(St-PepN) from Streptococcus salivarius ssp. thermophilus CNRZ 302

Purification and Characterization of a General Aminopeptidase (St-PepN) from Streptococcus salivarius ssp. thermophilus CNRZ 302 FRANCOISE RUL, VERONIQUE MONNET, and JEAN-CLAUDE GRIPON

Unite d’Enzymologie Station de Recherches Laitieres lnstitut National de la Recherche Agronomique 78352 Jouy-en-Josas, Cedex, France

ABSTRACT

A general aminopeptidase (St-PepN) was purified from an intracellular extract of Streptococcus salivan’us ssp. thermophilus CNRZ 302 by ion-exchange chromatography and hydrophobic interaction chromatography. Gel electrophoresis of the purified enzyme in denaturing or nondenaturating conditions showed a single protein band. The enzyme is a monomer with a molecular mass of 97 kDa. Its activity is maximal at pH 7 and 36°C and is completely abolished by CuC12 and ZnC12. The enzyme is strongly inhibited by metalchelating reagents, such as EDTA and ophenanthroline, which suggests that StPepN is a metalloenzyme. The enzyme showed activity toward p-nitroanilide derivatives or dipeptides and tripeptides and showed a preference for hydrophobic or basic amino acids at the N-terminal position, Longer peptide chains, such as the B-chain of insulin, glucagon, or peptides generated by the hydrolysis of caseins, were degraded, too. The sequence of the first 21 residues of the mature enzyme was determined and showed high homology with that of the aminopeptidase PepN isolated from Lactococcus lactis ssp. cremoris Wg2. The properties of the enzyme are compared with those of corresponding enzymes of other species of lactic acid bacteria. (Key words: aminopeptidase, Streptococcus salivarius ssp. thennophilus, lactic acid bacteria)

Received November 22, 1993 Accepted May 2, 1994. 1994 J Dairy Sci 77:2880-2889

Abbreviation key: FPLC = fast protein liquid chromatography, LAB = lactic acid bacteria, p-Na = p-nitroanilide, TEA = triethanolamine, TFA = mfluoroacetic acid. INTRODUCTION

Streptococcus salivarius ssp. thennophilus is a common constituent of thermophilic lactic starters used to make yogurt and cooked cheese. Similar to other lactic acid bacteria GAB), S. salivarius ssp. themphilus requires an exogenous N source for optimal growth in milk (14, 28). However, milk is relatively poor in free amino acids and small peptides. Therefore, optimal growth of LAB depends on their proteolytic system, which consists of proteinases and peptidases that degrade milk caseins to peptides and free amino acids (12, 13). Furthermore, this proteolytic system contributes to texture and flavor development in dairy products. The proteolytic system of Lactococcus sp. has been widely studied and characterized [reviews, (19, 26)], but, despite its wide utilization in the dairy industry, few data are available about the proteolytic enzymes of S. salivarius ssp. thennophilus. Generally, S. salivarius ssp. thennophilus presents very low proteinase activity and develops in milk in symbiotic association with Lactobacillus species, which provide peptide substrates for its peptidases. Several of these peptidases have been described. Two metallopeptidases (a Leu aminopeptidase and a dipeptidase) were purified and characterized from S. salivarius ssp. thennophilus CNRZ 160 by Rabier and Desmazeaud (20). The Leu aminopeptidase had a molecular mass of 62 kDa and broad specificity. The dipeptidase exhibited a specificity toward dipeptides with a large and hydrophobic amino acid residue at the N-terminus. Des2880

STREPTOCOCCAL AMINOPEPTIDASE

mazeaud (5) reported the purification of a neutral intracellular endopeptidase from S. salivanus ssp. thennophilus CNRZ 160. Meyer and Jordi (16) have investigated an Xprolyl-dipeptidyl aminopeptidase from a mixed culture starter used for Gruykre cheese manufacture. Recently, Tsakalidou and Kalantzopoulos (30) described the purification and characterization of an intracellular aminopeptidase from a strain of S. saiivan'us ssp. thermophilus isolated from traditional Greek yogurt. During a preliminary study, we were able to detect four aminopeptidases in an intracellular extract of S. salivanus ssp. thennophilus CNRZ 302. To investigate further its potential role in N metabolism and organoleptic development of yogurt and cheese, the present work describes the purification and characterization of one of these aminopeptidases, StPepN, which showed homology to the aminopeptidase PepN from lactococci.

288 1

method of Bradford (4); bovine serum albumin (Pierce, Rockford, JL) was used as a standard. Aminopeptidase Activity Assay

Activity of St-PepN was assayed during the purification by monitoring the release of p nitroaniline @-Na) from Lys-p-Na (Sigma Chemical Co., St. Louis, MO) at 410 nm as described by Zevaco et al. (33). The activity of the purified enzyme was tested against several oligopeptides (Sigma Chemical Co.). Fragment 58-68 of /3-casein was provided by F. Mulholland (Agricultural Food Research Council, Reading, England). Aljquots of the purified enzyme were incubated at 37°C in the presence of .23 mM substrate in 50 mM TEA, pH 7. After 30 min of incubation (or 1 h for fragment 58-68 of @casein or 7 h for glucagon), the reaction was stopped by the addition of trifluoroacetic acid FFA) to 1% final concentration. The samples were analyzed by reversed-phase HPLC using MATERIALS AND METHODS a C 18 column (Nucleosil C 18, 250 x 4.6 mm; Shandon, Eragny, France) in a TFABacterial Strain and Culture Conditions acetonitrile solvent system (solvent A = .115% Streptococcus salivarius ssp. thennophilus "FA; solvent B = .l% TFA, 60% acetonitrile) CNRZ 302 (from the collection of the Station with a linear gradient from 20 to 80% solvent de Recherches Laitihres) was grown at 37°C in B in 15 min and recording at 214 nm. The 5 L of M17 broth (Biokar, Beauvais, France) collected fractions were then identified with a containing 5 g of IactoseL. Growth was as- pulse-liquid sequencer (model 477A; Applied sessed by measurement of the absorbance at Biosystems, Foster City, CA). The St-PepN activity was also tested 650 nm. against dipeptides and tripeptides (Sigma Chemical Co.), and the hydrolyzates were Intracellular Extract Preparation directly analyzed with an amino acid analyzer Late logarithmic phase cells (after 4 h) were (LC3000; Biotronik, Maintal, Germany). collected by centrifugation (6OOO x g for 15 min at 4°C). The pellet was washed twice with 50 mM @-glycerophosphateand then incubated Aminopeptidase Purlficatlon for 2 h at 30°C in 50 mM triethanolamine Nucleic acids in the intracellular extract VEA) buffer, pH 7, containing 30% (wt/vol) were hydrolyzed by addition of RNAse (2.75 sucrose, .1 mg/ml of lysozyme, and 42 U/ml of mg/100 ml of extract; Sigma Chemical Co.) mutanolysine. The spheroplasts obtained were and DNAse (.05 mg/100 ml of extract; Sigma collected by centrifugation (12,000 x g for 30 Chemical Co.) in the presence of MgCl2 (1.66 min at 4'C) and resuspended in 50 mM TEA mg1100 ml of extract) according to Rabier and buffer, pH 7. The supernatant obtained after Desmazeaud (20). The solution was then centrifugation (20,000 x g for 30 min at 4°C) is designated below as the intracellular extract. precipitated in the presence of 60 mM MnS04 and centrifuged at 10,OOO x g for 30 min. The extract was then dialyzed overnight against 10 Protein Assay mM TEA, pH 7, and filtered through a .45-pm Protein amounts in the extracts and in the membrane filter (type HVLP; Millipore Co., purified fractions were determined by the Bedford, MA). Journal of Dairy Science Vol. 77, No. 10, 1994

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RUL ET AL.

The first ion-exchange chromatography was done by fast protein liquid chromatography (FPLC) on a Q-Sepharose Fast Flow XK 26/40 column (2.6 x 15 cm; Pharmacia, Uppsala, Sweden). The column was equilibrated with 50 mM TEA, pH 7. The extract (317 mg of proteins) was applied to the column and eluted with a linear NaCl gradient from 0 to .5 M in 5 h at a flow rate of 5 d m i n , and 10-ml fractions were collected. Fractions active against Lys-p-Na were recovered and dialyzed overnight against 10 mM TEA, pH 7.5. The second ion-exchange chromatography was performed by FPLC with a MonoQ HR 10/10 column (1 x 10 cm; Pharmacia) equilibrated with 50 mM TEA, pH 7.5. The elution was achieved with a linear NaCl gradient from 0 to .5 M in 3 h at a flow rate of 3 mumin. Three-milliliter fractions were collected, and the fractions that were active against Lys-p-Na were pooled. The hydrophobic interaction chromatography was performed with an Alkyl Superose HR5/5 column (.5 x 5 cm; Pharmacia). The column was equilibrated with 50 mM Na phosphate buffer @H 7.5)-1.7 M (NH4hSO4. Then, 1.7 M ( N H 4 h S O 4 was added to the sample, which was applied on the column and eluted with a linear gradient from 1.7 to .51 M (NH4hSO4 per 50 mM Na phosphate @H 7.5) for 1 h and 30 min at a flow rate of .5 d m i n ; .5-ml fractions were collected. Fractions active against Lys-p-Na were recovered and dialyzed against 10 mM Na phosphate buffer. Amlnopeptldase Characterizatlon

Electrophoresis. The purified fraction was analyzed by PAGE in 10% acrylamide gels with and without SDS in presence of 8mercaptoethanol, followed by staining with Coomassie blue. The SDS-PAGE was used to determine molecular sizes with the following markers: myosin (205 kDa), @-galactosidase (116.5 m a ) , phosphorylase b (97.4 m a ) , bovine serum albumin (66.2 m a ) , and ovalbumin (45 kDa). After PAGE in the absence of SDS, the enzyme activity was detected directly in the gel according to Miller and McKinnon (17) using Lys-@-naphthylamide as a substrate. Hydrolysis of the substrate yields 8naphthylamine, which reacts with Fast Garnet to produce a dark red color at the level of the enzyme. Journal of Dairy Science Vol. 77, No. 10, 1994

Gel Filtration. The molecular size of the active enzyme was estimated by gel filtration on a Superose 12 column (Pharmacia) in .15 M NaCl in 50 mM TEA @H 7.5) at a flow rate of .25 mumin. The column was calibrated with thyroglobulin (670 m a ) , gamma globulin (158 m a ) , ovalbumin (44 ma), myoglobin (17 ma), and vitamin B12 (1350 Da). Effect of pH on Enzyme Activiry. The effect of pH on the St-PepN activity was determined at 37°C over a pH range of 4 to 10 using the following .1 M buffers: sodium acetate, pH 4 to 5.5; bis-Tris, pH 6 to 7; TEA. pH 7.5 to 8; Tns, pH 8.5 to 9.5; and 3-(cyclohexylamino)-1propanesulfonic acid, pH 10. Effect of Temperature on Enzyme Activity. The effect of temperature on enzyme activity was determined at temperatures ranging from 20 to 60°C. After incubation at the desired temperature, the reaction mixture was equilibrated at room temperature (25°C) before absorbance was measured. Effect of Bivalent Cations on Enzyme Activity. The enzyme was preincubated in the presence or absence of various bivalent cations: CaC12, CuC12, CoC12, MgC12, and ZnC12 for 20 min at 37'C at final concentrations of .1 and 1 mM in .2 M Tris.HC1 buffer, pH 7.5. The remaining enzyme activity was assayed at 37'C for 20 min using Lys-p-Na as a substrate. The rate of hydrolysis of Lys-p-Na in the absence of metal ion was taken as 100%. The enzyme was also preincubated with .1 mM EDTA in .2 M Tris.HC1 buffer, pH 7.5, for 20 min at 37°C followed by incubation in 1 mM solutions of divalent cations: Ca2*, Cu2+, Co2+, Mg2+, and Zn2+ at 37°C for 20 min. Effect of Inhibitors on Enzyme Activiry. The enzyme was preincubated with phenylmethanesulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), iodoacetic acid, iodoacetamide, p-hydroxymercuribenzoate, EDTA, o-phenanthroline, or bestatin for 20 min at 37'C at various concentrations in .2 M Tris-HC1buffer, pH 7.5. Enzyme activity was measured following the standard enzyme assay procedure, and the rate of hydrolysis of Lys-pNa in the absence of any inhibitor of metal chelator was taken as 100%. N-Terminal Amino Acid Sequence. The protein sequence was determined by using a pulse-liquid sequencer (model 477A; Applied Biosystems). The enzyme was electroblotted

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STREPTOCOCCAL AMINOPEPTIDASE TABLE 1. Purification of aminopeptidase from Streptococcus Purification step Cell-free extract Q-Sepharosel MonoQ' Alkyl Superose'

Total

Total

protein

salivarius ssp. rhermophilus CNRZ

302.

activity

Specific activity

Yield activity

Purification

(m

(h)

(Ww)

(W

(-fold)

317 40.5 1.6 .33

348 152

100 44 24 19.5

3.4 48 I87

as

68

1.1 3.8

52.5 206

1

'Pharmacia, Uppsala, Sweden.

onto a polyvinylidene difluoride membrane as described by Matsudaira (15). The membrane was stained with Coomassie blue and then directly used in the sequencer. Isoelectric pH. Chromatofocusing was performed with a Mono P HR 5/20 column (.5 x 20 cm; Pharmacia). The column was equilibrated with -025 M imidazole @H 7.4). The purified enzyme was injected into the column and eluted with polybuffer 74 (Pharmacia) @H 3.98). The isoelectric pH was the pH of the fraction active on Phe-p-Na. RESULTS St-PepN Purification

The enzyme was purified about 190-fold from the cell-free extract by three steps of chromatography; yield of activity recovery was 19.5% (Table 1). The last purification step (alkyl Superose HR5/5, Pharmacia) resulted in one peak, which showed a single protein band upon examination by SDS-PAGE and PAGE; after PAGE, enzyme activity was detected at the same position as the protein band (Figure 1).

substrate in 50 mM TEA buffer, pH 7 (Figure 3). The optimal temperature was around 36'C with remaining activity at 20 and 48°C of about 45% of maximal activity. Enzyme activity was measured in the presence of different classes of inhibitors (Table 2). Inhibitors of cysteine proteases (iodoacetamide, iodoacetic acid, and p-hydromercuribenzoate), as well as inhibitors of serine proteases (diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride), had no effect on peptidase activity. Incubation with bestatin, an aminopeptidase inhibitor, resulted in inhibition of St-PepN activity. Metal-chelating reagents, such as 1 mM EDTA or o-phenanthroline, strongly inhibited the enzyme activity, suggesting St-PepN is a metallopeptidase. After inhibition by .I mM EDTA, the enzyme activity was partially restored (45%) by addition of 1 mM CaC12 (Table 3); ZnC12 and MgCl2 gave a slight activity (Table 3). Without EDTA pretreatment, St-PepN was totally inhibited by I mM CuC12 and ZnCl2; CoCl2 left slight activity, CaC12 slightly decreased it, and MgC12 increased it slightly (Table 4). Substrate Specificity

St-PepN Properties

The molecular mass was estimated to be 96.5 kDa by gel filtration and 97 kDa by SDSPAGE. The similar values obtained under native and denaturating conditions indicate that this St-PepN is probably monomeric. The isoelectric point of St-PepN was pH 5.1. The St-PepN appeared to be active over the range of pH 5 to 9.5; maximal hydrolysis of Lys-p-Na was at pH 7 (Figure 2). Activity of St-PepN was measured in the range of 20 to 60°C using Lys-p-Na as a

The specificity of the purified St-PepN was tested with several substrates (Table 5). Specificity was broad toward p-Na amino acid derivatives, but acidic amino acid derivatives were not released. Dipeptides and tripeptides were also hydrolyzed except for those containing Pro at the penultimate position. Dipeptides containing hydrophobic or basic amino acids at the N-terminus were preferentially degraded. The amino acid residue at position P'1 [according to the nomenclature of Schechter and Berger (21)]was also important. When the Journal of Dairy Science Vol. 77, No. 10, 1994

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RUL ET AL.

A 2

1

205

3

4

5

t

66.2

45

6

--

-

Figure 1. A. The SDS-PAGE profiles of the active products obtained after the different purification steps and the purified aminopeptidase St-PepN from Streptococcus salivarius ssp. thermophilus CNRZ 302. Lanes 1 and 6, molecular mass standard proteins (kilodaltons); lane 2, cell-free extract; lanes 3 and 4, first and second anion-exchange chromatography, respectively; and lane 5, hydrophobic interaction chromatography. B. The PAGE of the purified aminopeptidase St-PepN in the absence of SDS.Lane 1, Coomassie blue staining; lane 2, activity detected directly in the gel with Lys 8-naphthylamide as substrate

Figure 2. Effect of pH on activity of the purified aminopeptidase St-PepN of Streptococcus salivarius ssp. thermophilus CNRZ 302 on Lys-p-nitroanilide.

Journal of Dairy Science Vol. 77, No. 10, 1994

amino acid at P1' position was a basic or an aromatic amino acid, rates of hydrolysis were high compared with those of peptides having a small, uncharged amino acid (Gly, Ala). Larger peptides were hydrolyzed also; the first 5 residues of the 22-30 fragment of insulin Bchain, the first 7 residues of glucagon, and the first 3 residues of fragment 58-68 of &casein were liberated; aminopeptidase activity was then stopped by a prolyl or an acidic residue. This result and the fact that X-Pro dipeptides, bradykinin, and substance P were not hydrolyzed showed that St-PepN did not have the capacity to cleave X-Pro bonds. Furthermore, these data emphasized the broad specificity of

2885


TABLE 3. Effect of bivalent ions (1 on the activity of 1 the purified aminopeptidase of Streptococcus salivarius mM)

loo

ssp. thermphilus CNRZ 302 after inactivation with .1 mM EDTA. Metal ion

Relative activity ~~

CUCl2 CaC12 coc12 MgClZ znc12 Control

Figure 3. Effect of temperature on the activity of the purified aminopeptidase St-PepN of Streptococcus salivarius ssp. thermophilus CNRZ 302 on Lys-pnitroanilide in 50 mM triethanolamine buffer.

St-PepN because of the great variety of amino acids liberated from these peptides. No carboxypeptidase activity was detected with the substrates Cbz-Gly-Leu and Cbz-Phe-Leu nor endopeptidase activity on insulin B-chain, glucagon, fragment 58-68 of &casein, netenkephalin, bradykinin, substance P, or neurotensin.

TABLE 2. Effect of inhibitors on St-PepN activity of Streptococcus salivarius ssp. thermophilus CNRZ 302. Reagent

Iodoacetamide Iodoacetic acid p-Hydroxymercuribenzoate Diisopropylfluorophosphate Phenylmethanesulfonyl fluoride EDTA

.001 .01 .1 1

1 1 1 1

1 .1 1

10 o-Phenanthroline Control

N-Terminal Sequence

From a stained protein blot, the N-terminal sequence of St-PepN was determined as HzN-Lys-Ala-Ser-Val-Ala-Arg-Phe-Ile-GluSer-Phe-ne-FYo-Glu-(Thror Ser)-Tyr-Am-Leu?-Gly-Asp. DISCUSSION

In the present study, a general aminopeptidase, St-PepN, was purified and characterized from the intracellular extract of S. sulivan'us ssp. rhemphilus CNRZ 302. The enzyme was purified to homogeneity by a three-step procedure that led to a 190-fold purification with a yield of activity recovery of 19.5%. The St-PepN had a monomeric structure with a molecular mass of about 97 kDa as determined by SDS-PAGE and gel filtration,

wnal concentration Inhibition

(W Bestatin

0 45 0 7 13 100

.I 1

(94 0 3

55 100

0 3 0 0 0 83 83 100

TABLE 4. Effect of bivalent ions on the aminopeptidase St-PepN activity of Streptococcus salivarius ssp. thermophilus CNRZ 302. Ion

Concentration

Relative activity

(W

(%)

.1

cuc12 CaClz

.1 1

coc12

.1 1

MgC12

1

ZnCl2

0

Control

93 79 10 12

.1

70 98

20 0

1

.1 1

112

115 17 0 100


RUL ET AL.



which is similar to that of aminopeptidases N from Lactococcus lactis ssp. cremoris (9, 24), Lactobacillus helveticus (2, 10, 18), Lactobacillus delbrueckii ssp. bulgaricus (1. 3, 29) or ssp. lactis (ll), and S. salivarius ssp. thermophilus (30). The St-PepN was active in a broad pH range between pH 5 and 9.5 and optimal at pH 7, which is similar to the optimal pH range from 6.5 to 7.2 for other aminopeptidases N of LAB mentioned (1, 2, 3, 9, 10, 11, 18, 24, 29, 30). Variability was greater at optimal temperature; activity of St-PepN on Lys-p-Na was maximum at 36'C as for PepN of lactococci (7, 9, 24) or S. salivarius ssp. thennophilus ACA-DC 114 (31); however, several aminopeptidases N from lactobacilli (1, 3, 8, 10, 11). described until now, exhibited higher optimal temperature (at 45 to 50'C). Sulfhydryl and serine reagents had no effect on the enzyme activity. The St-PepN from S. salivarius ssp. themphilus CNRZ 302 is a metalloenzyme because it is inhibited by metal-chelating reagents (EDTA and ophenanthroline). As for the aminopeptidase characterized from lactobacilli (2, 8, 10, 18, 29) and from lactococci (24). St-PepN was completely inhibited by Cu*+. None of the bivalent ions tested had a positive effect on StPepN activity, and Co2+ was a good activator for most of the other aminopeptidases of LAB. Contrary to the aminopeptidase of S. salivarius ssp. thermuphilus ACA-DC 114, which was irreversibly inactivated by EDTA, the activity of St-PepN was partially restored with addition of Ca2+ after inactivation by EDTA. Our data on the N-terminal amino acid sequence of PepN of S. salivarius ssp. thermophilus compared with those of L. lactis ssp. cremoris Wg2 (26) and Lb. delbrueckii ssp. lactis (11) showed 43% identity (and about 67 and 62% homology, respectively, if analogous amino acids are considered) in the first 21 amino acid residues (Figure 4). However, comparison with the N-terminal sequence of the aminopeptidase N from Lb. bulgaricus (3) showed only slight identity with that of S. salivarius ssp. thennophilus, and research in Genbank database gave no other strong homology with other sequences. Usually, consensus sequences are found in the active site of the enzymes; here, lactococcal, lactobacillal, and streptococcal aminopeptidases present an im-

2887

portant homology in N-terminal sequences, which suggests that great similarities exist between the three aminopeptidases. On immunoblots made by Tan and Konings (Z), specific antibodies raised against the purified endopeptidase (70 kDa) of L. lactis ssp. cremoris Wg2 reacted with a protein of S. salivarius ssp. thennophilus CNRZ 302 with the same molecular mass, indicating the presence of an equivalent endopeptidase in s. salivarius ssp. thennophilus CNRZ 302. Our results directly show the existence of strong similarities among peptidases of Lactococcus, Lactobacillus, and Streptococcus species. Concerning optimal temperature of aminopeptidase, we observed that St-PepN from S. salivarius ssp. themphilus was more closely related to PepN from Lactococcus species than to PepN from Lactobacillus species. Homology between N-terminal amino acid sequence of St-PepN from S. salivarius ssp. thermophilus and PepN from L. lactis ssp. cremoris was higher than that between S. salivarius ssp. themphilus and Lactobacillus delbrueckii ssp. luctis. These results can be related to those from molecular taxonomy. Studies based on comparison of 16s rRNA have indeed shown that S. salivarius ssp. thermophilus presents a phylogenic position closer to Lactococcus species than to Lactobacillus species (23); S. salivarius ssp. thermuphilus and Lactococcus species have a common ancestor that is not shared by the Lactobacillus group. The cell fractionation protocol followed here to extract the aminopeptidase St-PepN, the similarity of N-terminal sequence of StPepN and that of PepN from L. luctis ssp. cremoris and from Lb. delbrueckii ssp. lactis, and more generally the several properties of St-PepN in common with other intracellular aminopeptidases from other LAB are indicative of an intracellular location of St-PepN from S. salivarius ssp. themphilus CNRZ 302. The similar biochemical properties, Nterminal amino acid sequence, and cellular location of St-PepN from S. salivarius s s ~ther. mophilus CNRZ 302 and PepN from lactococci, led us to name the aminopeptidase from the former, St-PepN. The aminopeptidase St-PepN had broad substrate specificity. Indeed, St-PepN could Journal of Dairy Science Val. 77, No. 10, 1994

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RUL ET AL.

Y

L. lactis ssp. cremoris Wg2 S.salivarius ssp. thermophilus CNRZ 302

K

A

1

Lb. delbrueckii ssp. lactis DSM7290

Y Y -

20

Figure 4. Comparison of the amino acid sequence of the aminopeptidases N of Streptococcus salivarius ssp. thermophilus CNRZ 302, Lactococcus lactis ssp. cremoris Wg2, and Lactobacillus delbrueckii ssp. lactis DSM7290. Black boxes correspond to the conserved regions in sequences. Numbers below the sequences refer to the amino acid positions in the aminopeptidases; + = amino acids with structural homology.

hydrolyze p-Na derivatives, dipeptides, and tripeptides (Table 5); it can also cleave sequentially amino acid residues from the N terminus of larger peptides such as fragment 22-30 of the insulin B-chain, glucagon, or peptides generated by the enzymatic digestion of caseins such as fragment 58-68 of @-casein. Cleavage to either side of a Pro does not occur as for many other proteases. Because S. salivarius ssp. rhernwphilus does not possess substantial extracellular proteolytic activity (22). the peptides released from caseins could appear after the action of the cell-wall-associated proteinase of lactobacilli, or after the action of rennet in the case of cheese manufacturing, or both. These peptides are rich in hydrophobic amino acids and Pro (31,32) and can be transported into the cell. Indeed, it has been suggested that S. salivarius ssp. thennophilus possesses an oligopeptide transport system (6). By its broad specificity, St-PepN should be able to degrade these peptides and thus be involved in N supply of bacteria during growth in milk. Nevertheless, the action of St-PepN must be complemented by that of other peptidases that can release prolyl or acidic residues. Such peptidases have been found in S. salivarius ssp. rhernwphilus CNRZ 302; an X-prolyldipeptidyl-aminopeptidase (X-PDAP) (16) able to release the N-terminal dipeptide X-Pro from X-Pro-Y- has been characterized as well as an aminopeptidase specific for acidic amino acids and Ser (Rul, 1993, unpublished data). The combined actions of these three peptidases should lead to a good utilization of peptides. The general aminopeptidase, St-PepN, of S. salivarius ssp. thernwphilus CNRZ 302 may contribute to flavor development during cheese ripening through its role in production of free amino acids. In milk products, the hydrophobic Journal of Dairy Science Vol. 77. No. 10, 1994

peptides liberated from caseins are often responsible for bitterness (31). Tan et al. (27) have shown that PepN of L. lactis ssp. cremoris could reduce bitter flavor of a trypsic hydrolysate of @-casein.This reduction of bitterness was related to the decrease of peptides sharing strong hydrophobicity. Thus, the ability of St-PepN, the aminopeptidase homologous to lactococcal PepN, to process such hydrophobic peptides may improve the taste of milk products by reducing bitter flavor. ACKNOWLEDGMENTS

This work was supported by the French Ministry of Research and Technology (Programme Aliment 2002, contract 9030927). We thank P. Anglade and G. Brignon for the N-terminal sequencing of the enzyme and peptides. REFERENCES

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