Characterisation of a Peptidase from Lactococcus lactis ssp* cremoris HP that Hydrolyses Di- and Tripeptides Containing Proline or Hydrophobic Residues as the Aminoterminal Amino Acid

System. App!. Microbial. 14, 317-323 (1991) © Gustav Fischer Verlag, StuttgartlNew York

Characterisation of a Peptidase from Lactococcus lactis ssp. cremons HP that Hydrolyses Di- and Tripeptides Containing Proline or Hydrophobic Residues as the Aminoterminal Amino Acid RONALD BAANKREIS and FRED A. EXTERKA TE"· Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), 6710 BA Ede, The Netherlands

Received February 15, 1991

Summary An intracellular peptidase, showing highest catalytic activity towards di- and tripeptides containing proline and to a lesser extent other hydrophobic residues as the amino-terminal amino acid, was purified from cell-free extracts of Lactococcus lactis ssp. cremoris HP. On SDS-PAGE the enzyme exhibited a molecular mass of 50 kDa. In HPLC gel filtration experiments, an apparent molecular mass of approximately 110 kDa was observed. The activity of the enzyme was inhibited by EDTA, dithiothreirol and some metal ions, but was not affected by PMSF, aprotinin or pepstatin. After inhibition with EDT A the activity could be restored by Co+ + and Nln++. The optima for pH, temperature and NaCl concentration are 8.5, 37°C and 100 mM respectiyely. The Michaelis constant (Km) and Vmax for several proline-containing di- and tripeptides were determined.

Key words: Lactococcus lactis ssp. cremoris - Peptidase - Enzyme purification - Cheese ripening

Introduction

Lactococcus lac tis ssp. cremoris is an important organism present in mixed-strain starter cultures used by the Dutch dairy industry in cheese production. The proteolytic activities of this starter organism are essential to the process of cheese ripening (Thomas and Pritchard, 1987). The pep tides and amino acids liberated from the milk protein casein by the concerted action of rennet and starter proteolytic enzymes contribute to flavour development during cheese ripening, either directly or as precursors. Therefore, if full insight in the process of cheese ripening is to be gained, a study of these proteolytic enzymes is necessary. Lactococci possess an intricate system of endo- and exopeptidases to provide them with those amino acids that are essential for growth (Thomas and Pritchard, 1987; Cliffe and Law, 1979; Exterkate, 1984). The system has been reported to consist of a cell envelope bound proteinase and a number of intra- and extracellular en do- and exopeptidases of varying specificity (Kolstad and Law, 1985; Law, 1979). Peptide bonds in which proline is involved are less sus" Corresponding author

ceptible to the action of aminopeptidases that remove most of the other amino acid residues. A number of proline-specific peptidases is known (see Tabe 1). Several of these enzymes occur in Lactococcus lactis ssp. cremoris. Among these are a reportedly extracellular dipeptidyl peptidase IV (Casey and Meyer, 1985; Meyer and Jordi, 1987; Kiefer-Partsch et al., 1989; Booth et al., 1990) and an intracellular prolidase (Kaminogawa et al., 1984). Hydrolysis of proline-p-nitroanilide as reported by several authors (Mou et al., 1975; Kamaly and Marth, 1988) and of Pro-X type dipeptides (Kaminogawa et al., 1984) indicates that a prolinase (imino-dipeptidase) activity could also be present in this organism. Since casein is a prolinerich protein, peptidases that are able to degrade prolinecontaining peptides can playa vital role in the process of proteolysis in ripening cheese. In th~s paper we describe the purification and partial characterisation of a peptidase that, although not specific for proline is able to hydrolyse di- and tripeptides that contain proline, as the amino-terminal amino acid. We have, for the reasons outlined above, focused our attention on the proline-releasing capabilities of the enzyme.

318

R. Baankreis and F. A. Exterkate

Table 1. Summary of known proline-specific amino- and endo peptidases and their specificities towards peptide substrates. The peptide bond to be cleaved is indicated (--) Peptidase

Natural substrate

prolinase prolidase proline endopeptidase proline iminopeptidase aminopeptidase P dipeptidyl peptidase IV

Pro--Y X--Pro -X-Pro--YPro-- YX--Pro-YX-Pro--Y-

The overall efficiency of the purification procedure up to this point was about 30%, while a ISO-fold purification was achieved. The activity of this preparation was about 166 ~lmoles Pro-Gly-Gly hydrolysed per mg protein per hour (at pH 8.5, 2mM Pro-Gly-Gly, 35°C and 100 mM NaCI present in the buffer). Purified peptidase was obtained from this fraction by preparative PAGE or HPLC gel filtration. The purification procedure is summarized in Table 2. Table 2. Summary of the purification procedure for peptidase from Lactococcus lactis ssp. cremoris HP. Specific activity is expressed as ~mol substrate hydrolysed per hour per mg protein. (2mM Pro-Gly-Gly, 100 mM NaCi, 50 mM imidazole pH 8.0, 35°C). n.d. = not determined

Material and Methods 01-ganisms and growth. Lactococcus lactis ssp. cremoris HP was stored and grown in reconstituted non-fat milk as described earlier (Exterkate, 1984). Cells were grown overnight at 30°e. For measuring peptidase activity towards N-terminal prolinecontaining peptides (iminopeptidase-activity) Lactococcus lactis ssp. cremoris strains AMI, SKll, ML1, SCR, E8, FD27, TR and HP, Lactococcus lactis ssp. lactis strain MG 1820 (plasmid free), Lactobacillus helveticus strain 37H and L. delbruckii ssp. bulgaricus strain 1B were grown in RFP medium (Exterkate, 1979) containing 1 % casiton as the nitrogen source. Enzyme purification. Cells from an overnight grown milk culture were harvested, washed in imidazole buffer (25 mM, pH 6.3) and resuspended to an 0.0. (650 nm) of 40 in 25 mM imidazole buffer pH 6.3, containing 100 mM NaCI and 550 mM sucrose. The suspension was supplemented with lysozyme (5 mg/ml) and incubated at 25 °C f~;r 90 min. This procedure converted the cells into spheroplasts as revealed by electron microscopy (not shown). The suspension was centrifuged and the pellet was resuspended in 50 mM imidazole buffer pH 6.3 containing 100 mM NaC!. Completely lysed spheroplasts were obtained by sonication (5x30 s); the debris was removed by centrifugation (20 min, 15000 g). The resulting cell-free extract, containing mainly cytosolic proteins, contained all the Pro-Gly-Gly hydrolysing activity. When performing fractionation experiments the cell wall fraction (the supernatant obtained after centrifugation of the intact spheroplasts), the membrane fraction (the pellet obtained after lysis of the spheroplasts, centrifugation and resuspension) and the cytosolic fraction (the supernatant obtained after centrifugation of the lysed spheroplasts) were dialysed overnight against water. The resulting preparations were used for activity measurements. The cytosolic fraction was further fractionated by ammoniumsulphate precipitation, with the peptidase activity appearing in the 28-33% (w/v) fraction. During this and all subsequent steps of the purification procedure the preparation was kept at 4°e. The active fraction was dialysed against water and applied to a DEAE-Sepharose CL-6B (Pharmacia) ion-exchange column equilibrated in 25 mM imidazole pH 6.3. Elution was performed by stepwise increasing the NaCi concentration, with the peptidase activity eluting from the column at a NaCI concentration of 250-275 mM. The active fractions from the DEAE-column were dialysed against water, solid ammonium sulphate was added to a concentration of 1.5 mM and the fractions were applied to a Fractogel, TSK-butyl-650 (S) (Merck) hydrophobic interaction chromatography column. The bound protein was eluted by stepwise lowering the ammoniuJ:lL. sulphate concentration, with the peptidase activity eluting from the column between 1100 and 1050 mM ammonium sulphate.

Purification stage

Yield (%)

Protein (mg)

cytosolic fraction

100% (by def.)

906

1.1

ammonium sulphate fraction (28-33%)

95%

452

2.1

DEAE-Sepharose CL-6B fraction (250-275 mM)

75%

67

11.4

TSK-Butyl fraction (1100-1050 mM)

30%

1.9

3%

n.d.

HPLC gel filtration fraction (TSK3000 + TSK2000)

Spec. activity

166 n.d.

Measurement of enzyme activity and protein concentration. The iminopeptidase (proline-liberating) activity of the peptidase was determined using the method of Troll and Lindsley (1955) modified for use with proline iminopeptidase (Sarid et aI., 1959; Sarid et al. 1962). Enzyme activity was routinely measured by incubating samples with Pro-Gly-Gly (2 mM in 25 mM imidazole pH 8.5 + 100 mM NaCI) at 35°C for 30 min. The reaction was stopped by adding 2.5 vol. glacial acetic acid and subsequently 2.5 vol. ninhydrine reagent were added. The samples were heated for 30 min at 100°C and the red colour, which develops if free proline is present in the sample, was measured at 480 nm. 2 mM Pro-Gly-Gly was used because this concentration sufficed for measuring reasonable enzyme activities throughout the purification process. This method was also used to determine the Krn and Vrnax for the hydrolysis of several proline-containing substrates by the peptidase. Hydrolysis of peptides not containing proline as the aminoterminal amino acid was assayed by incubating 2 mM substrate solutions under the conditions described above. Samples were analysed using thin-layer chromatography on Merck HPTLC plates, (Kieselgel 40 F254 ). The plates were developed using a 14: 4: 14: 8 mixture of n-butanol, acetic acid, acetone and water. The resulting peptide and amino acid patterns were visualised by spraying the developed and dried plates with ninhydrin solution (0.4% ninhydrin in n-butanol: acetic acid 96: 4) and allowing the color to develop at 70°C for 15 min. Inhibitors and other compounds were tested by a 30 min preincubation at 30°C prior to addition of the substrate. For the determination of the heat stability of the enzyme samples were incubated at different temperatures for 30 min and subsequently cooled on ice for at least 15 min. The remaining enzyme activity was then measured at 35 °C as described above. Lactate and glycerol dehydrogenase activity in the various cell fractions were measured by incubating the fractions in sodium

Lactococcus lactis ssp. cremoris Iminopeptidase Activity

phosphate buffer (50 mM, pH 7.0). NADH (final concentration 100 !tg/ml) and substrate (pyruvate and dihydroxyacetone respectively, final concentration 10 mM) were added and the decrease in absorbance at 340 nm (35°C) was followed. Dipeptidyl peptidase IV activity was measured by following the hydrolysis.of the substrate Ala-Pro-p-nitroanilide (Bachem, Switzerland). The release of nitroaniline (pH 8.0, 35°C) was measured at 410 nm. Protein was estimated by the method of Bradford (1976) using crystalline bovine serum albumin (fraction V; BDH, Poole, England) as the standard. SDS-preparative PAGE, IEF and HPLC gel filtration. SDSPAGE was performed using the Bio-Rad Mini-Protean cell and the Laemmli buffer-system (Laemli, 1970) with 12.5% polyacrylamide gels. Preparative PAGE was performed using the LKB 2117 Multiphor system with 8% polyacrylamide gels and a 50 mM imidazole buffer adjusted to pH 6.5 or 7.0. Isoelectric focusing was performed using precast LKB Ampholine PAG plates (pH range 4.0-5.0) and the LKB Ultrophor unit. HPLC gel filtration was performed using a Waters M510 HPLC pump (flow 0.8 mVmin), two coupled columns (Varian TSK3000 and TSK2000, 300 x 7.5 mm), a Waters Lambda Max 481 detector (280 nm) and a Gilson model 2311401 injector. The eluent buffer used was 0.25 mM potassium phosphate pH 7.25. For determination of the molecular mass of the peptidase the column was calibrated with HPLC molecular weight standards (Molecular Weight Standard Kit HPLC, United States Biochemical Corporation).

319

Table 3. Aminopeptidase activities in cytosolic, membrane and cell wall fractions of Lactococcus lactis ssp. cremoris HP. Activities are defined as mmol substrate hydrolysed per hour. Absence of detectable activity is denoted by Enzyme

Cell wall

Membrane Cytosol

lactate dehydrogenase glycerol dehydrogenase peptidase dipeptidyl peptidase IV

0.12

0.83 0.07 0.55 4.98

0.66

17.7 3.2 2.6 88.7

PAGE a single band was observed with an apparent molecular mass of approximately 50 kDa (see Fig. 1). After isoelectric focusing of a HPLC gel filtration fraction a single band was observed (data not shown) corresponding to an isoelectric point of 4.55. The enzyme seems to be a metallopeptidase with Co++ or Mn+ + as the essential ion since after inhibition of the enzymatic activity with 1 mM EDTA (see Table 4) the enzyme can be restored to full activity by addition of CoCI 2 or MnCI 2 to a final concentration of 1.5 mM. No such reactivation was observed upon addition of Zn+ +- Mg++- Ca++- Ni++- Cu++ or Fe ++ -ions. " "

Results

92.5 66.2

Location of the enzyme Hydrolysis of Pro-Gly-Gly could not be detected with whole cells. In order to further establish the location of the enzyme, cells were fractionated into a solubilized cell wall fraction, a membrane fraction and a fraction containing mainly cytosolic enzymes. As judged by the distribution of lactate ,and glycerol dehydrogenase between the different fractions (> 90% of the activity of both enzymes appeared in the supernatant only after lysis of the spheroplasts, the remainder occurring in the pellet, see Table 3) no significant lysis of the spheroplasts occurred during the preparation of the cell wall fraction, whereas almost all the spheroplasts were lysed after resuspending and sonication. These results were confirmed using electron microscopy (results not shown). The cell wall fraction contained no lactate dehydrogenase, glycerol dehydrogenase or Pro-Gly-Gly hydrolysing activity. The resuspended membrane fraction exhibited only very low activies for these enzymes. The peptidase activity cofractionated with lactate dehydrogenase and glycerol dehydrogenase into the cytosolic fraction indicating an intracellular location of the enzyme (Table 3). Dipeptidyl peptidase IV - although previously reported to be an extracellular enzyme (Kiefer-Partsch et aI., 1989) - mainly fractionated into the cytosolic fraction.

Molecular characteristics Judged by HPLC gel filtI;;ltion, yielding one single symmetrical peak (data not shown), the peptidase has an approximate relative molecular mass of 110 kD.a. On SDS-

50

45.0 31.0 21.5 14.4

00 Fig. 1. SDS-PAGE of purified peptidase. Lane 2: molecular weight markers (Bio-Rad Low Molecular Weight). Lane 1: HPLC gel filtration fraction.

Factors influencing enzyme activity The influence of various reagents on enzyme activity is shown in Table 4. The results indicate that a metal ion is essential for catalytic activity. Furthermore, since neither PMSF, pepstatin nor aprotinin seem to inhibit peptidase activitY, the enzyme probably is neither a serine peptidase nor an aspartic peptidase. The results obtained with dithiothreitol suggest that at least one disulfide bridge plays an important role in maintaining an active conformation of the enzyme. On the

320

R. Baankreis and F. A. Exterkate

Table 4. Effect of various compounds on peptidase activity. Activity is expressed as percentage relative to blank Compound used

Concentration used (mM)

Relative activity (%)

p-chloro-mercuribenzoate EDTA Phenanthroline Citrate Iodoacetic acid Iodoacetamide PMSF N-Ethylmaleimide Co++ Pepstatin Aprotinin Dithiothreitol

1 5 5

190 5

5

11 110 110 95 120 20 100 100 5

1 1 1 1 5 1 1 1

5

other hand it seems clear that other thiol groups in the molecule need to be maintained in the reduced state because addition of some thiogroup specific reagents does not inhibit or - as was the case with PCMB - even seems to stimulate enzyme activity. The optimum pH and temperature of the enzyme are near 8.5 and 3JOC respectively (Figs. 2, 3). The enzyme activity was stable when preincubated for 30 min at temperatures of up to 38 DC. At pH 8.5 and 35 DC the enzyme is maximally active towards Pro-Gy-Gly when 100 mM NaCl is present in the incubation mixture (Fig. 4). Therefore, under the conditions present in ripening Gouda type cheeses (4-4.5% NaC!, pH 5.0-5.4 and 13 DC) the enzyme most probably exhibits low activities, provided lysis of the cells and release of the peptidase has occurred. The specificity of the enzyme is shown in Table 5. The activities measured with the various substrates are shown relative to the activity of the peptidase towards Pro-GlyGly at the same concentration (for the pep tides containing· proline as the amino-terminal residue) or as + (hydrolysis) and - (no hydrolysis) for the other peptides (due to the low solubility of some peptides a concentration of 1 mM was used in this experiment). When both proline-containing and other pep tides were tested on TLC, the former almost invariably were hydrolysed faster. The Km and V max values for several proline-containing peptide substrates were measured to gain additional insight in the specificity of the enzyme. The results are shown in Table 6. These results seem to suggest that Km values are more a function of the residues in the second and third position of the substrate peptide rather than of chain length (di- vs. tripeptides). It is clear however that the V max values for tripeptides generally are higher than for dipeptides. Unfortunately we were unable to test this hypothesis with several peptides that might have shed some light on this subject (e. g. Pro-Val, Pro-Tyr-Gly) because it was impossible to dissolve said peptides to the concentrations necessary for reliable measurements using this method. The enzyme hydrolysed almost all di- and tripeptides containing proline as the amino-terminal, but did not

110 reI. act. (%) 100 90 80 70 60 50 40 30 20 10 O~~--~~--~~--~~--~~

5.005.506.006.507.007.508.008.509.009.50 pH

Fig. 2. Influence of pH on peptidase activity towards Pro-GlyGly. Activity is expressed relative to the optimal activity (at pH = 8.0). The reaction conditions were: 35°C, 2 mM Pro-Gly-Gly, 100 mM NaCI in 50 mM imidazole pH 8.0. •

Stability

T

Activity

1.1 0 rei. activity 0.88 0.66 0.44 0.22

,.-/

0.00 10

20

/

/

30

/

40

50

60

Fig. 3. Influence of temperature on the stability and hence activity of peptidase activity. (50 mM imidazole pH = 8.0,2 mM ProGly-Gly and 100 mM NaC!). Activities are expressed relative to the optimal activity (at 35 DC) for the optimum curve and relative to a non-preequilibrated sample for the stability curve. 120 A (% A max) 90 60 30

oL------'---......L..-...:==--......."'-----... o 400 800 1200 1600 2000 NaCI (mM)

Fig. 4. Influence of NaCI concentration on peptidase activity towards Pro-Gly-Gly. Activities are expressed relative to the activity at the optimal NaCI concentration (100 mM). The reaction mixture contained: 50 mM imidazole pH 8.0, 2 mM Pro-Gly-Gly and the indicated amount of NaC!. The incubation temperature was 35°C.

Lactococcus lactis ssp. cremoris Iminopeptidase Activity Table 5. Peptidase activity towards various peptides. Activities are expressed relative to the activity towards Pro-Gly-Gly (proline substrates) or as + (degraded as observed by TLC) and - (not degraded). All substrate , concentrations (except (Pro)n) 1 mM

321

Substrates

Relative activity

Substrates

Relative activity

Pro-Gly Pro-Gly-Gly Pro-Gly-Phe Pro-Gly-Lys-Ala-Arg Pro-Glu Pw-Glu-Thr Pw-Glu-Pro-Ghl-Thr Pro-Phe Pro-Phe-Asp Pro-Phe-Lys Pro-Phe-Gly-Lys Pw-Tyr Pw- Tyr-Ala Pw-Met Leu-Leu-Leu Leu-Leu

0.4 1.0 1.1

Pro-Val-Gly Pro-His-Gly Pro-Lys Pro-Leu (Pro)n (n = 2,3,4,10 4 ) Proline-p-nitroanilide Ala-Glu Glu-Gly Glu-Ala-Ala Phe-Asp Lys-Gly-Gly Ala-Pro-Ala Asp-Phe Phe-Pro Gly-Gly-Glu-Ala Gly-Leu

1.4 0.2 0.5 0.4

o

0.08 0.3

o

0.4 0.6 1.0 0.2 0.8 1.6 0.3

+ +

o o

+/+/-

+

cleave X-Pro bonds (and therefore failed to hydrolyse ProPro and Pro-Pro-Pro). The enzyme was also able to hydrolyse di- and tripeptides with Leu or Ala as the aminoterminal residue (albeit more slowly than prolinecontaining substrates) and therefore is not proline-specific. The most striking feature of the enzyme is its inability to hydrolyse peptides longer than four residues. Even tetrapeptides are hydrolysed with vastly diminished efficiency compared to di- and tripeptides, the enzyme's preferred substrates.

The results are shown in Table 7. All Lactococcus strains tested exhibit peptidase activity but neither Lactobacillus helveticus 37H nor L. delbriickii ssp. bulgaricus IB hydrolysed Pro-Gly-Gly tripeptide. The peptidase activities observed varied strongly between various Lactococcus strains. Whether the observed correlation betWeen cell wall proteinase type and peptidase activity is significant and what may be the cause of such a correlation remains unclear.

Table 6. Michaelis constants (Km) and Vmax for the hydrolysis of some pwline-containing di- and tripeptides by the iminopeptidase-like activity from Lactococcus lactis ssp. cremoris HP (50 mM imidazole, pH 8.0, 35 DC, 100 mM NaCLpresent in the reaction mixture

Table 7. Activity of peptidase towards Pw-Gly-Gly in various strains of lactic acid bacteria. Activities shown are relative to the activities measured in strain HP, grown in milk. Strains are ordered according to cell envelope pwteinase type as described by Viss er et al. (26). For strain HP the specific activity did not seem to differ significantly when the organism was grown in either milk or RFP medium

Substrate used

Km values (mM)

V max values (Il moV(h.mg))

Pro-Gly-Gly Pro-Val-Gly Pro-Glu-Thr Pro-Lys Pro-Met Pro-Tyr

6.7 2.6 >125 2.3 20.3 13.8

740 634

Organism

Strain

Cell wall proteinase type

Proline tripeptidase activity

Lactococcus lactis ssp. cremoris

AMI SKll

III III

3.19 3.48

MLl SCR

I, excreted ?

2.20 0.33

E8 FD27

(III + I) I+(III)

1.61 0.5

77

35 18 114

TR HP HP (milk)

1.09 0.85 1.00

Lactococcus lactis ssp. lac!is

MG 1820

2.18

Lactobacillus helveticus

37H

0

Occurrence of peptidase activity in lactic acid bacteria Several strains of Lactococcus lactis ssp. cremoris and a Lactococcus lactis ssp. lactis strain, and also other lactic acid bacteria were grown overnight at 30 DC in RFP medium containing bactocasiton as the amino acid source. Subsequently lysozyme-treated, disrupted cell suspensions were tested for peptidase activity. RFP medium was chosen because of the inability of some of the tested strains to grow in un supplemented milk. 22 System. AppJ. Microbiol. Vol. 14/4

Lactobacillus del1B briickii ssp. bulgaricus

0

322

R. Baankreis and F. A. Exterkate

Discussion The proline iminopeptidase-like (E.C. 3.4.11.5) activity from Lactococcus lactis ssp. cremoris HP differs in a number of respects from other proline peptidases purified and characterised so far (Sarid et aI., 1959; Sarid et aI., 1962). The most obvious differences are the preference for :di- and tripeptides shown by the lactococcal enzyme and Ithe ability to hydrolyse peptides containing amino-terminal amino acids other than proline (see Table 5). The enzyme, however does not hydrolyse proline-p-nitroanilide. The low activities of intact cells observed with this substrate (Exterkate, 1984) therefore should be attributed to another enzyme. Various features of the enzyme e. g. the pH-, temperature and inhibitor influenc~ on activity and the dependency on Co++ and Mn+ + ions, show the enzyme to be a typical lactococcal metallopeptidase, exhibiting many similarities with the lactococcal aminopeptidase A (Exterkate and de Veer, 1987). the intracellular Leu-Leu aminopeptidase (van Boven et aI., 1988) and a recently described tripeptidase from Lactococcus lactis ssp. cremoris Wg2 (Bosman et aI., 1990). We detected the present peptidase activity in all Lactococcus strains tested so far, including a plasmid-free strain. This finding suggests that the gene for the peptidase is located on the Lactococcus chromosome and that the peptidase activity described here possibly plays an important role in the lactococcal amino acid metabolism. The concerted action of peptidases of the starter culture is believed to generate components which contribute either directly or indirectly to flavour development during the proces of cheese ripening. The role of the peptidase described here in this process is not fully understood but probably is connected to its ability to degrade prolinecontaining peptides. Although the enzyme is able to degrade peptides with amino acids other than proline in the N-terminal position, this activity might be of relatively minor importance considering the large number of peptidases presently known to be capable of hydrolysing such substrates (van Boven et aI., 1988; Bosman et aI., 1990; Tan et aI., 1990; Neviani et aI., 1989). The enzyme described by Bosman et ai. reportedly is able to degrade ProGly-Gly but, until now no peptidase has been reported to hydrolyse Pro-X dipeptides. Although under our experimental conditions prolinecontaining tripeptides generally are hydrolysed somewhat faster than proline-containing dipeptides, the sometimes low Km values determined for the hydrolysis of some of the dipeptide substrates seem to suggest that under natural conditions (low substrate concentrations) dipeptides also are good substrates. Hence the imino-dipeptidase (prolinase) activity of the enzyme may be an important metabolic function of the enzyme. In principle exopeptidases that are able to hydrolyse proline-containing peptides in cheese playa very important role, both directly by degrading proline containing peptides that often possess a bitter taste (Kato et aI., 1986) and indirectly by making peptides that were inaccessible to other peptidases accessible again by removing the proline

residues that blocked further degradation by broadspecificity aminopeptidases. Several of the features of the peptidase however, namely pH- and salt optimum, but above all its intracellular location make such an important role for this peptidase difficult to imagine. This does not exclude an important role of this enzyme in the lactococcal amino acid metabolism. The relatively high activities of the enzyme measured with Pro-X(Y) peptides, in spite of substrate concentrations well below the calculated Km values for these substrates, together with the fact that all Lactococcus strains tested so far seem to possess this enzyme suggests that in fact it plays a significant role in the amino acid metabolism of lactococci. The main problem concerning the role of this peptidase in the lactococcal metabolism is the fact that it is not known how the preferred substrates for this enzyme (diand tripeptides with proline in the amino-terminal position) are produced. Moreover proline-containing peptides that are possible sources of such peptides are rapidly degraded by dipeptidyl peptidase IV (Kiefer-Partsch et aI., 1989; Booth et aI., 1990) yielding X-Pro dipeptides that are not degraded by this peptidase. In order to account for the constitutive peptidase activity observed one must assume that the enzyme activity is tuned to the preferred substrate production during growth. This hypothesis suggests that at least one but possibly several as yet unknown proline specific peptidases may be present in lactococci in addition to the already known prolidase and dipeptidyl peptidase IV (Booth et aI., 1990). Possible candidates are aminopeptidase P (Yaron and Mlynar, 1968) and endopeptidases that cleave N-terminal rather than C-terminal of proline residues in peptide chains. The presence of such enzymes would constitute an alternative pathway of proline liberation and uptake, parallel to the currently known pathway formed by the concerted action of dipeptidyl peptidase IV and prolidase. The possible existence and biological significance of such an alternative pathway is being investigated. Acknowledgements. We would like to thank Prof. Dr. K. van Dam, C. J. C. M. de Veer and A. C. Alting for invaluable discussions, C. Slangen for help with the HPLC experiments and P. Both for performing the electron microscopy experiments.

References Booth, M., Donnely, W. J., Fhaolain, I. N., Vincent Jennings, P., O'Cuin, C.: Proline-specific peptidases of Streptococcus cremoris AM2. J. Dairy Res. 57, 79-88 (1990) Booth, M., Fhaolain, T. N. , Vincent Jennings, P., O 'Cuin, C.: Purification and characterisation of a post-proline dipeptidyl aminopeptidase from Streptococcus cremoris AM2. J. Dairy Res. 57, 89-99 (1990) Bosman, B. W., Tan, P. S. T., Konings, W. N.: Purification and characterization of a tripeptidase from Lactococcus lactis subsp. cremoris Wg2. App!. Environ. Microbio!. 56, 1839-1843 (1990)

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Dr. Fred. A. Exterkate, Dept. of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), P.O. Box. 20, 6710 BA Ede, The Netherlands