Appl Microbiol Biotechnol (1991) 36:196-204

Appfl Microbiology Biotechnology © Springer-Verlag 1991

Cell-wall-associated proteinase of Lactobacillus deibrueckii subsp. bulyaricus CNRZ 397: differential extraction, purification and properties of the enzyme Patrick Laloi, Dani~le Atlan, Brigitte Blanc, Christophe Gilbert, and Raymond Portalier Laboratoire de Microbiologie et G~n&ique Molrculaire, (CNRS UMR 106) Bg~timent405, Universit~ Claude Bernard - Lyon I, F-69622 Villeurbanne Cedex, France Received 26 April 1991/Accepted 17 July 1991

Summary. Whole cells of Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 were able to hydro!yse ~t- and //-caseins. Irrespective of the growth medium used, milk or De Man-Rogosa-Sharpe (MRS) broth, identical patterns of a- and//-casein hydrolytic products, respectively, were visualized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. A soluble proteinase present in cell-wall extracts was active on caseins and displayed the same hydrolytic patterns as whole cells. It was purified from cell-wall extract to homogeneity by ultrafiltration and ion exchange chromatography. The enzyme is a monomer with a ,molecular mass of 170 kDa, an optimum temperature of 42°C and an optimum pH of 5.5. It was strongly activated by dithiothreitol and partially inhibited by E-64. These properties indicate that cysteine residues play an important role in the enzyme mechanism. The purified proteinase was not able to hydrolyse di- or tripeptides.

Introduction Lactobacillus delbrueckii subsp, bulgarieus, like most lactic bacteria species, is auxotrophic for many amino acids (Morishita et al. 1981), and milk contains low concentrations of these compounds (Thomas and Mills 1981). Therefore, growth of L. delbrueekii subsp~ bulgaricus in milk up to high cell densities requires a complex proteolytic system composed of extra- and intracytoplasmic proteinases and peptidases able to produce small peptides and free amino acids from milk caseins (Desmazeaud 1983). The organization of the L. delbrueckii subsp, bulgarieus proteolytic system is beginning to emerge. L. delbrueekii subsp, bulgaricus (strain CNRZ 397) synthesizes at least four aminopeptidases (API-IV). One of these, named APII, is located at the cell surface and specifically assayed by measuring lysyl-para-niOffprint requests to: P. Laloi

troaniline (pNA) hydrolysis (Atlan et al. 1989). A second type of peptidase able to catalyse the cleavage of Pro~X bonds, has beenidentified (Atlan et al. 1990): this dipeptidylaminopeptidase (X-Pro-DPAP) is located in the cytoplasm. The APII and X-Pro-DPAP are synthesized during growth in milk or De Man-RogosaSharpe broth (MRS) medium and their biosynthesis is co-regulated (Atlan et al. 1989, 1990). Part of the proteolytic activity of L. delbrueckii subsp. bulgaricus strain CNRZ 397 can be released from the cell wall by washing cells with buffer and has been resolved into three peaks by ion exchange chromatography (Ezzat et al. 1985, 1987). The proteolytic activity of the most active peak was partially characterized. Optimum proteolytic activity was found at pH 5.5 and 30°C. Serine- and thiol-proteinase inhibitors had no significant effect on this proteolytic activity, but its inhibition was observed in the presence of EDTA. The proteolytic activity of L. delbrueckii subsp, bulgaricus strain NCDO 1498 has been previously studied by Argyle et al. (1976). In this work,.the presence of a single proteinase associated with the cell envelope has been demonstrated. All these experiments were made with cells grown in rich broth medium. The properties of lactococcal proteinases have been more precisely analysed than those of lactobacilli species. They have a limited substrate specificity and Visser et al. (1986) distinguished two main types of proteinases: those able to degrade only fl-casein and those able to degrade asland to-caseins. Exterkate (1990) described a synthetic chromogenic substrate, MeO-Arg-Pro-Tyr-pNA, that is hydrolysed by both types of proteinase. Many lactococcal proteinases are located in the cell envelope and released by washing bacteria with a Ca2+-free buffer. Ca 2+ treatment has been used by several groups as the first step in proteinase purification (Exterkate and de Veer 1985, 1989; Geis et al. 1985; Hugenholtz et al. 1987; Monnet et al. 1987). Their molecular masses ranged from 80 to 145 kDa. All these proteinases were classified as serine proteinases. As proteinases synthesized by starter bacteria qualitatively and quantitatively depend on the composition

197 of the g r o w t h m e d i u m , e x p e r i m e n t s with cells g r o w n i n m i l k s h o u l d b e carried o u t for a better u n d e r s t a n d i n g o f the o r g a n i z a t i o n o f the L. delbrueckii subsp, bulgaricus p r o t e o l y t i c system ( T h o m a s a n d P r i t c h a r d 1987). I n the p r e s e n t article we show that a c a s e i n o l y t i c activity is located at the cell surface o f L. delbrueckii subsp, buloaricus. I n o r d e r to p r e p a r e a s o l u b l e cell-wall extract cont a i n i n g the enzyme(s) i n v o l v e d i n this c a s e i n o l y t i c activity, we h a v e d e v e l o p e d a m o d i f i e d p r o c e d u r e o f the m e t h o d a l l o w i n g release o f the A P I I ( A t l a n et al. 1989). T h e p r o t e i n a s e r e s p o n s i b l e for the first step o f fl-casein h y d r o l y s i s has b e e n p u r i f i e d a n d characterized. Its p r o p e r t i e s h a v e b e e n c o m p a r e d to those o f lactococcal proteinases.

Subcellular fractionation F1, F2 and F3 extracts were prepared as previously described (Atlan et al. 1990). F1 extracts were obtained from whole cells treated with lysozyme and F2 extracts after osmotic shock of the lysozyme-treated cells. These extracts were only slightly contaminated by cytoplasmic proteins. Cell lysis was estimated by measuring of the fl-galactosidase activity of the cell-wall extracts: fl-galactosidase was used as a cytoplasmic marker and assayed as described by Miller (1972).

Assay of proteinase activity

MonoQ columns were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Dephosphorylated a-, fl-casein and peptides were purchased from Sigma (St. Louis, Mo., USA). Succinyl-alanyl-alanyl-alanyl-prolylmethionyl-pNAwas obtained from Bachem (Bubendorf, Switzerland); D-isoleucyl-prolyl-arginyl-pNA and methoxy-succinyl-arginyl-prolyl-tyrosyl-pNAwere obtained from Kabi Diagnostica (Stockholm, Sweden).

14C-fl-Casein was used as the substrate; labelled casein was prepared as described by Rice and Means (1971). The reaction mixture contained 60 lxl substrate stock solution (3.5 mg/ml, 2000 dpm/~tg), 15 p~l enzyme extract and 70 ~tl of 200 mM 2-[N-morpholino]ethanesulphonic acid (MES), pH 6.0, or TRIS-maleate as indicated. The mixture was incubated at 42° C; i00 Ixl aliquots were taken at different times and diluted into 100 lxl of cold 12% trichloroacetic acid (TCA). After at least 60 min, the reaction mixture was centrifuged (15 min, 13 000 9). The radioactivity of 100 ~tl of TCA-soluble supernatants was determined ~y liquid scintillation counting in 10 ml of scintillant (Ready-solv EP, Beckman, Palo Alto, Calif., USA) with a Beckman LS 1800 scintillation counter. All assays were made in triplicate. One unit (U) of proteinase was defined as the amount of enzyme that released one dpm/min per millilitre.

Bacterial strains

Electrophoretic analysis of casein hydrolysis

L. delbrueckii subsp, bulgarieus parental strain CNZR 397 was obtained from the Centre National de Recherches Zootechniques (INRA, Jouy en Josas, France).

The method used was based on the procedure described by Hill and Gasson (1986).

Materials and methods

Chemicals

Sample preparations Growth conditions, media and harvestin9 of bacteria Bacteria were routinely grown anaerobically at 40°C in 125-ml flasks containing 100 ml MRS broth (Difco, West Mosley, Surrey, UK) (De Man et al. 1960) or skim milk. Reconstituted 10% (w/v) skim milk medium was prepared as described previously (Atlan et al. 1989). After 4 h of growth in skim milk or MRS, bacteria were collected as described previously (Atlan et al. 1989). Cell pellets were washed with 25 ml of 50 mM KH2PO4 - KOH, pH 7.0, and resuspended in 10 ml of the same buffer.

Biomass Growth was estimated by measuring the increase in cell suspension turbidity (SP 320 spectrophotometer; Jouan, St Nazaire, France): 1.0 unit of absorbance at 600 nm corresponds to 250 ixg bacterial dry weight/ml or 108 cells/ml.

Whole cells. Bacterial ceils were washed and concentrated in 100 mM NaH2PO4-NaOH, pH 7.2, containing 10 mM CaC12, then a- or fl-casein (200 lxl of a 5 mg/ml solution) solution was added to 3 ml of a suspension containing 7 × 108 cells/ml and incubated at 40°C. Aliquots (150 p,l) were taken at different times and diluted into 32 p~l solubilization buffer [2 ml of 20% w/v sodium dodecyl sulphate (SDS), 2.5 ml of 25 mM TRIS-192 mM glycine buffer, pH 8.3, and 4.5 ml of distilled water mixed extemporaneously with I ml of 2-mercaptoethanol]. The mixture were incubated at 80° C for 10 rain and centrifuged (9000 g, 10 min). An aliquot (50 l~l) of the supernatants was mixed with 10 Ixl of 30% glycerol containing 0.04% bromophenol blue and heated at 100° C for 2 rain before electrophoresis. F1 and F2 extracts. A volume of 70 Ixl of F1 or F2 extracts (concentrated tenfold) was raixed with 140 ktl of a 5 mg/ml a- or flcasein solution and 2 ml of 100 mM NaHzPO4-NaOH, pH 7.2, buffer solution containing 10 mM CaCI2. Reaction mixtures were incubated at 40° C and treated as described above. F1 and F2 extracts corresponded to suspensions with a density of 7 × 10s cells/ ml.

Determinaton of protein concentration Electrophoretic analysis Protein concentration was measured by the method of Bradford (1976), using bovine serum albumin as the standard. For the ion exchange purification step, protein concentration was monitored by measuring the absorbance at 220 nm.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with the buffer system described by Laemmli (1970) using 15% acrylamide slab gels. Gels were run at 13 mA per slab gel

198 until bromphenol blue tracking dye had moved to 0.5 cm from the bottom of the gel. Proteins were stained with Coomassie Brilliant Blue R250 (Prolabo, Paris, France) diluted into methanol/acetic acid/water (50/10/40 v/v/v). Dye in excess was eliminated by successive washings in methanol/acetic acid/water (5/10/85 v/v/v). When indicated, proteins were silver-stained using the Amersham quicksilver kit (Amersham, Bucks, UK).

(20 i11), 20 mM TRIS-HCI, pH 7.5, (100 lxl) and the substrate at a final concentration of 1 raM. After at least 24 h, enzyme activity was detected by the development of a yellow colour. Hydrolysis of non-chromogenic peptides was analysed by TLC as described by Tan and Konings (1990).

lsoelectric focusing (IEF) in polyacrylamide gel

Ultrafiltration. The F2 extracts prepared from 150 mg dried cells were used as crude extracts. They were fractionated using a YM 100 membrane from Amicon (Danvers, Mass., USA). The operating pressure was 2 bars and the membrane was prepared as described by the manufacturer. F2 extracts (55 ml) were washed with 200 ml of 20 mM TRIS-HC1 buffer, pH 7.5, and finally reduced to 5 ml.

Ultrathin gels (0.5 mm, 6% acrylamide) were prepared with the Ultromould system (Pharmacia LKB, Uppsala, Sweden) as described by the manufacturer, except that 3.7 ml of ampholine pH 4-6 (LKB) was used. Before electrophoresis, cell-wall extracts were dialysed at 4°C during 24 h against distilled water using membrane tubings with a relative molecular weight cut-off of 12000-14000 and were concentrated by lyophilization. Dried samples were dissolved in i00 Ixl of 10 mM TRIS-HCI, pH 7.0. Suitable adsorbents (LKB) laid on the gel surface were loaded with samples obtained from 108 bacterial cells. Electrophoresis was performed at 10°C during 2 h with the Multiphor II system (LKB) under 25 W constant power; 40 mM glutamate was used as anolyte and 0.2 M histidine as catholyte. After electrophoresis, gels were fixed and stained with Coomassie Brilliant Blue R250 as described by the manufacturer. The following proteins were used as pI markers: carbonic anhydrase A from human erythrocytes (Sigma, pI 6.57); carbonic anhydrase B from bovine erythrocytes (Boehringer, Mannheim, FRG; pI 5.85); fllactoglobulin A from bovine milk (Sigma, pI 5.20); trypsin inhibitor from soybean (Sigma, pI 4.55). On a pH 3-10 gradient of ampholines, aspartic acid (40 raM) and NaOH (1 mM) were used as analyte and catholyte, respectively, and a broad pI calibration kit from Pharmacia supplied with appropriate pI markers (3.5-9.5 pI range).

Hydrolysis of peptide substrates For testing chromogenic peptides (para-nitroanilide derivatives) as substrates, the reaction mixture contained the enzyme sample

Purification procedure of the proteinase

Chromatography on MonoQ. Ultrafiltred extracts were applied to a column of Monobeads (MonoQ HR 5/5) that had been equilibrated with 20 mM TRIS-HC1 buffer, pH 7.5. The column was washed with 3.5 ml of the same buffer and enzyme activity was eluted with a 16-ml linear gradient of 0.0-0.4 M NaC1 in 20 mM TRIS-HC1 buffer, pH 7.5. The flow rate was 1 ml/min and 1 ml fractions were collected. The fractions containing the enzyme activity (5 ml) were pooled and concentrated before electrophoretic analysis by ultrafiltration on a YM 100 membrane (Amicon) in 20 mM TRIS-HCI buffer.

Results

Casein hydrolysis by whole cells of L. delbrueckii subsp. bulgaricus The kinetics o f fl- a n d a - c a s e i n h y d r o l y s i s b y w h o l e cells o f s t r a i n C N R Z 397 are s h o w n i n Fig. 1. A n equiv a l e n t o f 9.106 cells g r o w n i n m i l k c o m p l e t e l y h y d r o lysed 13 lxg o f a - or fl-casein i n less t h a n 15 rain (Fig. 1, l a n e c). H o w e v e r , rates o f fl- or a - c a s e i n h y d r o l y s i s b y

Growih medium

Fig. 1. Electrophoretic analysis of fland a-casein hydrolysis by whole cells of strain CNRZ 397 grown on milk or De Man-Rogosa-Sharpe (MRS) medium. Incubation times: 0 rnin (lanes a, ~, 5 rain (lanes b, g), 15 min (lanes e, h), 30 min (lanes d, i) and 60 rain (lanes e, j). Enzyme preparations were from 9 x l 0 6 cells, b l - 4 and al, 2 are peptide products

199

Fig. 2. Electrophoreticanalysis of fland a-casein hydrolysisby F1 extracts from strain CNRZ 397. Incubation times: 0 rain (lanes a, f), 5 min (lanes b, g), 15 rain (lanes c, h), 30 rain (lanes d, i) and 60 min (lanes e, j). F1 extracts prepared from 9 × 10 6 cells were used as enzyme preparations

cells grown in MRS medium were significantly lower than those observed with cells grown in milk medium. Irrespective of the growth medium, fl-casein was hydrolysed more rapidly than a-casein. The hydrolytic patterns of fl- and a-caseins by cells grown either in milk or MRS medium were similar. The a-casein pattern was characterized by the al and a2 peptide products with molecular masses of 23 kDa and 22 kDa respectively. The clevage of fl-casein released two major products b2 and b4 (about 19 kDa and 18 kDa, respectively) and two minor products, bl (lane g) and b3 (lane c), themselves quickly hydrolysed. Under prolonged incubation times, all products were further hydrolysed into smaller peptides that were undetectable on 15% acrylamide gels (data not shown). Using the same electrophoretic method, we did not detect any hydrolysis of ;c-casein by whole cells (data not shown).

milk were much more active on caseins than extracts from cells grown in MRS medium. F2 extracts prepared from 9.106 milk-grown cells completely hydrolysed 13 ~tg of fl-casein in less than 30 min (Fig. 3, lane d). Casein hydrolytic products (bl-4 and al-2) released by soluble proteinase(s) present in F2 extracts were identical to those detected with whole cells (Fig. 1). However, proteolytic activity was very weak in F1 extracts, which could not completely hydrolyse 13 Ixg of fl-casein in 60 min, and only released b 1 and b2 products. F1 and F2 extracts from cells grown in MRS or milk medium contained about 50% of the total caseinolytic activity detected in crude extracts but less than 3% of cytoplasmic fl-galactosidase activity, which indicated that no significant lysis occurred during cell fractionation.

Protein composition o f F1 and F2 extracts Subcellular localization o f caseinolytic activity No extracellular caseinolytic activity was detected in culture supernatants even after concentration by lyophilization and no caseinolytic activity was released by washing cells with buffer as described by Ezzat et al. (1985). In order to release the caseinolytic enzyme(s) from the cell wall we used a previously described procedure (Atlan et al. 1990, see Materials and methods). As shown in Figs. 2 and 3, F1 and F2 extracts obtained from strain CNRZ397 grown in MRS or milk medium were able to hydrolyse fl- and a-caseins at different rates. Irrespective of the growth medium, fl-casein was hydrolysed faster than a-casein. On the other hand, F1 and F2 extracts prepared from cells grown in

The protein composition of F1 and F2 extracts prepared from strain CNRZ 397 grown either in milk or MRS medium was analysed by isoelectrofocusing (IEF) (Fig. 4). All the proteins identified in F1 and F2 extracts had a pI ranging form pH 4.5 to 4.75, except for protein 13, which had a pI near 10.0 and was characterized on independent gels using a gradient of ampholines from pH 3.0 to 10.0. Protein 13 was synthesized during growth in milk or MRS medium and was present in equal amounts in F1 and F2 extracts (data not shown). Proteins 2, 3, 5 and 11 were mainly released by lysozyme treatment (F1 extracts) whereas proteins 7, 8, 9 and 10 were recovered in both F1 and F2 extracts

200

Fig. 3. Electrophoretic analysis offland g-casein hydrolysis by F2 extracts from strain CNRZ 397. Incubation times: 0 min (lanes a,f), 5 min (lanes b, g), 15 rain (lanes c, h), 30 min (lanes d, i) and 60 min (lanes e, j). F2 extracts prepared from 9 x 106 cells were used as enzyme preparations

Fig. 4. Isoelectric focusing (IEF) analysis on polyacrylamide gel (pH gradient = 4.0-6.5) of the protein composition of F1 (lanes B, C) and F2 (lanes D, E) extracts from strain CNRZ 397. Cells were grown in milk (lanes B, D) or MRS medium (lanes C, E). Standard proteins were separated on lane A

(milk-grown cells) or specifically released in F2 extracts ( M R S - g r o w n cells). F1 proteins that were released b y a mild l y s o z y m e treatment were p r e s u m a b l y e x p o s e d nearer to the cell surface t h a n F2 proteins, w h i c h required an o s m o t i c s h o c k o f the lysozyme-treated cells to be extracted.

Proteinase purification F2 fluids f r o m milk-grown cells, w h i c h c o n t a i n e d only few proteins (Fig. 4) but were strongly active on flcasein (Fig. 3, lanes a-e), were used as crude extracts for purifying the surface proteinase. Proteinase activity was visualized a n d m e a s u r e d b y using fl-casein as the substrate. H y d r o l y t i c p r o d u c t s were directly detected

201 Table 1. Purification of the cell-wall associated proteinase from Lactobacillus delbrueckii subsp, bulfaricus Purification step

Enzyme Protein Specific PurlYield activity (mg) activity fication (%) (U × 103) (U x lO3/mg (fold) protein)

F2 extract 3954 Ultrafiltration 3558 Anion exchange 1530 chromatography (MonoQ HR 5/5)

10.25 0.61 0.17

385 5833 9000

1 15 23

100 90 39

Purification steps as well as determination of enzyme activity and protein concentration are described under Materials and methods. Proteinase activity was assayed with [~4C] //-casein as the substrate: U, units

b y S D S - P A G E a n d the levels o f caseinolytic activity were assayed b y using [~4C]casein as the substrate. As the first purification step, the proteinase was retained by an ultrafiltration m e m b r a n e with a cut-off o f 105 Da. This step allowed us to remove m o r e t h a n 90% o f the c o n t a m i n a t i n g proteins and to recover 90% o f total proteinase activity (Table 1). The proteinase was further purified by ion e x c h a n g e c h r o m a t o g r a p h y ( M o n o Q H R 5/5). Figure 5 shows the elution pattern o f proteins. The activity towards fl-casein detected b y S D S - P A G E electrophoresis was eluted at 0.3 M NaC1. The h o m o g e n e i t y o f the proteinase p r e p a r a t i o n was est i m a t e d b y S D S - P A G E (Fig. 6). The proteinase purification p r o c e d u r e is s u m m a r i z e d in Table 1. Using this p r o c e d u r e , the e n z y m e was purfied 23-fold with a yield o f 39%.

Fig. 6. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (15% acrylamide) of enzyme fractions during the purification of the//-casein hydrolysing enzyme. Proteins were silver-stained: lane A, 2 I~g of proteins after the ultrafiltration step; lane B, purified proteinase from active fractions after monoQ chromatography (the protein concentration was lower than the detection limit of the Bradford method); lane C High molelcular weight standard mixture (SDS-6H, Sigma) (numbers indicate molecular masses in kDa)

U .1600 A220nm 1 NaCI

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Fig. $. Ion exchange chromatography of proteinase. Proteins (150 Ixg) from the first step of purification were applied to a MonoQ HR 5/5 column. The proteinase was eluted at a flow rate of 1 ml/min with a linear (0.0-0.4 M) gradient of NaC1 (. . . . . ) in 20 mM TRIS-HCi buffer (pH 7.5). Fractions of 1.0 ml were collected. Proteins ( - - ) were monitored by absorbance at 220 nm (A22o). Proteinase activity was assayed with [14C]//-casein as the substrate ( - - - - - )

~l~l .200"4°° o 35

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202

Properties o f the purified cell surface proteinase

1O0

Molecular mass and subunit composition of the proteinase. The proteinase had a molecular mass of about 170 kDa as estimated by gel filtration on a TSKG3000SW column (data not shown). On SDS-PAGE gels, the proteinase gave only one protein band, which corresponded to a molecular mass of 170 kDa. These results strongly suggest that the proteinase is a monomer. Effect of temperature and pH. Proteinase activity was measured on/]-casein at different temperatures: maximum activity was obtained between 42 and 45°C (Fig. 7). Proteinase activity was optimal between pH 5.5 and 6.0 (Fig. 8). Above pH 6.0, proteinase activity decreased rapidly. Effect of various chemical reagents and metal ions. Proteinase activity was not inhibited by the inhibitors of metallo- and aspartic-proteinases such as EDTA (0.1 mM), 1,10-phenanthroline (20mM) and bestatin (0.033 mM). On the other hand, 45% and 79% of the activity were recovered after incubation with 1 mM phenylmethylsulphonyl fluoride (PMSF) and 0.5 mM E-64, components known to be inhibitors of serine- and cysteine-proteinases, respectively. Proteinase activity was activated by dithiothreitol (DTT), and the inhibition by PMSF was partially reversed in the presence of 10 mM DTT. The addition of various divalent cations (Ca 2÷, Co 3+, Mg 2+ and Mn 2+) at 5 mM had no significant effect on proteinase activity. Proteinase activity on various substrates. The a- and/]but not x-caseins were hyrolysed by the purified protei-

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pH Fig. 8. Effect of pH on proteinase activity. Experiments were carried out at 42°C with 200 mM TRISomaleate buffer, using [14C] r-casein as the substrate

nase (Table 2). SDS-PAGE showed that the peptide hydrolytic pattern of/]-casein obtained with the purified proteinase was identical to that observed with F2 extracts (Fig. 9). None of the peptides tested (Table 2) was hydrolysed by the purified proteinase.

Discussion

-I-

1O0

80

Fig. 7. Effect of temperature on proteinase activity

In this study, we have shown that the caseinolytic system of L. delbrueckii subsp, bulgaricus CNRZ 397 is at least partially localized in the bacterial cell envelope. Casein hydrolysis by whole cells indeed demonstrates that milk proteins are accessible to cell surface proteinases. Furthermore, the release of half caseinolytic activity after lysozyme treatment (F1 extracts) and osmotic shock (F2 extracts) of milk-grown cells shows that most caseinolytic activity is located in the cell wall. The IEF analysis of the protein composition of F1 and F2 extracts showed that many proteins are present in the cell wall of strain CNRZ 397. Proteins identified in F1 extracts are very likely closer to the outer side of the cell envelope than those recovered in F2 extracts. Irrespective of the growth medium, while cells as well as F2 extracts quickly hydrolysed caseins into the same primary products. So, the F2 extracts correspond to prepurified preparations of the cell-wall proteinase. Much more activity was present in cell-wall F1 and F2 extracts after growth in milk than in MRS broth. These results show that milk cultures overproduce surface protein(s) with caseinolytic activity to reach high cell

203 Talkie 2. Substrate specificity of the proteinase purified form cellwall extracts of strain CNRZ 397 Substrate Caseinsa a-Casein fl-Casein to-Casein Peptidesb Leu-Gly Leu-Ala Leu-Leu-Leu Ala-Met Ala-Phe Ala-Leu Ala-His Val-Leu Val-Gly Val-Gly-Gly Val-Pro Gly-Tyr Gly-Phe Gly-Trp Gly-Val Pro-Leu His-Ser Lys-Ser Benzyloxycarbonyl-Gly-Leu Benzyloxycarbonyl-Gly-Phe Chromogenic substrate~ Succinyl-Ala-Ala-Ala-Pro-Met-pNA D-Ile-Pro-Arg-pNA Methoxy-succinyl-Arg-Pro-Tyr-pNA

Activityd ÷ +

pNA, para-nitroanilide a Casein hydrolysis was analysed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis b Petide hydrolysis was analysed by TLC ° Hydrolysis of chromogenic substrates was visually estimated d +, hydrolysis; - , no hydrolysis

washing them with buffer and these enzymes have been separated by ion exchange chromatography (Ezzat et al. 1987). When extracts prepared by this method were incubated with caseins, 20 h were necessary to detect the primary hydrolytic products by electrophoresis (Blanchard 1984). Using our method, F2 extracts are able to completely hydrolyse fl-casein into small TCAsoluble peptides that cannot be detected by SDS-PAGE in less than 30 rain. Therefore, the extraction of cellwall proteins by lysozyme treatment and osmotic shock is a much more efficient procedure than cell washing. We have isolated a proteinase from F2 extracts of L. delbrueckii subsp, bul#aricus C N R Z 397. The enzyme was purified to homogeneity by a two-step procedure. The results obtained with the method used to detect proteinase activity during the purification allowed us to propose a role in the cell for the isolated enzyme: the proteinase could be responsible for the first step of casein hydrolysis. This cell-wall associated proteinase of L. delbrueckii subsp, bul#aricus showed an optimum temperature of about 42 ° C and an optimum pH of 5.5. Like Argyle et al. (1976) we fouffd a single proteinase associated with the cell envelope. The proteinase was strongly activated by DTT and partly inhibited by E-64, a specific inhibitor of cysteine proteinases. These properties indicated that cysteine residues play a major role in the enzyme mechanism. The enzyme described in this work was unable to degrade MeO-Arg-Pro-Tyr-pNA, a synthetic chromogenic substrate that was hydrolysed by both types of lactococcal proteinases (Exterkate 1990). The primary hydrolytic products released from caseins by the purified proteinase and detected by SDS-PAGE have molecular masses very similar to those of caseins, suggesting that fl-casein proteolysis was initiated from one end of these molecules. The purified proteinase will be used to pre-

Fig. 9. Electrophoretic analysis offl-casein hydrolysis by F2 extracts (lanes a, b, c, d) and purified proteinase (lanes e, f, 9, h) from strain CNRZ397. Incubation times: 0 min (lanes a, e), 5 rain (lanes b, f), 15 rain (lanes c, 9) and 60 min (lanes d, h). Extracts prepared from 107 cells 'were used as enzyme preparations

densities despite the low initial concentrations of free amino acids in this medium. Three cell wall proteinases have been released from cells of L. delbrueckii subsp, bulgaricus (CNRZ 397) by

pare fl-casein primary hydrolytic products and their Nterminal amino acid sequences will be characterized in order to determine if the enzyme has a peptide bond preference. These peptides will also be used to identify

204 the p r o t e o l y t i c e n z y m e s a b l e to h y d r o l y s e t h e m i n t o smaller peptides. Acknowledgements. We are grateful to Simone Rouzirs and Bernard Dequatre for excellent technical assistance. This work was supported by grants from the Centre National de la Recherche Scientifique (UMR 106 and Action Thrmatique programmre 8316), the Universit6 Claude Bernard and the Centre International de Recherches Daniel Carasso (BSN/Gervais -Danone).

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