Purification and Characterization of an Endopeptidase from Propionibacterium freudenreichii

Purification and Characterization of an Endopeptidase from Propionibacterium freudenreichii R. 0. TOBIASSEN, A. H. PRIPP, L. STEPANIAK, and T. SORHAUG Department of Food Science, Agricultural University of Norway, N-1432 As, Norway

Abbreviation key: LAB = lactic acid bacteria, PAB = propionic acid bacteria, PEP = Propionibacterium endopeptidase, p-NA = p-nitroanilide, RP = reversedphase.

development of typical taste and flavor of Swiss-type cheeses and Norwegian Jarlsberg cheese are dependent on the contribution of PAB. The PAB are considered to be weakly proteolytic, and degradation of casein is observed only after prolonged incubation in milk ( 12 ) . Peptidase activities in PAl3 have been reported earlier, but emphasis has been on proline iminopeptidases and production of proline (4, 7, 13, 14, 15, 23, 25, 26). Recent reports indicate that PAB contain more than one proteinase and that one of these enzymes may be associated with the cell wall (6, 27). Noncaseinolytic, intracellular, lactococcal endopeptidases PepF and PepO have been quite well characterized ( 10, 20). Characterization of endopeptidases from propionibacteria has not yet been reported. Exterkate and Alting ( 8 ) , utilizing characteristic differences in specificities of proteinases associated with the lactococcal cell envelope and intracellular lactococcal PepO, demonstrated that PepO degrades (r,lCN (fl-23) during the ripening of Gouda cheese. The fragment is a major peptide that is released from a,l-CN by chymosin, and cleavage sites of this peptide by different lactococcal endopeptidases have been determined ( 1 0 ) . The objective of this study was to purify and characterize intracellular endopeptidase from Propionibacterium freudenreichii ATCC 9614.

INTRODUCTION

MATERIALS AND METHODS

ABSTRACT

A 44-kDa endopeptidase, isolated by lysozyme and sonic treatment from the cytoplasm of Propionibacterium freudenreichii ATCC 9614, was purified t o homogeneity. Ion-exchange chromatography of the cytoplasmic fraction separated the enzyme from several fractions with caseinolytic activities and one fraction with proline iminopeptidase activity. The endopeptidase was subsequently purified by chromatography and by gel filtration. The enzyme was a monomer with a PI of 3.8, and the enzyme hydrolyzed bradykinin most actively between pH 6.5 and 8 and between 45 and 50°C. The specificity of the Propionibacterium endopeptidase on bradykinin, methionine enkephalin, angiotensin I, and (r,l-CN (fl-23) was determined; specificity on (r,l-CN (fl-23) was different from that of the 70-kDa endopeptidase ( P e p o ) from Lactococcus spp. The activity on oxidized insulin @-chainwas very low. The enzyme was inhibited more by EDTA than by 1,lO-phenanthroline and was not sensitive to phosphoramidon. ( Key words: Propionibacterium sp., endopeptidase, cheese)

Proteinases and peptidases from lactic acid bacteria (LAB) contribute to primary and secondary proteolysis during cheese ripening and are important for the development of texture and flavor ( 1 0 ) . Relatively little is known about the proteolytic system of propionic acid bacteria ( PAB) compared with that of LAB (15, 30); PAl3 is the predominant secondary microflora of Swiss-type cheeses, and populations reach approximately 109/g when the cheese is removed from a warm room (16). Eye formation and

Received November 2, 1995. Accepted June 24, 1996. 1996 J Dairy Sci 79:212%2136

Microorganism and Culture Conditions

Propionibacterium freudenreichii ATCC 9614 (previously Propionibacterium shermanii ATCC 9614) was obtained from the American Type Culture Collection (Rockville, MD). Stock cultures were stored at -80°C in sodium lactate broth ( 2 5 containing 15% (vol/vol) glycerol. All media were autoclaved for 20 min at 121°C. An active culture was used to inoculate ( 2 % ) 10 L of sodium lactate broth, and the culture was then incubated at 30°C for 36 h without shaking. The cells were harvested by centrifugation for 10 min at 13,000 x g at 4°C and then washed twice in 0.5 A4 NaCl at 4°C (25).

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Materials

Unless otherwise stated, all chemicals were analytical or HPLC grade and were obtained from Sigma Chemical Co. (St. Louis, MO). Preparation of Subcellular Fractions

The washed cells from 10 L of medium were incubated in 200 ml of 0.05 M Tris buffer a t pH 7.5 for 30 min at 30°C with shaking on a n orbital shaker a t 150 rpm. Cells were then removed by centrifugation and resuspended in 20% sucrose at 4°C for 1 h to facilitate the release of material associated with the cell wall ( 2 5 1. Centrifugation followed, and the cells were resuspended in a spheroplast buffer, pH 7.8, consisting of 0.01 M Tris, 0.5 M sucrose, 0.3 M NaC1, 0.05 M MgS04, and 0.01 M KC1 (which prohibits cell lysis a t this stage) containing lysozyme (1 mg/ml); the sample was then incubated at 30°C for 5 h with slow mechanical shaking ( 2 5 1. The spheroplasts were separated by centrifugation at 20°C and resuspended in 5 mM sodium phosphate buffer, pH 7.5 ( 2 5 ) . After samples were held on ice overnight, the spheroplasts were sonicated at 7 to 8 pm (MSE 7100 Ultrasonic Disintegrator M200; Measuring Scientific Equipment Ltd., London, England). Sonication was in the presence of 2% (wt/vol) glass beads (150 to 200 pm) for a total of 30 min, and the glass tube was immersed in ice, alternating 3-min sonication periods and 5-min resting periods. After sonication, RNase A (Type l-A) and DNase I (Type IV), both a t 0.02 mg/ ml, were added, and the suspension was incubated for 30 min a t 37°C before centrifugation at 48,000 x g and 4°C for 20 min. The supernatant, designated as the cytoplasmic fraction, was dialyzed against 0.05 M sodium phosphate buffer, pH 6.0. Protein Content

The concentration of protein was determined with the Bio-Rad protein assay (Bio-Rad Laboratories GmbH, Munich, Germany), using BSA as the standard. Enzyme Assays

Endopeptidase activity. Endopeptidase activity was monitored with bradykinin as the substrate. The reaction mixture [25 p1 of substrate stock solution ( 1: 3:1, vol/vol/vol, of 5 mM bradykinin, HzO, and 0.5 M sodium phosphate buffer, pH 6.21, 3 to 30 pl of enzyme solution, and HzO to make a total of 100 p11 was incubated at 30°C for 5 to 30 min. The amount of enzyme and the incubation time were selected to be within the linear range of hydrolysis and to obtain Journal of Dairy Science Vol. 79, No. 12, 1996

measurable results. Then, 1.2 ml of 0.2% trifluoroacetic acid (Rathburn Chemicals Ltd., Walkerburn, Scotland) were added to stop the reaction before analysis by reversed-phase ( R P ) HPLC with a PepRPC Hr5/5 column (Pharmacia Biotechnology, Uppsala, Sweden) and FPLCa equipment (Pharmacia Biotechnology) with a detector operating at 2 14 nm. The peptides were eluted by a n acetonitrile (Rathburn Chemicals Ltd.) gradient as described by Stepaniak and Fox (28). The activity was expressed as micromoles of bradykinin hydrolyzed per minute per milliliter of enzyme solution, based on the rate of reduction of the substrate peak area. Caseinolytic activity. The caseinolytic activity was assayed with a fluorescent casein as described by Twining (32). The incubation mixture consisted of 30 p1 of enzyme solution, 60 p1 of 50 mM phosphate buffer, pH 6.2, 60 pl of 0.5% fluorescent casein, and 3 p1 of 10% NaN3; the mixture was incubated for 24 h a t 30°C. The increase in fluorescence was measured a t an excitation wavelength of 490 nm and a n emission wavelength of 525 nm (LS-5 Luminescence Spectrophotometer; Perkin Elmer Corp., Norwalk, CT). Aminopeptidase activity. The following substrates were used to study the aminopeptidase activities: Pro-p-nitroanilide ( p-NA) to determine the proline iminopeptidase activity, Leu-p-NA for general aminopeptidase activity, and Gly-Pro-p-NA for the Xprolyl-dipeptidyl aminopeptidase activity. Ten microliters of 20 mM substrate were dissolved in methanol, 100 p1 of 50 mM sodium phosphate buffer at pH 6.2, enzyme solution (10 to 50 pl), and HzO made a total volume of 200 pl; samples were incubated up to 2 h at 30°C or until a yellow color was evident. The reaction was stopped by addition of 1.0 ml 10% acetic acid. The absorbance of the p-NA was read a t 410 nm using a Shimadzu double-beam spectrophotometer (UV-21OA; Shimadzu Seisakusho Ltd., Kyoto, Japan). Purification of Enzyme

Purification was carried out with FPLCID equipment. The dialyzed cytoplasmic fraction was applied to a Fast Flow Q Sepharose column (1.6 by 49.0 cm; Pharmacia Biotechnology) pre-equilibrated with 50 mM sodium phosphate buffer, pH 6.0. The proteins were eluted with a multiple-step NaCl gradient of 0 to 2.0 A4 in the same buffer at a rate of 2 mumin, and 8-ml fractions were collected. The fractions containing the highest activity on bradykinin were pooled and dialyzed against 10 mM sodium phosphate buffer, pH 6.0, a t 4°C. Further purification on hydroxyapatite

ENDOPEPTlDASE FROM PROPlONBACTERlUM

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Effect of inhibitors on the endopeptidase activity. The purified endopeptidase was tested with the following inhibitors: EDTA (Merck, Darmstadt, Germany), p-chloromercuribenzoic acid (Fluka Chemie AG, Buchs, Switzerland), phenylmethylsulfonyl fluoride, 1,lO-phenanthroline, and phosphoramidon. To 0.5 pl of the enzyme solution (concentrated lox by lyophilization), 5 p1 of inhibitor solution were added by mixing; the mixture was incubated for 10 min at 20°C. Twenty-five microliters of Properties of PEP standard bradykinin stock and 65 pl of HzO were then added, and the incubation was continued for 20 Determination of molecular mass and PI. The min at 30°C before the residual substrate was anaSDS-PAGE was carried out with ready-cast 12.5% lyzed. The effect of @-CN(f58-721, a peptide inhibitor gels in the Phast System (Pharmacia Biotechnology) of Pep0 from Lactococcus lactis ssp. lactis MG1363 as described by the manufacturer. (291, was also determined by incubating the enzyme For gel filtration, a prepacked Superose 12 HR 16/ solution in 50 p1 containing 0.025 mM 0-CN (f58-721, 50 column was equilibrated with 50 mM sodium phos25 mM sodium phosphate buffer pH 6.0, and 0.25 mM phate buffer, pH 6.8, containing 0.15 M NaC1. bradykinin. Molecular mass markers for protein and PEP were Specificity of purified PEP. The specificity of the eluted with the same buffer. endopeptidase was tested on the following substrates: The PI was determined using ready-cast gels for electrofocusing, pH range 3.5 to 9.0, and the Pharma- bradykinin, methionine enkephalin, angiotensin I, oxcia Phast System. After separation, the gels were idized insulin P-chain, and a,l-CN (fl-23). Twenty stained with Coomassie Blue R. Electrofocusing and microliters of the enzyme solution were incubated staining were performed as described by the manufac- with 1 mM substrate and 33 mM sodium phosphate buffer, pH 6.0 (total 80 pl), for 4 h at 30°C. The turer. Effect of pH and temperature. All activity meas- hydrolysates were separated by RP FPLC@ as urements were made with bradykinin as substrate, described herein. Eluted peaks were collected and the reactions were stopped with trifluoroacetic acid, freeze-dried. An automated Edman degradation was and the residual substrate was analyzed by R P performed at the Biotechnology Centre of Oslo FPLC@ as described. (University of Oslo, Norway), using an Applied BioActivity a t different pH values was determined by systems 470A protein sequencer (Applied Biosystem, incubating 10 p1 of enzyme solution, 2.5 p1 of 5.0 mM Division of Perkin-Elmer Corporation, Foster City, bradykinin, and 37.5 pl of universal buffer ( 31)for 20 CA). The phenylthiohydantion derivatives of the min at 30°C. The pH stability of the enzyme was amino acids were identified by HPLC (Applied Bidetermined by incubation at different pH values in osystems 120A Analyzer). universal buffers at 4°C for 30 min. The pH was then Amino acid analysis. The PEP was freeze-dried adjusted to 6.0, substrate was added, and the incuba- and then hydrolyzed in 6.0 M HC1 in evacuated tubes tion was continued for 20 rnin a t 30°C. for 24 h at 110°C. Amino acid composition was anaThe effect of temperatures between 5 and 60°C was lyzed with an AB1 421 amino acid analyzer (Applied studied at pH 6.2, and the enzyme was incubated for Biosystems). 20 min. The thermal stability was examined by incubating the enzyme solution for 10 min at different RESULTS temperatures. After being cooled in an ice bath, the enzyme solution was tempered a t 30"C, and the residual activity was measured as described. Partial Separation of Proteolytic Activities Michaelis-Menten constant. The peptidase ac- on Fast Flow Q Sepharose and tivity toward bradykinin was determined for different Purification of PEP concentrations of substrate (0.05 to 0.5 W. The After separation of the cytoplasmic fraction on Fast substrate decrease was measured after 20 min of Flow Q Sepharose (Figure 11, three fractions that incubation at 30°C and pH 6.2. The Michaelis-Menten constant and maximum velocity were estimated with were active on casein were eluted at different concena Lineweaver-Burk plot. trations of NaC1. Fast Flow Q Sepharose separated ( 1.6 by 11.0 cm column; Sigma Chemical Co.), Mono Q HR 5/5 (Pharmacia Biotechnology), and Superose 12 HR 16/50 (Pharmacia Biotechnology) was as described by Stepaniak and Fox ( 2 8 ) . Monitoring of activity continued with bradykinin as the substrate, and the final purified preparation was dialyzed against 1 mM sodium phosphate buffer, pH 7.2. The purified enzyme was designated Propionibacterium endopeptidase, or PEP.

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TABLE 1. Purification of Propionibacterium endopeptidase from Propionibacterium freudenreichii ATCC 9614. Purification step1

Total protein

Total activity

(mg) 488.5 37.3 2.16 0.37 0.05

( pmol/min)

Crude extract Fast Flow Q Sepharose Hydroxyapatite Mono Q Superose 12

43.1 9.1 2.0 0.63 0.16

Specific activity2 0.09 0.24 0.93 1.7 3.2

Purification

Yield

(-fold)

(960) 100 21 4.6 1.5 0.4

1.0 2.67 10.3 18.9 35.6

'Fast Flow Q Sepharose, Mono Q, and Superose 12 from Pharmacia Biotechnology (Uppsala, Sweden). 2Micromoles of bradykinin hydrolyzed per minute per milligram of protein.

most of the Pro-p-NA activity from activity on lar mass of PEP, determined by SDS-PAGE and gel bradykinin. The main endopeptidase fraction that filtration chromatography, was 44 and 55 kDa, was active on bradykinin was superimposed on one of respectively, indicating that the enzyme is a the activities on casein and the activity on Leu-pNA. monomer. The PI of PEP was 3.8; the MichaelisThis fraction was purified further on hydroxyapatite Menten constant and the maximum velocity for and resulted in separate peaks with activity on hydrolysis of bradyhnin were 2.20 mM and 6 pmol/ bradykinin, casein, and Leu-p-NA, respectively (data mg per min, respectively. not shown). Table 1 summarizes the purification of PEP. The Characterization of the Purified Enzyme final yield of enzyme activity was very low. The puriEffect of temperature and pH on the activity fied PEP was free from aminopeptidase activity on and stability of PEP. The PEP was active on Leu-p-NA, Pro-p-NA, and Gly-Pro-p-NA, but traces of bradykinin in the range of 5 to 65°C; activity was activity on fluorescent casein were detectable after 24 45 and 50°C. At 5 and 65"C, the maximal between h of incubation. The enzyme, as found by SDS-PAGE, activity of PEP was approximately 10% of the maxwas purified to homogeneity (Figure 2). The molecuimal activity. The enzyme was rather stable up to 40°C. The enzyme showed a broad pH optimum and hydrolyzed bradykinin with maximal activity between pH 6.5 to 8.0. The enzyme appeared to be x stable in the same pH range. The PEP activity at pH 4 5.0 was approximately 10% of maximum. > U N-Terminal sequence and amino acid compoU C m0 sition. The N-terminal sequence analysis of PEP in.5 cluded seven residues: X-Gln-(Phe)-Pro-Phe-Ala-Ala-. m L The first residue could not be identified. The amino c .o 8 acid composition is given in Table 2. C 0 Substrate specificity. Figure 3 shows the specificU ity of PEP on four peptides. The PEP hydrolyzed the .5; Gly-Phe bond of methionine enkephalin. The Gly-Phe, z Y Phe-Ser, and Pro-Phe bonds of bradykinin were x U hydrolyzed; PEP cleaved the Phe-His bond of anm 0 L m giotensin I and the Pro-Ile, Gln-Gly, and Glu-Val F r a c t i o n number bonds of aS1-CN (fl-23). The enzyme expressed very little activity on the oxidized insulin @-chain. Figure 1. Separation of the crude cytoplasmic extract of PropiEffect of inhibitors. The PEP was inhibited by onibacterium freudenreichii ATCC 9614 on Fast Flow Q Sepharose, EDTA and 1,lO-phenanthroline (Table 3 ) and marusing a 0 to 2.0 M NaCl gradient (- - - - - -1 in 50 mM sodium phosphate buffer pH 6.0. Protein (-1 was monitored a t 280 nm. kedly inhibited by the thiolprotease inhibitor Propionibacterium endopeptidase activity ( ). was assayed as p-chloromercuribenzoic acid. Phenylmethylsulfonyl described in Materials and Methods. Arrows indicate fractions with maximal caseinolytic ( C N ) , general aminopeptidase ( P e p N ) , and fluoride, phosphoramidon, NaC1, and 6-CN (f58-72) had little or no effect on the activity of the enzyme. proline iminopeptidase ( P I P 1 activities. .d -rl

Y

.d

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ENDOPEPTIDASE FROM PROPIONIBACTERIUM

DISCUSSION

TABLE 2. Amino acid composition of Propionibacterium endopeptidase from Propionibacterium freudenreichii ATCC 9614.

The separation of the cytoplasmic fraction by Fast Molar Flow Q Sepharose demonstrated the existence of Amino acid Quantity ratio several fractions with activity on casein or bradyki(pmol) (moll100 mol) nin, implying the existence of multiple intracellular AsplAsn 47 8.3 enzymes that are capable of hydrolyzing proteins and GldGln 81 14.4 56 9.9 oligopeptides. This result indicates that PAB, like Ser 87 15.4 GlY LAB, have a complex proteolytic system consisting of His 8 1.4 several proteolytic enzymes. Arg 26 4.6 22 3.9 Endopeptidases from lactococci with molecular Thr 41 7.3 masses of 98 kDa (341, 80 kDa (351, 180 kDa (21, 70 Ala 25 4.4 Pro kDa (2, 31), 93 kDa (211, 66 kDa (201, and 49.5 Tyr 18 3.2 32 5.7 kDa ( 5 ) have been reported. These endopeptidases Val ... NDz were neutral metalloenzymes that were active on Met1 5 0.9 CYS oligopeptides and showed very low or no caseinolytic Ile 29 5.1 activity. Intracellular cysteine proteinase, metal- Leu 62 11.0 25 4.4 loproteinase, and serine proteinase with activity Phe LY s 19 3.4 on benzyloxycarbonyl-L-phenylalanyl-L-arginine-7ND2 . . . Trp3 (4-methyl) coumarylamide, on casein, or on both, 1Below the detection limit. were demonstrated in L. Eactis ssp. lactis ( 1 ) . For Es2Not determined. cherichia coli, the majority of the characterized pro3Destroyed during hydrolysis. teinases are of serine type ( 18). In the present study, the major fraction that was active on bradykinin from Fast Flow Q Sepharose was separated from a minor fraction that was active on bradykinin. The main endopeptidase fraction was superimposed on fractions ther purification of this fraction on hydroxyapatite that were active both on casein and Leu-p-NA. Fur- resulted in separation of a protein peak with high activity on bradykinin, low activity on casein, and no activity on Leu-p-NA. The presence of a 70-kDa lactococcal endopeptidase, Pepo, that does not hydrolyze caseins was con1 2 3 4 5 6 7 firmed by several independent studies (2, 24, 28, 3 1). kDa This enzyme appears to be the best characterized lactococcal endopeptidase. The PEP that was purified in this study had a molecular mass that was clearly different from the endopeptidases reported for LAB.

-

66 -.

45TABLE 3. Effect of inhibitors on Propionibacterium endopeptidase from Propionibacterium freuderireichii ATCC 9614.

29 -. Inhibitor

Concentration

EDTA 1,lO-Phenanthroline Figure 2. The SDS-PAGE showing purification of an endopeptidase from Propionibacterium freudenreichii ATCC 9614. Lanes 1 and 7, molecular mass protein markers; lane 2, crude cell extract; lane 3, bradykinin hydrolyzing fractions from Fast Flow Q Sepharose; lane 4, bradykinin hydrolyzing fractions from hydroxyapatite; lane 5, bradykinin hydrolyzing fractions from Mono Q column; and lane 6, purified Propionibacterium endopeptidase from Superose 12. Fast Flow Q Sepharose, Mono Q column, and Superose 12 from Pharmacia Biotechnology (Uppsala, Sweden).

p-Chloromercuribenzoic acid Phenylmethylsulfonyl fluoride Phosphoramidon (3-CN(f58-72) NaC1, %) 5 10

(mM) 0.1 1.0 0.1 1.0 0.1 1.0 0.1 1.0 0.5 0.25 0.85 x 103 1.7 x 103

Relative activity (%)

52 33 71 40 69 55 111 92 95 100 90 81

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Figure 3. Specificity of Propionibacterium endopeptidase from Propionibacterium freudenreichiz ATCC 9614 on selected peptides. The arrows indicate hydrolytic activity recorded after 4 h of hydrolysis of 80 pl of reaction mixture, containing 1.0 mM of peptide and 20 pl of enzyme. Peptide

Bonds hydrolyzed

Methionine-enkephalin

Tyr-Gly-Gly-Phe-Met

Bradykinin

Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Angiotensin I

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu

CY,,-CN (fl-23)

Arg-Pro-Lys-His-Pro-Ile-Lys-His-Gln-Gly-Leu-Pro-Gln-Glu-Val-Leu-Asn-Glu-Asn-Leu-Leu-Arg-Phe

-1

1

1

1 1

1

1

Proteinase purified by Desmazeaud and Zevaco ( 5 ) from citrate-utilizing L. lactococcus ssp. lactococcus has a molecular mass of 49.5 kDa and a temperature optimum of 45”C, which is closest to that of PEP. The N-terminal sequence of the purified PEP was different from those published for lactococcal endopeptidases (19, 35). Although most active between pH 6.5 and 8.0 and 45 and 50°C, PEP may remain quite active at pH of cheeses and at temperatures during cheese ripening. The 44-kDa PEP showed no general aminopeptidase activity, proline iminopeptidase activity, or Xprolyl-dipeptidyl aminopeptidase activity. The enzyme was very active on peptides containing between 5 and 23 amino acid residues and had very low activity on substrates with higher molecular mass such as oxidized insulin P-chain (30 amino acid residues) and casein. Barret and Rawling ( 3 ) proposed a nomenclature including “oligopeptidase” to distinguish endopeptidases that do not hydrolyze proteins. Although we favor such a distinction, we are reluctant to classify PEP as an oligopeptidase because the enzyme, like the lactococcal intracellular endopeptidase reported by Desmazeaud and Zevaco ( 5 ) and Muset et al. (211, showed measurable activity on casein. The specificity of PEP on crwsl-CN (fl-23) was different from that of lactococcal oligopeptidase PepO ( 2 8 ) , endopeptidases LEP-I ( 3 4 ) and LEP-I1 ( 3 5 ) , and neutral oligopeptidase NOP (2,31).The specificity of PEP was also different from that of proteinases PI and PIII, which were associated with the lactococcal cell envelope (9, 10). Unlike the 70-kDa PepO (281, the 44-kDa PEP had little activity on the insulin P-chain. Similar to PepO and LEP-I1 (28, 351, PEP hydrolyzed the Gly-Phe bond of methionine enkephalin. The PepF from Lactococcus Zactis was not able to hydrolyze peptides that were shorter than seven amino acids ( 2 0 1. PepO preferentially hydrolyzed the Gly-Phe and Pro-Phe bonds of bradykinin. These bonds are also hydrolyzed by PEP; however, Journal of Dairy Science Vol. 79, No. 12, 1996

1

similar to PepF (20), PEP also hydrolyzes the PheSer bond. Similar t o the angiotensin-converting enzyme (171, PEP cleaved the Phe-His bond of angiotensin I. The PEP was inhibited more by EDTA than by 1,lO-phenanthroline;however, PEP was not inhibited by phosphoramidon or 0-CN (f58-721, a hydrophobic peptide identified in Cheddar cheese (29). Lactococcal 70-kDa PepO was more sensitive to 1 , l O phenanthroline than to EDTA and was strongly inhibited both by phosphoramidon and by @-CN (f58-72) (24, 28). The inhibition by p chloromercuribenzoic acid indicates that cysteine may be present in or near the active site of the PEP, which is likely to be a metalloenzyme. Further studies, including cheese-making experiments, are necessary t o determine the significance of PEP for cheese ripening. Different specificity on aS1CN (fl-23) of this enzyme and lactococcal PepO indicates that a PAB endopeptidase or endopeptidases may contribute to the formation of unique peptide profiles in ripened Swiss-type cheeses. The contribution of intracellular enzymes to secondary proteolysis in cheese depends also on the sensitivity of starter microflora to autolysis ( 2 2 ) . 0stlie et al. ( 2 2 ) , who investigated the autolytic properties of a number of PAB strains, found that the strain used for isolation of PEP in this study is highly autolytic at the ripening temperature of Swiss-type cheeses. Another factor is that PAB have been shown to grow in synergism with thermophilic LAB starters in Emmental-type cheese; thus, it is difficult to distinguish the contribution of the individual starter bacteria and enzymes to proteolysis. The significance of PEP for cell physiology remains unknown. It has been suggested that endopeptidases may function in degradation of signal peptides ( 3 5 or modulate or destroy peptide messenger molecules ( 3 ) . In general, intracellular protein turnover is a n

ENDOPEPTIDASE FROM fROPlONlBACTERlUM

indispensable activity of any living cell ( 111. Depletion of 70-kDa PepO from L. lactis ssp. cremoris ( 19 1 and depletion of proteinase from Salmonella typhimurium ( 3 3 ) did not affect growth of these microorganisms. The experiments indicate that complex intracellular proteolytic systems of bacteria offer alternative pathways for protein turnover and peptide degradation. CONCLUSIONS

Propionibacteria have a complex intracellular proteolytic system that probably comprises more than one endopeptidase and several proteinases and aminopeptidases. Propionibacterium freudenreichii ATCC 9614 produces a major 44-kDa intracellular endopeptidase that is sensitive to metal chelators and that has specificity and other properties that are different from that of the 70-kDa PepO from lactococci. The enzyme was insensitive to 6-CN (f58-72) and therefore may be more active in cheese than the lactococcal PepO. ACKNOWLEDGMENTS

The financial support from the Research Council is acknowledged.

Norwegian

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