Vol. 171, No. 10JOURNAL OF BACTERIOLOGY, OCt. 1989, p. 5630-56370021-9193/89/105630-08$02.00/0

Isolation, Purification, and Properties of Penicillium charlesiiAlkaline Proteaset

C. A. ABBAS, S. GROVES, AND J. E. GANDER*

Department ofMicrobiology and Cell Science, University of Florida, Gainesville, Florida 32611

Received 17 February 1989/Accepted 5 July 1989

A serine protease with a pH optimum from 7 to 9 and activity over the range of pH 3 to 10 was isolated andpurified from culture filtrates ofPenici;ium charlesii 16 days after inoculation. The enzyme was purified by thefollowing sequence of procedures: (i) gel permeation chromatography through Sephacryl S-200, (il) DEAE-Sepharose anion-exchange chromatography, and (iii) fast protein liquid chromatography (FPLC) over

Superose 12. Anion-exchange chromatography separated the protease activity into a major activity (proteasePIH, 82%) and two minor activities (proteases PI and PM, 10 and 8%, respectively, of the total activity).Protease PII has a molecular mass of 44 kilodaltons. Purified preparations of this enzyme are susceptible toautodegradation. FPLC of heat-treated PII gave one major species (PIIa), whereas untreated enzyme resultedin three species (PUb, PUc, and PIHd). PIIb and PHc also catalyzed the hydrolysis of protein (hide powderazure). PUb and PUc were in the molecular mass range of 10 to 20 kilodaltons. Protease PIT is completelyinhibited by phenylmethylsulfonyl fluoride (PMSF). The protease has primary substrate specificity forphenylalanyl or arginyl amino acyl residues attached to amines. The enzyme has amidase, but no esteraseactivity toward similar synthetic substrates such as occurs with trypsinlike microbial serine proteases. Theaddition of PMSF (final concentration, 10-4 M) to 1- and 2-day-old cultures of P. charlesii inhibited theproduction of extracellular peptidophosphogalactomannan (pPGM) by 41 and 34%, respectively, and inhibitedthe alkaline protease activity by 85%. These results suggest that the production and release of pPGM may beaffected by alkaline protease.

Representatives of each of the major fungal taxonomicgroups examined are capable of protease production duringthe nonautolytic phase of growth (7). Among these groups,members of the classes Basidiomycetes and Phycomycetesshow a narrow capacity to secrete proteases, whereas manyAscomycetes and Fungi Imperfecti spp. release all classes ofproteases.

In general, Penicillium species and other acid-tolerantfungi produce acid proteases but show limited capacity toproduce alkaline and neutral proteases (19). In contrast,members of the genus Aspergillus are known to produce allthree types of proteases (4, 7, 27). Metallo-proteases, whichhave optimal activity at neutral pH, are secreted by Penicil-lium, Aspergillus, and some Basidiomycetes spp. (18). Thiol-proteases are limited in fungi (18, 21), although Aspergillusoryzae and A. sclerotiorum are known to secrete proteaseswhich are inhibited by thiol reagents. Serine proteases arewidely distributed in nature and have been reported fromPenicillium cyaneo-fulvum, Penicillium notatum, and Peni-cillium cyclopium (3, 8, 18).We have reported previously (22) that the activities of two

glycohydrolases produced by Penicillium charlesii first ap-pear only after depletion of glucose when the pH of theculture is 4.5 or greater. The medium contained an acidprotease with optimum activity around pH 3 and little or noactivity above pH 4.5 to 5. It was concluded that the acidprotease inactivated the glycohydrolases. The medium alsocontained an alkaline protease, which apparently did notdestroy glycohydrolase activity. More recently, it was ob-served that acid protease occurs in the culture filtrate only in

* Corresponding author.t Publication 9440 of the Florida Agricultural Experiment Station,

Gainesville, FL 32611.

those cultures which have been derived from conidiumsuspensions which have been stored for over a month in 150mM NaCl4.125% Tween 20 (C. Abbas, unpublished data).We conclude that extracellular acid protease activity inculture filtrates is not a physiologically relevant phenome-non. However, the role of alkaline protease in the physiol-ogy of the organism needs to be examined to determinewhether it is of importance in regulating cellular events.

P. charlesii produces a glycopeptide (peptidophosphoga-lactomannan [pPGM]) which may be a constituent part of itscell wall or may be derived from a series of fungal glycopro-teins elaborated into the culture medium (25). We haveshown previously that cell walls prepared by the procedureof Mahadevan and Tatum (16) contained little or no pPGM(11). However, pPGM may have been released from the cellwall preparation during its treatment with sodium dodecylsulfate (SDS). One function of the fungal proteases may beto hydrolyze amino acyl-containing cell wall polymers. Thispaper describes a serine protease secreted by P. charlesiiand demonstrates that upon the addition of phenylmethyl-sulfonyl fluoride (PMSF) to cultures, this enzyme is inhibitedby 85 to 95%. Accumulation of pPGM in the culture filtrateafter the addition of PMSF is inhibited 35 to 40%.

(This research was conducted by C. A. Abbas in partialfulfillment of the requirements for the Ph.D. degree fromUniversity of Florida, Gainesville, 1987.)

MATERIALS AND METHODSOrganism and culture conditions. P. charlesii G Smith

NRRL 1887 was maintained in sterile soil at -20°C. Theorganism was subcultured on 2% Bacto-Agar (Difco Labo-ratories, Detroit, Mich.) in Czapek Dox medium (15) con-taining 5% glucose and mineral salts. Conidia were har-vested after approximately 3 weeks by elution with sterile

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150 mM NaCl-0.125% Tween 20 (wt/vol). P. charlesii conid-iospores (107) in 200 ml of modified Raulin-Thom medium(23) or low-phosphate standard growth (LPSG) medium (25)were agitated at 20°C under constant light on a gyrotoryshaker. Cultures were routinely sampled on days 3, 5, 7, and16 after inoculation.Chemical analyses. The total carbohydrate content was

determined by the phenol-sulfuric acid method (9). Proteinlevels were estimated by the mini procedure of the BCAprotein assay (Pierce Biochemicals). Bovine serum albumin(BSA) (Sigma Chemical Co., St. Louis, Mo.) was used toestablish the reference curve. The phosphate content ofglycopeptides was estimated by using the technique of Amesand Dubin (2) and the color reagent of Ames (1) as previ-ously described (25).

Determination of enzyme activities. Protease activity was

determined by a modified procedure of Rinderknecht et al.(24), in which hide powder azure (HPA) (10 mg per tube) wasincubated in 2.0 ml of 100 mM citrate, phosphate, Trishydrochloride or borate buffer in the pH range of 2.0 to 10.0.The preparations were incubated at 37°C for 12 to 24 h, andthe A595 was measured. The values were converted topercent activity and plotted against the corresponding pHvalues.The influence of PMSF on alkaline protease (PII) activity

was determined in 100 mM citrate buffer (pH 6.0) andvariable quantities of PMSF. Protease, buffer, PMSF, and 10mg of HPA were incubated at 37°C for 48 h. A595 was

measured against a blank to which no enzyme or inhibitorwas added. Preparations to which alkaline protease but no

PMSF was added were used to determine 100% activity. Allof the readings were converted to percent activity andplotted against the corresponding pH values.The activities of various hydrolases (phosphatases and

glycohydrolases) were determined by measurement of theA410 of p-nitrophenol released by cleavage of p-nitrophenylesters or glycosides. Reaction mixtures contained 1.0 or 10,umol of p-nitrophenyl substrate in 100 ,ul of distilled deion-ized water, 100 ,ul of crude culture filtrate, and 1.0 ml of 100mM sodium citrate buffer (pH 5). Incubations were per-

formed at 22 to 24°C or in a constant temperature bath at37°C for 1 to 24 h. Reactions were stopped by the addition of1.0 ml of 0.2 N NaOH. The A410 was determined, and thep-nitrophenol released was quantified.

Purified protease PII was assayed by using amino acylderivatives of the p-nitroanilides to measure amidase activityand by using ethyl esters of benzoyl tyrosine and benzoylarginine to determine esterase activity. The quantities ofp-nitrophenyl amine and benzoic acid released were moni-tored at 410 and 253 nm, respectively. In these assays, 1 to

10 mM solutions of substrates were incubated with variabledilutions of PII (80 jig/ml) in 100 mM Tris hydrochloride 10mM CaCl2 buffer (pH 7.8). Trypsin and chymotrypsin were

dissolved in concentrations of 1 mg/ml and diluted further to

obtain a change of 0.01 to 0.02 absorbance units per min withthe appropriate substrates.

Gel permeation chromatography. Gel permeation chroma-tography was performed on a column (2.5 by 97 cm) ofSephacryl S-200 (superfine) in 50 mM Tris hydrochloride-100 mM NaCl (pH 7.2) at a rate of 0.75 ml/min. Seventy-twofractions of 5.2 ml each were collected, and the A280 was

measured. The protease activity in each fraction was thendetermined.

Anion-exchange chromatography. DEAE-Sepharose (fastflow; Pharmacia, Inc., Piscataway, N.J.) was used for frac-tionation of proteins obtained from gel permeation chroma-

tography. The pooled fractions from Sephacryl S-200 whichcontained protease activity were dialyzed against distilleddeionized water and then freeze-dried. The powder wasdissolved in 20 ml of 50 mM Tris hydrochloride (pH 9.6) andapplied to a column (2.5 by 28 cm) of DEAE-Sepharose. TheDEAE-Sepharose was washed with the Tris hydrochloridebuffer, followed by irrigation with a salt and pH gradient (50mM Tris hydrochloride [pH 9.6] to 50 mM Tris hydrochlo-ride [pH 7.6]-100 mM NaCl). The column was then washedwith 100 mM NaCl. A total of 144 fractions (21 ml perfraction) were collected, and the A280 was monitored. Frac-tions 1 through 72 were assayed for protein and proteaseactivity.

Fractionation of protein by desorption ion-exchange chro-matography was carried out on a column (2.5 by 35 cm) ofDEAE-Sepharose with the sample applied in and eluted with50 mM Tris hydrochloride-100 mM NaCl (pH 8.0). A total of144 fractions were collected, and the A280 was measured.The first 50 fractions were analyzed for protein and proteaseactivity.

Fast protein liquid chromatography. Fast protein liquidchromatography of protease Pll was carried out on a Super-ose 12 (Pharmacia) column; the effluent was monitored at280 nm. The protease (1 to 10 ,ug) was applied in 100 to 200pJ of 50 mM Tris hydrochloride-100 mM NaCl (pH 8.0). Thecolumn was washed with the same buffer at a flow rate of0.25 ml/min. Proteins of known molecular mass were ana-lyzed in the same manner to establish a relationship betweenelution position and molecular mass of the protein.

Isolation and chemical modification of pPGM. pPGM wasisolated from culture filtrates by procedures routinely usedin this laboratory (25). This procedure involves precipitationof pPGM as its borate complex with alkyltrimethylammo-nium cation and fractionation of the polymer on DEAE-cellulose-borate (12). A major species of pPGM (60 to 70%)and a minor species (30 to 40%) were recovered from theabove column in 0.01 N HCI-0.06 M LiCl and 0.01 NHCl-0.4 M LiCl washes, respectively.The phosphogalactomannan and manno-oligosaccharide

were released from the polypeptide by treatment with 0.4 NKOH at ambient temperature for 8 h. The reaction wasterminated by neutralization with HCI. Phosphogalactoman-nan was separated from the low-molecular-mass compo-nents by dialysis in cellulose membranes (cutoff, 3.5 kDa)against distilled, deionized water. The fraction was assayedfor carbohydrate, protein, and phosphate after removal oflow-molecular-mass substances and used in the proteaseassay.

Galactofuranosyl residues were released from pPGM byits treatment in 0.01 N HCl for 90 min at 100°C. Thegalactose released was separated from the remaining pepti-dophosphomannan by dialysis as described above.

Polyacrylamide gel electrophoresis. Polyacrylamide gelelectrophoresis of proteins was performed, using a mini slabgel apparatus. Gels were cast in glass plates (7 x 10 cm).Running and stacking gels contained 12 and 4% acrylamide,respectively. Buffer systems were used as previously de-scribed (5). Samples of approximately 0.3 to 1 ,ug of proteinwere applied to gels in SDS sample buffer (pH 6.8) (50 mMTris hydrochloride plus 10% glycerol, 2% SDS, 5% beta-mercaptoethanol, and 0.002% bromophenol blue). Variablecurrent at 120 V was applied across the gels until the trackingdye approached the end of the running gels. The gels werefixed and stained by using the silver staining kit of Bio-RadLaboratories, Richmond, Calif. The stained gel was photo-

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graphed with a Polaroid DS-34 direct screen camera contain-ing Polaroid type 65 black-and-white film.

Substrate gel electrophoresis in which the gels contained150 ,ug of BSA per ml was carried out on glass plates (7 by 10cm) for approximately 2 h at constant current (10 or 16 mA)and variable voltage (116 to 138 V). The gels were placed in2.5% Triton X-100 for 30 to 60 min with constant shaking andsubsequently washed thoroughly with distilled deionizedwater. The washed gels were placed in 50 mM Tris hydro-chloride-10 mM CaCl2 buffer (pH 7.8) and incubated for 12to 16 h at ambient temperature. After incubation, the gelswere stained with Coomassie blue dye and photographed asdescribed above. The following reference proteins were usedfor comparative purposes: BSA (66 kDa), 100 ng; ovalbumin(45 kDa), 100 ng; glyceraldehyde-3-phosphate dehydroge-nase (36 kDa), 100 ng; carbonic anhydrase (27 kDa), 100 ng;trypsinogen (24 kDa), 100 ng; trypsin inhibitor (20.1 kDa),100 ng; and o-lactalbumin (14.2 kDa), 100 ng. In SDS-polyacrylamide gels, ovalbumin (36 ,ug), glyceraldehyde-3-phosphate dehydrogenase (16 Rxg), carbonic anhydrase (10p.g), oa-lactalbumin (10 jig), and phosphorylase b (25 jig)were used as reference proteins in the experiment employing10.5% polyacrylamide gel (see Fig. 3B).

RESULTS

The activity of alkaline protease(s) in P. charlesii culturefiltrates increased steadily (sevenfold) between days 3 and 16when the organism was cultured in Raulin-Thom medium(data not shown). Acid protease activity, measured at pH3.0, did not increase significantly. When cultured in LPSGmedium, alkaline protease activity was insignificant and acidprotease activity was only slightly greater. Samples of cul-ture filtrates, obtained from cultures 16 days after inocula-tion, were concentrated fivefold by using a concentrator(Amicon Corp., Lexington, Mass.), followed by fractiona-tion on Sephacryl S-200. Alkaline protease activity waspresent in all fractions in the molecular mass range of 20 to60 kDa (data not shown). The proteins in the 20- to 60-kDarange were fractionated on Sephacryl S-200, the fractionswere pooled, and the proteins were further fractionated intothree proteolytically active fractions (PI, PIT, and PIII) onDEAE-Sepharose (Fig. 1). Fraction Pll contained 82% of thetotal alkaline protease activity obtained from the column.About 50% of the protein applied to the column was eluted inthe scheme shown. The remainder was eluted during regen-eration of the column. Attempted fractionation of the prote-ase(s) by desorption chromatography (pH 8.0 and 100 mMNaCl) was not useful in purification of the proteases (datanot shown).The degree of homogeneity of proteases PI and Pll was

examined by SDS-polyacrylamide gel electrophoresis of theproteins (Fig. 2). Fraction Pll provided only one major bandwhich stained with silver, whereas fraction PI gave twobands. Fraction PI appeared to be a mixture of PIT (mass, 44kDa) and a slightly smaller species (mass, 40 kDa). ProteasePll was examined further by substrate gel electrophoresis on12% polyacrylamide gels to which had been added 150 ,ug ofheated BSA per ml of gel (Fig. 3A). Trypsin (lanes 1 and 2)was also examined for comparative purposes. Lanes 3 and 7contained reference proteins to establish molecular masslocation markers. Lanes 4 through 6 contained protease PIT.Protease activity, as measured by the disappearance ofCoomassie blue-staining BSA was detected in the region ofless than 20 kDa for lanes containing trypsin or PII and in the22-kDa region for trypsin. Penicillium protease PIT activity

1.25

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0.75

0.50

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:-i

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FRACTION NUMBER

FIG. 1. Elution pattern of proteases on DEAE-Sepharose anion-

exchange column (adsorption mode) after gel filtration. Fractions

containing proteolytic activity (20 to 60 kDa) recovered from gelfiltration (Sephacryl S-200 superfine) column chromatography, as

described in Materials and Methods, were subjected to anion-

exchange chromatography, as described in Materials and Methods.Protease activity (optical density at 595 nm [OD 595]) and NaCl

concentration (molarity) are shown.

was also observed at the origin (lanes 4 to 6) as well. It isunclear whether the Penicillium protease PIT is too large to

penetrate the 12% cross-linked gel, whether the proteaseaggregates into nonpenetrating species, or whether it binds

tightly to the heat-inactivated BSA. The size of the small

protease active species in protease PIT preparation is of

about the same mass as those from trypsin. The experimentwas repeated with a 10.5% polyacrylamide gel containingheat-inactivated BSA (Fig. 3B). Protease Pll penetrated the

gel with destaining apparent up to about 45 kDa. However,unlike the lanes (1 and 2) containing trypsin, protease Pllappeared to bind very tightly to BSA, resulting in a contin-

uous loss of BSA from the origin to a region where proteinsof 45 kDa migrate (lanes 4 and 5). Unlike the 12.0% gels, no

clearing areas corresponding to the lower-molecular-mass

proteolytically active fragments were observed in lane 1, 2,4, and 5.

Protease PIT was analyzed by fast protein liquid chroma-

tography on a column of Superose 12 gel. Heat-inactivated

PII showed one major peak of 280 nm absorbing material

(Fig. 4A) in the region of 44 kDa. In contrast, unheated

protease PIT (Fig. 4B) and Pll incubated overnight at 4°C(Fig. 4C) were partially degraded, as indicated by the mul-

tiple peaks in Fig. 4B and C. Proteolytic activity was found

in substances eluting in fractions from peaks b and c; peak d

contained negligible protease activity.Characterization of protease PI. Protease PIT had a pH-

activity profile characteristic of neutral or alkaline proteases(Fig. 5). Optimum activity was observed over a pH of 7 to 9,and activity was observed over a pH range of 3 to 10.

The results of an inhibition study using PMSF showed that

the enzyme was completely inhibited by PMSF (Fig. 6). The

temperature optimum for PIT protease activity was deter-

mined to be about 45°C (data not shown). Thermal stabilityof the enzyme was a function of incubation period (Table 1).

11

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PENICILLIUM ALKALINE PROTEASE 5633

1 2 3 4

a

f ........ .. + 1:.:: :.

2 3 4 5 6 7

a

b

C

cI

e

f

g

bc

e

f

2 4 5 6 7

FIG. 2. Slab gel electrophoregram of alkaline protease(s) PI andPll recovered after DEAE-Sepharose column chromatography.Samples containing 0.1 to 1.0 ,ug of protein were diluted 1:3 (vol/vol)in SDS sample buffer and heated for 5 min at 95°C before theirapplication to gels. The standard mixture, lanes 1 and 4, containedthe following: a, BSA (66.0 kDa); b, egg albumin (45.0 kDa); c,

glyceraldehyde-3-phosphate dehydrogenase (36.0 kDa); d, carbonicanhydrase (27.0 kDa); e, trypsinogen (24.0 kDa); f, trypsin inhibitor(20.1 kDa); g, oa-lactalbumin (14.2 kDa). Lanes 2 and 3 containedalkaline proteases PII and PI, respectively.

Little loss in activity occurred over the temperature range of5 to 55°C when the enzyme incubation time was limited to 30min (data not shown). In contrast, 90-min incubation periodsresulted in 23% inactivation at 25°C and even greater loss ofactivity was observed at higher temperatures. The proteaseactivity appeared to be stable over a pH range of 6 to 10, butit was rapidly inactivated at pH 3 or less.

Effects of various proteins on PIT activity. The effects ofvarious polymers from P. charlesii on the activity of prote-ase PIT on HPA was examined. In the presence of crudepPGM, protease PIT did not hydrolyze HPA during a 12-hincubation (Table 2). This ability to block proteolytic degra-dation of HPA was lost upon purification of pPGM, treat-ment of crude pPGM with KOH, or with 0.01 N HCI,suggesting that the protease binds to a protein fractionassociated with pPGM.

Activity of protease PIH on synthetic substrates. It is appar-

ent that PII has a strong affinity for heated BSA and some

components present in crude preparations of pPGM. How-ever, the specificity of Penicillium protease PIT has not beencompletely established. The ability of Pll to hydrolyze sixsynthetic substrates was compared with those of trypsin and

FIG. 3. Substrate gel electrophoregram of alkaline protease (PII)and trypsin. (A) 12.0% polyacrylamide gel containing Heat-inacti-vated BSA; (B) 10.5% polyacrylamide gel containing heat-inacti-vated BSA. Samples containing 10 ng or 1 ,ug of trypsin or 18 to 54ng of protease (Pll) or 18 ng of protease (Pll) heated for 5 min at95°C were diluted 1:3 (vol/vol) in SDS sample buffer and applied togels. The reference proteins that are described in the legend to Fig.2 were heated to inactivate contaminating proteases and were usedfor comparative purposes. Lanes in panel A: 1, trypsin (100 ng); 2,trypsin (1 ,ug); 3, reference protein (see Fig. 2 legend); 4, proteasePll (18 ng); 5, PII (36 ng); 6, PII (54 ng); 7, reference proteins. Lanesin panel B: 1, trypsin (100 ng); 2, trypsin (1 p,g); 3, phosphorylase b(97.4 kDa); 4, PII (18 ng); 5, Pll (36 ng); 6, reference proteins; 7,heat-inactivated Pll (18 ng). Reference proteins: a, ovalbumin (45kDa); b, carbonic anhydrase (29 kDa); c, at-lactalbumin (14.2 kDa).

chymotrypsin (Table 3). Protease PII hydrolyzed p-nitroanil-ides of arginine and phenylalanine but not the ethyl esters ofeither arginine or tyrosine. Both trypsin and chymotrypsinhydrolyze both esters and amides.

Influence of adding PMSF to Penicillium cultures on theproduction of pPGM and activity of protease PII. A series ofexperiments was done to determine whether PMSF influ-enced production of pPGM and extracellular protease PIIactivity and activity of other extracellular hydrolases. PMSF(3.4 mg/200-ml culture) was added to cultures of P. charlesiieither 24 or 48 h after inoculation of flasks with conidio-spores. The culture was continued until day 7 at which timethe culture filtrate was obtained by filtration. The culturefiltrates from the control and two PMSF treatments wereexamined for quantity of pPGM and its percentages ofcarbohydrate, protein, and phosphate (Table 4). The pPGM

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time(min)FIG. 4. Fast protein liquid chromatographic analysis of alkaline

protease (Pll). (A) Heat-inactivated enzyme; (B) untreated enzyme;(C) untreated enzyme after overnight incubation at 4°C. Equalaliquots of heat-inactivated and untreated alkaline protease (Pll)were applied to a Superose 12 gel filtration column, and thesubstances which were eluted from the column were monitored at280 nm and assayed for proteolytic activity using the HPA assay(see Materials and Methods for details).

in cultures to which PMSF was added at 24 or 48 h contained41 and 34% less pPGM than a control which was not treatedwith PMSF. The percentages of carbohydrate, protein, andphosphate in pPGM isolated from cultures to which PMSFwas added were not significantly altered when comparedwith those of the control. In a similar experiment, modifiedonly by extending the culture period to 16 days, it was foundthat the activity of protease PII in culture filtrates from flasksto which PMSF was added at 24 to 48 h was 15 and 5%,respectively, compared with that of the control to which noPMSF was added (data not shown). When assayed for anumber of other enzyme activities, some glycohydrolasesand phosphatases in the filtrates of PMSF-treated cultureshad greater activities than those of the untreated (control)cultures (Table 5). The filtrates of P. charlesii cultures towhich PMSF was added at 48 h showed substantially greaterenzyme activities than either the untreated or culturestreated at 24 h.

80

60

40

0 2 4 6 8 10

pHFIG. 5. pH activity profile of alkaline protease (Pll). Procedures

for measurements of protease activity are given in Materials andMethods.

DISCUSSION

An alkaline protease, PII, isolated from culture filtrates ofP. charlesii at day 16, was purified and shown to contain onemajor protein species by SDS-polyacrylamide gel electro-phoresis and fast protein liquid chromatography. Of thePenicillium spp., only the alkaline protease secreted by P.cyaneo-fulvum has been previously described in detail (3, 14,17, 26). Similarities exist between the alkaline protease fromP. cyaneo-fulvum and P. charlesii; that from P. cyaneo-fulvum has a mass of 45 kDa, a pl in the range of 9 to 10.5,and an optimum pH range of 6.5 to 8.5. The Penicilliumprotease has a mass of approximately 44 kDa and a pl in thepH range of 8.5 to 9.0 and is active over a pH range of 3 to

1.00

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100

75

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50 -

25

0 20 40 60 80 100

PMSF (pg)FIG. 6. Inhibition of alkaline protease (Pll) by PMSF. This

procedure is described in Materials and Methods. A595 was mea-sured against a blank to which no enzyme or inhibitor was added.Preparations to which alkaline protease but no PMSF was addedwere used to determine 100% activity. All of the readings wereconverted to percent activity and plotted against the correspondingpH values.

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PENICILLIUM ALKALINE PROTEASE 5635

TABLE 1. Thermal stability of alkaline proteaseaTemp (°C) of % Activityincubation

5 ...................................................... 10025 ........................................ 7835 ........................................ 5145 ........................................ 4755 ........................................ 2365 .......................................... . . . . . . . . . . . . 575 .......................................... . . . . . . . . . . . . 3

a After incubation of 3 or 6 for incubaton temperatures of 55, 65, and 75°Cp.g of alkaline protease (Pll) in 50 mM Tris hydrochloride-10 mM CaCl2 (pH7.8) for 90 min, alkaline protease activity was determined at 37°C as describedin Materials and Methods.

10. Both enzymes are inhibited by PMSF, which suggeststhat both are serine proteases. The optimum pH of PII is 7 to9. Although PII has no specific cation requirement, Ca2+appears to stabilize the enzyme and to somewhat enhance itsactivity (data not shown). This protease is extremely labileand autolytic, especially in the purified concentrated state,as shown by its rapid conversion to lower-mass forms (Fig.4).

Unlike the genus Penicillium, the closely related genus,Aspergillus, contains a number of species which secretealkaline serine proteases (4, 6, 18, 20, 27, 28). Examples ofthese are aspergillopeptidases B, C, and F, which havemasses in the range of 20 to 25 kDa and similar amino acylsequences at their active sites. Also, a serine protease wasisolated from culture filtrate of P. cyclopium (18). Theproperties of this protease bear little resemblance to those ofP. charlesii or P. cyaneo-fulvum; it has a mass of 35 kDa, apI of 5.0, and a pH optimum of about 7.0.

P. charlesii protease PII has some unusual propertieswhich may relate to its function(s). The protease catalyzesthe hydrolysis of phenylalanyl and to a lesser extent that ofargininyl amide linkages, but not those of arginyl or tyrosinylesters (Table 3). This lack of esterase activity is uncommonto the trypsinlike or subtilisinlike microbial serine proteases.However, the primary specificity of PII resembles those ofthe subtilisinlike alkaline proteases of Aspergillus spp. andP. cyaneo-fulvum. Furthermore, PII has unusual affinity forthose proteins to which it binds; PII was completely inhib-ited for up to 12 h by the addition of a crude preparation ofpPGM which contained some bound Penicillium proteins.This was not an irreversible inactivation, as shown by theexpression of protease activity by 48 h. This inhibition wasnot observed when pPGM purified to remove noncovalentlybound protein(s) was used. Neither casein nor BSA wereeffective inhibitors of PII. It may also be significant that PII

TABLE 2. Hydrolysis of HPA by alkaline protease in thepresence of various proteins and glycopeptidesa

Preincubation preparation mg of HPA

Crude pPGM (3.8 mg) .......................................... 0Major pPGM species (5.6 mg) .................................... 3.6Autoclaved mycelial extract (1-14 kDa) (5.0 mg) ........... 3.0Casein (2.0 mg) .......................................... 3.9BSA (2.0 mg) .......................................... 4.0Reprecipitated, major pPGM (5.6 mg) ............. ............ 3.3Control (no protein added) ........................................ 3.6

a Samples (50 ,ug) of altkaline protease (PII) were incubated with theindicated protein-containing preparations for 30 min, and the experiment wasperformed as described in footnote a of Table 1.

TABLE 3. Comparison of alkaline protease (PII) activity withtrypsin and chymotrypsin against synthetic substratesa

Substrate' ActivitycTrypsin Chymotrypsin Alkaline protease

BAEE + (+) -BTEE (+) + -PPLGPdA - - -SAAPPpNA ND + +BtpNA ND + +BPVApNA + (+) +

a Assay conditions are described in Materials and Methods.b Abbreviations: BAEE, N-benzoyl-L-arginine ethyl ester; BTEE, N-ben-

zoyl-L-tyrosine ethyl ester; PPLGPdA, 4-phenylazobenzyl-oxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-arginine; SAAPPpNA, succinyl L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl p-nitroanilide; BTpNA, N-benzoyl-L-tyrosinep-nitroanilide; BPVApNA, N-benzoyl-L-phenylalanyl-L-valyl-L-arginine p-ni-troanilide.

c Symbols: +, rapid hydrolysis; (+), slow hydrolysis by contaminatingprotease(s); +, incomplete hydrolysis due to substrate insolubility in aqueoussolutions; -, no hydrolysis of substrate. ND, Not done.

appears to bind much more tightly to denatured BSA thantrypsin does (Fig. 3). Thus, after gel electrophoresis in 10.5%acrylamide gels, the lanes containing nanogram quantities ofPII failed to stain with Coomassie blue in the region of thewell from which the sample was placed down to a region ofabout 45 kDa (Fig. 3B). This lack of blue stain occurred onlywhere most of the protein had been digested. The lanescontaining similar quantities of trypsin showed protein di-gestion only in the region of 22 to 25 and 10 to 20 kDa. Also,unlike trypsin, PII did not penetrate 12% acrylamide gels(Fig. 3A); in contrast, the 10- to 20-kDa catalytically activePII-lytic degradation products did not bind tightly to thedenatured protein. These data show that PII is particularlyeffective in binding to and degrading denatured proteins. Thefunction of PII may be to degrade unfolded regions ofproteins located in the region of the cell wall or in theexocellular space.

Preliminary evidence suggests that in P. charlesii, anendoproteolytic event which is partially inhibited by PMSFmay be involved in the release of pPGM, as shown by the41% decrease in pPGM in cultures filtrates to which PMSFwas added at day 1. Although PMSF inhibited pPGM accu-mulation in the growth medium, it has negligible influence onthe growth rate, acid production, or total mycelial massproduced by these cultures. PMSF did not influence thechemical composition of pPGM or the morphology of theorganism or inhibit the activity of its extracellular phos-phatases or glycohydrolases (Table 5).

Wrathal and Tatum (29) suggested that the cell wallglycopeptides in Neurospora crassa are attachment sites for

TABLE 4. Total quantities and percent composition ofmajor pPGM fractions isolated from PMSF-treated

and untreated cultures

Culture" ~Total %%Culture' pPGMb (mg) Carbohydrate Protein PhosphateUntreated 363 94.3 4.6 1.0Treated (24 h) 214 93.7 5.2 1.2Treated (48 h) 238 94.5 4.5 0.7aNo PMSF was added to untreated cultures; 3.4 mg of PMSF in 100 ,ug of

absolute ethanol was added to cultures 24 or 48 h after inoculation.b pPGM was isolated from culture filtrates after day 7 as previously

described (12).

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TABLE 5. Entracellular enzyme activities of PMSF-treated and untreated cultures of P. charlesiia

Enzyme activityb (mU/mg)Culture Phshrlhln-N-acetyl-p3- 13-Galacto-C3-Glucosidase ax-Glucosidase 1-Galactosidase Phosphoyicholine- Acid phosphatase glucosaminidase furanosidase

phosphodiesterase guoaiiae frnsds

Untreated 6.7 1.9 5.5 3.3 2.5 7.4 12.1Treated (24 h) 14.1 1.9 2.1 8.0 3.2 25.6 14.4Treated (48 h) 20.5 3.3 6.4 8.4 7.6 64.4 27.0

a Aliquots (3.4 mg) of PMSF were added 24 or 48 h after inoculation of P. charlesii conidiospores into a modified Raulin-Thom growth medium (200 ml per flask).The culture filtrate was obtained on day 18, and 100-,ul aliquots were assayed for hydrolase activity with the appropriate p-nitrophenyl derivative. PMSF wasadded as described in footnote a of Table 4.

b Measured in milliunits of enzyme activity per milligram of protein. One unit of activity represents 1 ,umol of substrate hydrolyzed per min under the givenconditions.

extracellular hydrolases before their release into the envi-ronment. If alkaline protease promoted the release of lyticenzymes bound to the glycopeptides, then a decrease inthese enzymes with decrease in alkaline protease would beexpected, but this was not observed.

In P. charlesii, pPGM precursors are attached to theplasma membrane (10) and extend out into the cell wallregion (13). The data presented here allow the possibilitythat release of the membrane- or cell wall-bound form ofpPGM is significantly inhibited by PMSF. This inhibitionmay be related to the approximately 10-fold decrease inalkaline protease Pll activity in the growth medium. How-ever, because inhibition of protease PII by PMSF does notsignificantly decrease the release of extracellular hydrolases,it seems unlikely that PII has a significant role in the releaseof these hydrolases into the culture filtrate and may insteadcontribute to their degradation. It is not known whetherthese hydrolases are truly secreted or whether they arereleased after cell wall autolysis.Numerous possibilities exist regarding the role(s) of pro-

tease PII in the release of pPGM from its cell-bound site(s).However, the appearance of Pll at about the onset ofammonium depletion, its great affinity for protein(s) associ-ated with pPGM, the increase in activity of extracellularhydrolases in culture filtrates of cultures to which PMSF hasbeen added, and the extracellular location of PII, all suggestthat PIT has a significant role in regulating the relationshipbetween the glycopeptide, pPGM, and the wall-bound andextracellular forms of the lytic enzymes.

ACKNOWLEDGMENTS

This work was supported by Public Health Service research grantGM 34068 from the National Institute of General Medical Sciencesand by the Institute of Food and Agricultural Science ExperimentStation (project FLA-MCS 02434).

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