strates of trypsin. Arch. Biochem. Biophys. 95:271-278.

10. Fischer, E. H., and E. A. Stein. 1961. a-Amylase fromhuman saliva. Biochem. Prep. 8:27-33.

11. Hofsten, B. V., and B. Reinhammer. 1965. An extracel-lular proteolytic enzyme from a strain of Arthrobacter.Ill. Stability and substrate specificity. Biochim. Bio-phys. Acta 110:599-607.

12. Lampen, J. 0. 1967. Cell-bound penicillinase of Bacilluslicheniformis. Properties and purification. J. Gen. Mi-crobiol. 48:249-259.

13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folin

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15. Matsubara, H., B. Hagihara, M. Nakai, T. Komaki, T.Yonetani, and K. Okunuki. 1958. Crystalline bacterialproteinase. II. General properties of crystalline protein-ase of Bacillus subtilis N. J. Biochem. (Tokyo) 45:25 1-258.

16. Morihara, K. 1967. The specificities of various neutral andalkaline proteinases from microorganisms. Biochem.Biophys. Res. Commun. 26:656-661.

17. Pollock, M. R. 1963. Exoenzymes, p. 121-178. In 1. C.Gunsalus and R. Y. Stanier (ed.), The bacteria, vol. 4.Academic Press Inc., New York.

18. Schwert, G. W., H. Neurath, S. Kaufman, and J. E.Snoke. 1948. The specific esterase activity of trypsin. J.Biol. Chem. 172:221-239.

19. Shaw, E., M. Mares-Gula, and W. Cohen. 1965. Evidencefor an active-centre histidine in trypsin through use of a

specific reagent, l-chloro-3-tosylamido-7-amino-2-hep-tanone, the chloromethyl ketone derived from N-a-tosyl-L-lysine. Biochemistry 4:2219-2224.

20. Spackman, D. H., W. H. Stein, and S. Moore. 1958. Au-tomatic recording apparatus for use in the chromatog-raphy of amino acids. Anal. Chem. 30:1190-1206.

21. Toom, P. N., T. N. Solie, and A. T. Tu. 1970. Characteri-zation of a non-proteolytic arginine ester-hydrolysingenzyme from snake venom. J. Biol. Chem. 245:2549-2555.

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