APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1984, p. 1105-11100099-2240/84/121105-06$02.00/0Copyright © 1984, American Society for Microbiology

The Proteolytic Systems of Streptococcus cremoris: an

Immunological AnalysisJEROEN HUGENHOLTZ,l FRED EXTERKATE,2 AND WIL N. KONINGSl*

Department of Microbiology, University of Groningen, 9751 NN Haren, 1 and Nederlands Instituut voor Zuivelonderzoek,6718 ZB Ede,2 The Netherlands

Received 24 May 1984/Accepted 28 August 1984

The cell-wall-associated proteolytic systems of several Streptococcus cremoris strains were analyzed bycrossed immunoelectrophoresis. At least four immunologically different components of the proteolytic system'swere found. One of these proteins was produced by all strains tested. The proteolytic activity of this enzyme wasdemonstrated with a zymogram staining technique which is based on the degradation of Coomassie-brilliant-blue-stainable casein. The crossed-immunoelectrophoresis patterns of the proteolytic systems of different S.cremoris strains indicated that each strain produces a characteristic combination of proteins. On the basis ofthese combinations, the different S. cremoris strains were classified into four groups.

The lactic acid Dacteria of mixed starter cultures play acentral role in cheese manufacturing (8, 14, 16, 18). Theseorganisms are very fastidious and require for growth a veryrich medium in which essential growth factors such as aminoacids, vitamins, and nucleotides must be present. Milk issuch a nutritious medium, but it contains too few free aminoacids to support substantial growth of these bacteria (8, 18).As the organisms produce exoproteases (5, 8, 19), the caseinin the milk can be degraded to peptides which are subse-quently hydrolyzed to amino acids by peptidases (3, 14). Asa result, growth and acidification of milk can proceed.

In the process of cheese making, curd is formed after themilk has been acidified and the casein has been coagulated.After the curd has been pressed, cut, and salted, theexoproteases of Streptococcus cremoris continue to hydro-lyze casein, and in time more and more amino acids andpeptides are produced. During this process, the flavor ofcheese is formed (8, 14, 16).

In Dutch starter cultures, S. cremoris is the dominantspecies. Many different strains and variants with sometimesquite different properties with respect to the cell-wall-associ-ated proteases are present. Exterkate (5) has studied theprotease activities in different S. cremoris strains and classi-fied the strains into five different groups on the basis of thepH and temperature optima of their proteases.On the basis of these results, two acid proteolytic activi-

ties with temperature optima of 30°C (PI,,) and 40°C (PI) andone neutral activity with 30°C as the optimal temperature(PI,) were distinguished. Different combinations of thesethree activities were reported to be present in the cell wallsof different S. cremoris strains (5). Many workers (8, 18, 19,21) have referred to these protease activities as being sepa-rate enzymes. This conclusion cannot be made as long as theproteases have not been purified and biochemically charac-terized. An attempt in that direction is described in thisstudy. Proteolytic systems, which contain proteins from thebacterial cell wall, which are specifically present in protease-producing strains, and in which all proteins responsible forthe degradation of the milk protein casein are present, wereisolated from different S. cremoris strains and partiallypurified. Several proteins of these proteolytic systems were

* Corresponding author.

identified with immunological methods, and the results led toanother classification of the proteolytic systems of differentS. cremoris strains.

MATERIALS AND METHODSBacterial strains. All S. cremoris strains were obtained

from The Netherlands Institute for Dairy Research, Ede,The Netherlands. The organisms were routinely maintainedin sterile 10% (wt/vol) reconstituted skimmed milk andstored at -20°C (12).

Prt- variants. Protease-negative (Prt-) variants of S.cremoris E8 and Wg2 were isolated as described previously(4). The Prt- variants were recognized by their inability tocoagulate skimmed milk within 24 h at 30°C.

Cultivation. Complex MRS medium (1) or chemicallydefined medium (13) with 0.8% sodium caseinate instead ofCasamino Acids was used for cultivation. The compositionsof both media were slightly adjusted for the isolation ofproteases with high CaCI2 concentrations (8 mM) and there-fore low phosphate concentrations (one-fifth the describedvalue in reference 1). Lactose (1%) was used as an energy

source, and incubation was always done overnight at 30°C.Isolation of the proteolytic systems. Proteolytic systems

were isolated from the cell wall as described previously (10)by incubation in Ca2+-free buffer. Cells were grown in 5liters of chemically defined medium (13) under pH control(pH 6.3) until all lactose was fermented (absorbance at 660nm, 2). Cells were harvested, washed in 500 ml of 50 mMsodium morpholineethanesulfonate (pH 6.3-10 mM CaCl,suspended in 500 ml of 50 mM potassium phosphate buffer(pH 7.3), and subsequently incubated for 30 min at 30°C. Theproteins of the proteolytic systems were released from thecell wall under these conditions (10). Cells were removed bycentrifugation (10 min, 5,500 x g), and the supernatantcontaining the proteins of the proteolytic system was usedfor analysis and further purification. The proteolytic systemof S. cremoris Wg2 was concentrated 100-fold by ultrafiltra-tion with Diaflo XM-50 and PM-10 filters (Amicon Corp.,Lexington, Mass.) and purified by elution in a Sephacryl S-300 column (length, 70 cm; diameter, 0.9 cm2) followed byelution in a DEAE-Sephacel column (length, 20 cm; diame-ter, 2.5 cm2) with a NaCl gradient of 0.1 to 0.3 M in 50 mMpotassium phosphate buffer (pH 7.0).Under these conditions, proteolytic activity was released

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from the ion-exchange material at a NaCl concentration of0.12 M. The fractions containing proteolytic activity werepooled and used for injection of rabbits (see below) and foranalysis by sodium dodecyl sulfate-10% polyacrylamide gelelectrophoresis (7). Two bands, representing molecularweights of ca. 165,000 and 180,000, appeared on the gel (datanot shown).

Partially purified proteolytic systems of S. cremoris E8,TR, and AM1 were supplied by The Netherlands Institutefor Dairy Research. Details of the purification procedurehave been described elsewhere (F. A. Exterkate and C. J. C.M. de Veer, submitted for publication). These include theisolation of crude cell wall proteolytic systems from milk-grown cells by three successive washings with Ca2+-freebuffer at pH 6.5 and 25°C, extraction of the freeze-driedcrude preparation with water, and gel filtration. The proteo-lytic system of S. cremoris ML1 was isolated directly fromthe culture medium by 60% (NH4)2SO4 precipitation.

Isolation of antibodies. Antibodies against the proteins ofthe proteolytic systems of S. cremoris Wg2, E8, TR, andAM1 were raised and isolated as described previously (2,20). The protein concentrations of the injected proteinsolutions were about 100 p.g/ml, and the concentrations ofthe isolated antibodies against the proteolytic systems of S.cremoris Wg2, E8, TR, and AM1 were 38, 60, 49, and 42mg/ml, respectively (in 20 mM barbital-hydrochloride buffer[pH 8.6]).CIE. Crossed immunoelectrophoresis (CIE) was carried

out as described previously (2, 20). The gels were run at 2.5V/cm for 200 min in the first dimension and at 1.5 V/cm for10 to 15 h in the second dimension. (In all figures, the anodeis at the top right-hand corner.) Various concentrations ofantigens and antibodies were used in all experiments. TheCIE gels showing all precipitation lines present were usedfor the figures.

Cell-free extracts. Cells grown overnight in 250 ml ofcomplex MRS medium containing 8 mM CaCl2 at 30°C werewashed in 50 mM sodium morpholineethanesulfonate (pH6.3)-10 mM CaCl2 and suspended in 2 ml of 50 mM potassi-um phosphate buffer (pH 7.3). Cells were disrupted bysonication (MSE Ltd., Crawley, Sussex, England) of the 2-ml cell suspension three times for 30 s each time at amaximal frequency and by cooling on an ice bath. Cell debriswas removed by centrifugation at a maximal speed for 15min in an Eppendorf model 5430 centrifuge. The cell-freeextracts were concentrated 20- to 50-fold with Minicon B15concentrators (15,000-molecular-weight cutoff; AmiconCorp.).

Determination of proteolytic activities. A 1% fluoresca-mine-labeled casein solution in 0.1 M borate-Na2CO3 buffer(pH 8.0) was used as the substrate (15). The fluorescence ofthe solution after precipitation of proteins with 1.63% tri-chloroacetic acid in sodium acetate (pH 4.0) was measuredin a Perkin-Elmer model 204 fluorescence spectrophotome-ter with an excitation wavelength of 395 nm and an emissionwavelength of 475 nm.

Standard assays were performed at 30°C for 60 min.Zymogram staining of proteolytic activities. The detection

of proteolytic activity on CIE gels was performed by swell-ing gels containing immunoprecipitates in a sodium caseinatesolution (0.1%; pH 7.0). The gels were incubated overnightat 30°C and stained with Coomassie brilliant blue proteinstain (0.5% in 45% methanol-10% acetic acid).

In some cases, a slightly different technique was used.Gels swollen in a 1% sodium caseinate solution were cov-ered with a highly concentrated CaC12 solution (1 M). This

resulted in the formation of a thick white precipitate in thegels by calcium caseinate micelles. The gels were incubatedovernight at 30°C and checked for clearing of the whiteprecipitate caused by casein hydrolysis.

Protein determination. Protein was determined by themethod of Lowry et al. (9) with bovine serum albumin as thestandard.

RESULTS

Proteolytic system of S. cremoris Wg2. CIE of the purifiedproteins of the proteolytic system of S. cremoris Wg2 withtheir specific antibodies showed two major precipitationlines in the center of the gel (Fig. 1A). These two proteinsseem to have similar molecular weights, as the electropho-retic mobilities of both proteins in the first dimension wereapproximately the same. This result is in agreement with theresults obtained with sodium dodecyl sulfate-polyacrylamidegel electrophoresis of the proteolytic system, which yieldedtwo protein bands with similar molecular weights (data notshown). A small, weak precipitation line which was found atthe right-hand side of the gel was probably not a componentof the proteolytic system (see below).CIE of the proteolytic systems of strains E8, TR, and AM1

against Wg2 antibodies. CIE of the purified proteolyticsystems of other S. cremoris strains with antibodies used inFig. 1A against the proteolytic system of strain Wg2 revealedsimilarities in the proteolytic systems of the different strains.Figures 1B, C, and D show the CIE patterns of the proteolyt-ic systems of the different strains. The preteolytic system ofstrain E8 (Fig. 1B) had only one major precipitation line, andthose of strains TR (Fig. 1C) and AM1 (Fig. 1D) both hadtwo precipitation lines. The two lines of the CIE pattern ofstrain AM1 fused at a certain point, indicating that althoughthe proteins were different, they had some component in

FIG. 1. CIE of the purified proteolytic systems of S. cremorisWg2 (A), S. cremoris E8 (B), S. cremoris TR (C), and S. cremorisAM1 (D). Protein solution (10 ,ul of a 230-,ug/ml solution) wasapplied to each gel. The second dimension contained 30 ,ul of a 38-mg/ml solution of antibody against the proteolytic system of strainWg2.

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common. The CIE pattern of strain TR looked almostidentical to that of strain Wg2."Tandem" CIE. One precipitation line seemed to be

present in all CIE patterns shown in Fig. 1. This suggeststhat the proteolytic systems of all strains have one protein incommon. To determine if this protein was indeed identical inall samples, we used a slightly different CIE technique, so-called tandem CIE (Fig. 2C). The proteolytic systems ofstrains Wg2 and E8 were applied on the same gel in adjacentsample holes, and CIE was performed against strain Wg2antibody in the second dimension. For comparison, the CIEpatterns of strain Wg2 alone (Fig. 2A = Fig. 1A), showingtwo major precipitation lines, and of strain E8 alone (Fig. 2B= Fig. 1B), showing one major precipitation line, are pre-sented again. In the tandem CIE pattern (Fig. 2C), one of thetwo lines of strain Wg2 fused with the only line of strain E8,indicating that these proteins are identical. Similar resultswere obtained with other combinations of proteolytic sys-tems and tandem CIE (data not shown). This commonprotein will be referred to as "protein A."Zymogram staining of proteolytic activity. To make sure

that protein A was indeed an enzyme with proteolyticactivity, we used zymogram techniques to identify enzymeactivity on the CIE gels. The techniques were based on the

FIG. 2. Tandem CIE (C) of the purified proteolytic systems of S.

cremoris E8 (applied to the right-hand well; B) and S. cremoris Wg2(applied to the left-hand well; A). The protein and antibody concen-trations used were the same as in Fig. 1.

FIG. 3. Zymogram staining of protease activity in the CIE pat-tern of the purified proteolytic system of S. cremoris E8 withantibody against the proteolytic system of strain Wg2. The gel wasswollen in a 0.1% sodium caseinate solution, incubated overnight at30°C, and stained with Coomassie brilliant blue.

caseinolytic properties of the proteins tested. Figure 3 showsa CIE gel of the strain E8 proteolytic system with Wg2antibody containing one precipitation line of protein A. Thegel was swollen in a sodium caseinate solution, incubatedovernight at 30°C, and subsequently stained for remainingprotein. The area around the protein A precipitation line wasless stained than the rest of the gel, indicating that caseinhydrolysis had taken place and that protein A was a proteo-lytic enzyme.

Proteolytic systems of other S. cremoris strains. The proteo-lytic systems of strains E8, TR, and AM1 were also used toraise specific antibodies. These antibodies were used in CIE

FIG. 4. CIE of the purified proteolytic systems of S. cremorisWg2 (A), S. cremoris E8 (B), S. cremoris TR (C), and S. cremorisAM1 (D) with their specific antibodies. The amount of proteinapplied to each gel was the same as in Fig. 1. Different antibodysolutions (30 ,ul; see text) were used in the second dimension of eachgel.

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experiments to see if other proteases besides protein A werepresent in the samples. Figure 4 shows the CIE pattern ofeach proteolytic system with its specific antibodies. The CIEpattern of strain Wg2 (Fig. 1) is shown here again forcomparison (Fig. 4A). The proteolytic system of strain E8(Fig. 4B) had a second protein besides protein A. Thisprecipitation line was positioned more to the left in the gel,suggesting a higher-molecular-weight protein. The CIE pat-terns of strains TR and AM1 (Fig. 4C and D, respectively)were both composed of at least four precipitation lines. Inthe CIE pattern of strain AM1 (Fig. 4D), one line fused withthe line of protein A, indicating that the proteins have acomponent in common. This protein is hereby termed "pro-tein A'."CIE of cell-free extracts of Prt- mutants. The zymogram

staining of proteolytic activity described above was ratherinsensitive. Only precipitation lines with high proteolyticactivity could be stained with this method. This was the casefor only protein A.To show that the newly revealed proteins in Fig. 4 were

part of the proteolytic systems, we used Prt- mutants ascontrols. These mutants have spontaneously lost geneticinformation coding for the proteolytic system (4, 11) andwere isolated from pure cultures of several S. cremorisstrains (4, 8, 16).

Cell-free extracts of Prt- mutants of S. cremoris Wg2 andE8 were prepared and used for CIE experiments (Fig. 5). InFig. 5A and B, the CIE pattern of the cell-free extract of thePrt- mutant of strain Wg2 against Wg2 antibodies is com-pared with that of the wild-type strain. The two proteinsfound in the cell-free extract of the wild-type strain werefound in proportionately the same amounts as in the purifiedproteolytic system (cf. with Fig. 1), indicating that noproteins formed by the cells were lost during the purificationprocedure. These two precipitates were absent in the CIE

FIG. 5. CIE of cell-free extracts of the Prt+ variant (A and C)and the Prt- variant (B and D) of S. cremoris Wg2 (A and B) and S.cremoris E8 (C and D). Cell-free extracts were used as described inthe text. Antibodies were used against the strain Wg2 proteolyticsystem (A and B) and the strain E8 proteolytic system (C and D).Proteins are labeled in panels A and C.

pattern of the Prt mutant. The only common precipitatewas the weak line at the right-hand side of the gel, indicatingthat this protein is not part of the proteolytic system. Weconclude that besides protein A, another protein in S.cremoris Wg2 is part of the proteolytic system. This proteinis hereby termed "protein B."The cell-free extract of S. cremoris E8 also had the same

CIE pattern as the isolated proteolytic system of this strain(Fig. SC and D), and again both precipitation lines disap-peared in the CIE pattern of the Prt- mutant. These resultsindicate that the proteolytic system of strain E8 is also madeup of two proteins, one of which is protein A. The secondprotein is clearly different from protein B, as it has a lowerelectrophoretic mobility. We have termed this protein "pro-tein C."

Identification of proteins of the proteolytic systems of strainsTR and AMI. Prt- mutants of S. cremoris TR and AM1 havenot yet been isolated. Tandem CIE experiments were per-formed with the proteolytic systems of these strains to checkfor similarities with the proteolytic systems of strains E8 andWg2. In Fig. 6A, C, and E, the CIE patterns of strains TRand Wg2 are compared. Tandem CIE showed that bothproteins A and B were present in both strains. The proteolyt-ic systems of strains E8 and AM1 also shared more than oneprotein (Fig. 6B, D, and F). Proteins A and C were present inboth strains, as both precipitation lines fused in the tandemCIE pattern. Protein A' appeared to be specific for S.cremoris AMI. Similar experiments revealed that one of theprecipitation lines in the tandem CIE pattern of strain TRcorresponded to protein C and that one in the tandem CIEpattern of strain AM1 (probably) corresponded to protein B(data not shown).

Classification of S. cremoris strains. The CIE studiespresented above showed that at least four different proteincomponents can be found in the proteolytic systems of S.cremoris strains. These components are found in differentcombinations in the different strains tested (Table 1). As thesame CIE patterns were found in cell-free extracts as inpurified proteolytic systems (Fig. 5), the purified systemsappear to contain all the protease components formed. Onthe basis of these results, the S. cremoris strains can beclassified into four groups.

Regulation of protease synthesis. When protease-positive(Prt+) S. cremoris Z8, AM1, KH, and TR were grown oncomplex MRS medium with 8 mM Ca2 , proteins of theproteolytic systems could not be detected with CIE, butsuch proteolytic activity was clearly present in these strainsduring growth on chemically defined medium with caseinand, of course. during growth on milk. The results indicatethat the synthesis of proteases in these strains is subject tometabolic regulation.

Proteolytic system of S. cremoris ML1. S. cremoris ML1,which is reported to have no cell-wall-associated proteolyticactivity and is thought to excrete all proteases into themedium (5, 19), produced the same proteolytic enzymes asstrains Wg2, HP, and C13. The proteins of this system wereisolated from the culture supernatant even when 10 mMCaCl2 was present in the growth medium, although someproteins could still be detected in cell extracts of washedcells (data not shown).

DISCUSSIONThe proteolytic system of S. cremoris is important during

the ripening of cheese (8, 14, 16). The flavor produced canvary from one strain to the other. Exterkate (5) distinguished

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distributed(Table 1).nalyze theof S. cre-fundamen-tion. Fourdentified in,Table 1).en found ininterestingIt has been

three different proteolytic activities that were 4among S. cremoris strains in various combinations

In this paper, an attempt has been made to aprotein compositions of the proteolytic systemsmoris strains with immunological methods. Thistally different approach led to a new classificacomponents of the proteolytic systems could be isfour different combinations in the strains tested (One component (protein A') has specifically bec

the proteolytic system of S. cremoris AML. It isthat this strain has unusual proteolytic activities. Ireported to hydrolyze effectively ox-casein as i

casein, but only 1-casein is degraded as an asource by most strains grown on milk (F. A.unpublished data).A general problem in the cheese industry is the

of bitter flavor in the end product. This flavor isso-called bitter peptides (23), which contain mosphobic amino acid residues (17-24). Some specsmoris strains are reported to be responsible for the

FIG. 6. Tandem CIE of the proteolytic systems of S. cremorisWg2 and TR (A, C, and E) and S. cremoris E8 and AM1 (B, D, andF). CIE of the proteolytic systems of S. cremoris Wg2 (A) and TR(C) and tandem CIE of both systems (E) with antibodies against theproteolytic system of strain Wg2. CIE of the proteolytic systems ofS. cremoris E8 (B) and AM1 (D) and tandem CIE of both systems(F) with antibodies against the proteolytic system of strain E8. The

protein concentrations used were the same as in Fig. 1 through 4.Proteins are labeled in all panels.

TABLE 1. Compositions of the proteolytic systems of different S.cremoris strains

S. cremoris strain Components of the Proteolyticproteolytic system activities"

Wg2, HP, C13, and ML1 A and B P1 and Pi,E8 A and C PITR, FD27, and US3 A, B, and C Pi, PI,, and PillAM1, SK11 A, (B), C, and A' Pill

" From reference 5.

well as 1- of these peptides in cheese (5, 22, 24). Exterkate suggestedmino acid (5) that the presence of proteolytic activity Pi, (pH optimum,Exterkate, 7.0; temperature optimum, 30°C) is correlated with bitter-

peptide production. S. cremoris strains which produce onlyformation PI or PI, and strain ML1 are known to be non-bitter-peptidecaused by producers. In this paper, we demonstrated that S. cremorisstly hydro- ML1 contains the same proteolytic system as the bitter-ific S. cre- peptide-producing strains Wg2, HP, and C13. As this strainformation excretes most of its proteases into the surrounding medium,

the proteases do not remain in the curd and do not contributeto bitter-peptide formation during cheese ripening.Which protein of the proteolytic system is the cause of

bitter-peptide production is difficult to say at this moment.Proteins A and C are certainly not responsible. Protein A ispresent in all strains, and protein C is present in the non-bitter-peptide-producing strains E8 and AML. The onlyremaining candidate is protein B, which is present in thebitter-peptide-producing strains Wg2 and TR. The situationfor strain AM1 (non-bitter-peptide producing) is still notclear. It is possible that protein B is present in smalleramounts than in strains Wg2 and TR, causing less bitter-peptide formation. A further purification of the differentcomponents of the proteolytic systems or a further analysiswith more specific antibodies (for instance, monoclonalantibodies) is needed to solve these problems.The regulation of proteolytic activity has been reported

previously in S. cremoris (5, 6, 8). Several strains show noproteolysis or reduced proteolysis under certain cultureconditions. In this paper, we showed that during growth oncomplex MRS medium in the presence of Ca- , some strainsdo not synthesize their proteolytic systems. A possibleexplanation for this is the regulation of the synthesis of cellwall proteases by amino acids or peptides at the level ofmRNA translation, as was suggested for S. cremoris AM1(6). It is striking that two closely related strains, AM1 andSK11, behave differently in this sense, as S. cremoris SK11produces its proteolytic system under all conditions, where-as the synthesis of the proteolytic system in strain AM1depends upon the composition of the growth medium (datanot shown). Which factor in the medium has an effect onprotease synthesis or activity or both and how the regulationworks are still questions that remain to be answered.

ACKNOWLEDGMENTSThis study was supported by The Netherlands Programme Com-

mittee on Biotechnology.We thank R. Otto for discussions and valuable suggestions during

the investigations.

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