Aminopeptidase and Dipeptidyl Peptidase Activity of Lactobacillus spp. and Non-starter Lactic Acid Bacteria (NSLAB) isolated from Cheddar Cheese

PII : S0958-6946(98)00047-8

Int. Dairy Journal 8 (1998) 255—266 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00

Aminopeptidase and Dipeptidyl Peptidase Activity of Lactobacillus spp. and Non-starter Lactic Acid Bacteria (NSLAB) isolated from Cheddar Cheese Alan G. Williamsa,*, Xavier Felipeb and Jean M. Banksa aHannah Research Institute, Ayr KA6 5HL, Scotland KA65HL, UK bUnit of Food Technology, Faculty of Veterinary Science, Universitat Autonoma de Barcelona, 08193 Barcelona, Spain (Received for publication 6 May 1998) ABSTRACT The complexity of the peptidolytic enzyme systems of a variety (67 strains) of non-lactococcal dairy lactic acid bacteria was established. Diagnostic substrate hydrolysis by cell lysates confirmed the widespread occurrence of general aminopeptidase and dipeptidyl peptidase activities; lower levels of other more specific aminopeptidases were also indicated. Inhibitor studies demonstrated that more than one type of leucyl aminopeptidase and dipeptidyl peptidase were present in cell lysates. Although both activities were susceptible to metallo- and serine-type inhibitors, the predominant aminopeptidase activity was associated with the metalloenzyme group whereas the principal dipeptidyl peptidase activity was mediated by serine-type protease action. Some characteristics of individual enzymes comprising the aminopeptidase and dipeptidyl peptidase activity were determined for components separated by polyacrylamide gel electrophoresis and located in situ in the gel by activity staining with amidomethylcoumarin substrates. Three individual aminopeptidases with apparent molecular masses of 95, 44 and 30 kDa and a multimer (5180 kDa) were detected. The 95 kDa and 44 kDa enzymes were metalloenzymes whereas the 30 kDa enzyme was a serine peptidase. The 95 kDa component thus resembled pepN while the 44 and 30 kDa enzymes exhibited some characteristics of an infrequently described LAB metallodipeptidase and serine prolyl aminopeptidase, respectively. Two serine dipeptidyl peptidases, with apparent molecular masses of 88 and 125 kDa, were also detected after PAGE. The 88 kDa enzyme resembled X-prolyl-dipeptidyl aminopeptidase (pepX); the 125 kDa enzyme, not previously characterized in LAB, occurred widely among bacteria from this group. The lactobacilli, and other dairy lactic acid bacteria examined, have a complex of enzymes involved in peptide turnover, several enzymes of which have a commonality within the group. ( 1998 Elsevier Science Ltd. All rights reserved Keywords: NSLAB; Lactobacillus; Weissella; aminopeptidase; dipeptidyl peptidase; PAGE

the populations examined from some cheeses (Peterson and Marshall, 1990; Martley and Crow, 1993; Williams and Banks, 1997). The role of NSLAB in ripening has yet to be resolved satisfactorily (Peterson and Marshall, 1990), although the inclusion of adjunct cultures of some strains of non-starter lactobacilli with the starter lactococci in cheese manufacture indicated that they were involved in the release of free amino acids (Lane and Fox, 1996; Lynch et al., 1996). The released amino acids not only contribute directly to the cheese flavour but also act as precursors of other important flavour and aroma components. Peptidolytic strains of NSLAB may, therefore, be considered for use as adjuncts in cheesemaking to both manipulate the overall flavour profile of the cheese and to accelerate the rate of flavour formation (Fox et al., 1996). Many of the peptidases of the starter ¸actococcus spp. have been purified and characterized (Kunji et al., 1996), whereas there is a lack of detailed information on the peptidolytic enzymes produced by the non-starter lactic acid bacteria. A wide range of peptidolytic activities has been detected in strains of a variety of NSLAB species isolated from cheese (Sasaki et al., 1995a; Williams and Banks, 1997), but even though peptidase profiling offers a means for the non-random selection of potential adjunct strains (Peterson et al., 1990; Williams and

INTRODUCTION The breakdown and metabolism of peptides derived from casein during cheese maturation is essential for flavour development. The peptides are formed principally from a -casein and b-casein through the pro41 teolytic action of milk, coagulant and bacterial enzymes (Fox et al., 1996). Peptide turnover in ripening Cheddar cheese is effected by the starter lactococcal and adventitious non-starter lactic acid bacterial populations. Although the NSLAB numbers are initially low in Cheddar cheese produced commercially from pasteurized milk, they multiply during the early stages of maturation so that the NSLAB are predominant in the viable population throughout most of the period of ripening (Peterson and Marshall, 1990). The NSLAB present in Cheddar cheese are principally strains of homo- and heterofermentative species of mesophilic lactobacilli (Broome et al., 1990; Jordan and Cogan, 1993; McSweeney et al., 1993) although there are reports of the presence of ¸euconostoc, Pedicoccus and ¼eissella spp. in *Corresponding author. Tel: #44 1292 674081 fax: #44 1292 674006 E-mail: [email protected] 255

256

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Banks, 1997) characterization of individual peptidases produced by strains of non-starter ¸actobacillus spp. isolated from cheese has been undertaken only infrequently (Bockelmann, 1995; Kunji et al., 1996; Law and Haandrikman, 1997). Information is generally not available on the complexity of the peptidolytic systems and the component enzymes that comprise the overall peptidase activity in non-starter microorganisms. The objective of this investigation was the partial characterization of the aminopeptidase and dipeptidyl peptidase profiles of a wide range of non-starter ¸actobacillus and ¼eissella spp. isolated from Cheddar cheese and other ¸actobacillus spp. associated with dairy products. The study has produced information on the range, component enzymes, inhibitor susceptibilities and apparent molecular mass of aminopeptidases and dipeptidyl peptidases produced by NSLAB and other lactobacilli. This type of information will enable the selection of adjunct strains possessing the enzyme potential to complement the activities present in the starter lactococci and influence flavour development during cheese maturation. MATERIALS AND METHODS Bacterial cultures The 67 strains examined were chosen to include a wide selection of dairy lactobacilli. The NSLAB isolates (32) were recovered from good-quality Cheddar cheese (Williams and Banks, 1997); the type strains studied were obtained from the National Collection of Food Bacteria (Reading, UK), the exceptions being ¸b. coryniformis subsp. coryniformis 9711, ¸b. rhamnosus 9537 and ¸b. helveticus 11071, which were purchased from the National Collection of Industrial and Marine Bacteria (Aberdeen, UK) and ¸b. casei subsp. casei 334 which was from the American Type Culture Collection (Rockville, USA). All of the strains used are listed in Table 1 and were described initially as ¸actobacillus spp., but some genus re-assignment has occurred subsequently (Hammes and Vogel, 1995; Schleifer and Ludwig, 1995; Dicks et al., 1996). Cultures were maintained on MRS agar and were subcultured regularly to maintain viability. Preparation of crude enzyme fractions Cultures were grown for 48 h at 30°C in MRS broth containing 2% (w/v) glucose. The cells were recovered by centrifugation (16,000 g for 30 min at 4°C), washed and resuspended in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 7.0, 4°C), and disrupted at 4°C with an MSE Soniprep 150 ultrasonic disintegrator fitted with an exponential probe (tip diameter 9.5 mm) using ten 30 s disruption cycles (10 km amplitude) with 30 s cooling intervals. The supernatant fraction of the cell lysate after centrifugation (10,000 g for 15 min at 4°C) was used as the crude enzyme preparation. Aliquots of the cell-free lysate were stored at !20°C prior to further characterization. Duplicate preparations were obtained from separate cultures for each strain examined. Enzyme assay procedures The lysates were screened for the presence of aminopeptidase and dipeptidyl peptidase activities using

the appropriate fluorogenic 7-amido-4-methylcoumarin (AMC) substrates (0.1 mM in 0.1 M MES buffer, pH 7). Substrate hydrolysis was detected qualitatively after incubation at 30°C for a maximum period of 120 min by fluorescence on UV illumination of the assay mixture using a UVF model TLW-200 transilluminator. Enzyme activities and inhibitor effects were measured using p-nitroanilide derivatives (0.5 mM in 0.1 M MES buffer, pH 7). The p-nitroaniline released after incubation at 30°C was determined spectrophotometrically at 405 nm or after diazotization of the reaction supernatant (Williams and Banks, 1997). Proline aminopeptidase activity was determined using bradykinin N-terminal pentapeptide as substrate (1 mM in 0.1 M MES, pH 7). Amino acid release after incubation at 30°C for 30 min was determined colorimetrically using a modified Cdninhydrin reaction (Doi et al., 1981). Protein in the cell lysates was quantified by the dye-binding method of Bradford (1976). Substrates were purchased from Sigma Chemical Co. Ltd (Poole, UK) and Bachem UK Ltd (Saffron Walden, UK). The effects of inhibitors were determined by preincubating the cell-free lysate with the inhibitor for 60 min at 30°C in 0.1 M MES (pH 7.0) prior to initiating the reaction by the addition of the substrates. The inhibitor concentration was 1 mM with the exception of 3,4-dichloroisocoumarin (DCI, 0.2 mM), pepstatin (0.1 mM) and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64, 0.01 mM). Stock solutions of phenylmethylsulfonyl fluoride (PMSF), 1,10-phenanthroline and pepstatin were dissolved in methanol and DCI was dissolved in dimethyl sulfoxide (DMSO); all other inhibitors were used as aqueous solutions. Control incubations established that, at the concentrations used, the solvents methanol and DMSO did not affect enzyme activities. Peptidase analysis by SDS-polyacrylamide gel electrophoresis Peptidases in the bacterial lysates were separated using non-denaturing SDS-PAGE and the characteristics of the resolved enzymes in situ in the gel were examined. Lysates were mixed with an equal volume of sample buffer (62.5 mM Tris buffer, pH 6.8, containing 2%, w/v, SDS, 5%, v/v, mercaptoethanol, 10%, w/v, glycerol and 0.004% bromophenol blue) and were resolved, without prior heat treatment, using a BioRad mini-Protean system, in 10% (w/v) polyacrylamide gels at 15 mA/gel with a 62.5 mM Tris buffer system containing 192 mM glycine and 0.1% SDS (Laemmli, 1970). The SDS was removed from the gels after electrophoresis by washing, with shaking, in 2.5% (v/v) Triton X-100 at 20°C. The location of the resolved enzymes in the washed gel was determined after incubation for 10—20 min at 30°C in 0.1M MES buffer (pH 7) containing the appropriate AMC substrate (0.1 mM). The sites of substrate hydrolysis were detected by fluorescence of the released AMC under UV illumination. The apparent molecular mass of the resolved peptidases was determined from their mobility relative to those of known pre-stained protein standards. The effects of inhibitors were determined by pre-incubation of washed gels in 0.1 M MES buffer (pH 7) containing the inhibitor for 30 min at 30°C prior to substrate addition; the inhibitor concentrations were as detailed for studies with the cell-free lysates. Following substrate inclusion,

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the incubation was allowed to continue for a further 15 min at 30°C; the extent of enzymic activity was then assessed by UV illumination to detect substrate hydrolysis. RESULTS Aminopeptidase activities in cell-free lysates The presence of a general, leucyl, aminopeptidase activity in all 67 strains was confirmed qualitatively using leucine 7-amido-4-methyl-coumarin (Leu-AMC) as substrate; fluorescence under UV illumination after incubation at 30°C for 5—10 min confirmed that enzymic hydrolysis of the substrate had occurred. The presence of type IV dipeptidyl peptidase activity (McDonald and Barrett, 1986) in all the lysates was likewise confirmed using Gly—Pro-AMC as the diagnostic substrate. Incubation of the lysates for 2 h at 30°C with the diagnostic substrates Asp-AMC, Pyroglu-AMC and

Pro-AMC indicated that the occurrence of enzymes potentially exhibiting an aspartyl, pyroglutamyl or prolyl aminopeptidase activity was less widespread amongst the strains examined (Table 1). The mean activities detected, and the range in the species activity means, measured with p-nitroanilide derivatives after 15 h at 30°C were only 3(0—29), 14(0—80) and 27(0—155) nmol p-nitroaniline released mg~1 protein for the aspartyl, pyroglutamyl and prolyl aminopeptidases, respectively. Inter-species and strain variations in the occurrence and level of enzymic activity were evident. The presence of a proline aminopeptidase with an apparent specificity for an XPro-Pro N-terminal sequence was indicated in some of the lysates which were active against the N-terminal pentapeptide of bradykinin (Table 1). These preliminary studies with diagnostic substrates are suggestive of the presence in ¸actobacillus spp. and NSLAB of both general aminopeptidase and other more specific aminopeptidase activities, which in the lactococci are effected by separate enzymes.

Table 1. The occurrence of various aminopeptidase activities in non-starter lactic acid bacteria isolated from Cheddar cheese and type strains of dairy ¸actobacillus Aminopeptidase ¸actobacillus spp.

Strains

Leucyl

Aspartyl

Pyroglutamyl

Prolyl

Proline

¸b. acidophilus ¸b. bifermentans ¸b. brevis ¸b. buchneri ¸b. casei ssp. alactosus ¸b. casei ssp. casei ¸b. collinoides ¸b. (syn ¼.) confusus ¸b. coryniformis ssp. coryniformis ¸b. coryniformis ssp. torquens ¸b. curvatus ¸b. delbrueckii ssp. bulgaricus ¸b. delbrueckii ssp. delbrueckii ¸b. delbrueckii ssp. lactis ¸b. farciminis ¸b. fermentum ¸b. gasseri ¸b. (syn ¼.) halotolerans ¸b. helveticus ¸b. kefir ¸b. maltaromicus (syn Carnobacterium piscicola) ¸b. (syn. ¼.) minor ¸b. parabuchneri ¸b. paracasei ssp. tolerans ¸b. paracasei ssp. paracasei

1748 2736, M7, J2 1749, AR8, M2 I10, K10, L7 2713 161, ATCC334, G3, O1 2805, L8 1586 NCIMB 9711 2740 2739, AR9, C8, D7, K4 1489 213

#! # # # # # # # # # # # #

# # # # ! $ # # # ! $ ! #

!" $# # # ! $ # # ! # $ ! #

# # # # # # # # # # # # #

NT$ ! ! ! NT $ ! NT NT NT $ NT NT

1438 230, C3, F4 1750, G9, 012 2233 2781, F9 2712, NCIMB 11971, I8, N6 2737, C13, L12 2382

# # # # # # # #

! # # ! $ $ # #

! # # # $ # # #

# # $ # # # # !

NT $ ! NT # $ $ NT

2780 2748, L2, M12 2774 151, 2743, AR5, F11, H6, L10, O3 363 1752, AC1, E2, G2, L9 243, NCIMB9537* 1655, D5

# # # #

! $ ! $

! # # #

! # # #

NT $ NT $

# # # #

! $ $ $

! # $ #

! $ # #

NT $ NT !

¸b. ¸b. ¸b. ¸b.

pentosus plantarum rhamnosus (*syn ¸b. zeae) (syn. W.) viridescens

!#, all strains positive, "!, No detectable activity in any of the strains tested. #$, some strains possess detectable activity. $NT, not tested. Aminopeptidase P determinations, using bradykinin N-terminal pentapeptide as substrate, were only made on NSLAB strains isolated from Cheddar cheese; type strains were not tested. Other aminopeptidase activities were detected using the appropriate amidomethylcoumarin derivaties.

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Effect of inhibitors on leucyl aminopeptidase activity The effects of protease inhibitors on the leucyl aminopeptidase (LAP) activity present in the cell-free lysates indicated that more than one type of enzyme was present in the NSLAB strains and ¸actobacillus type cultures examined; the activity was inhibited most effectively by the metalloprotease and serine protease inhibitors examined (Table 2). The most effective inhibitors of ¸actobacillus and NSLAB LAP activity were thus the metalloprotease inhibitors, 1,10-phenanthroline and EDTA, the mean inhibitory effect ($S.D.) of which for all the strains tested was 84.0$12.3 and 75.1$21.0%, respectively. The corresponding values for the serine protease inhibitors, 3,4-dichloroisocoumarin and PMSF, were 32.5$14.0 and 13.5$10.3%, respectively. The cysteine protease inhibitors, E64 and iodoacetic acid, had little effect (mean inhibition 2.9 and 3.9%, respectively). The general (LAP) aminopeptidase activity in the lactobacilli is thus comprised of at least two enzymes, the predominant activity residing in a metalloenzyme. The effects of inhibitors on the proline aminopeptidase-type activity were examined using the NSLAB strains ¸b. plantarum L9, ¸b. parabuchneri L2 and ¼. halotolerans F9. The metalloprotease inhibitors, 1,10phenanthroline and EDTA, were again the most effective, causing reductions in activity of 69.4$17.6 and 75.8$18.6%, respectively. Some inhibition was caused by the serine-type inhibitors, with PMSF having a

greater effect than 3,4-dichloroisocoumarin (39.3$10.9 and 8.0$7.2% inhibition, respectively). The dipeptidyl peptidase inhibitor, diprotin A, reduced activity by only 13.7$8.6% and the cysteine inhibitor, E64, had a negligible effect on the activity of the preparations. The enzyme principally responsible for initiating the hydrolysis of the pentapeptide substrate would appear to be a metallo-type aminopeptidase; its response to serine type inhibitors differed from that of the LAP activity in that PMSF had a greater effect than 3,4-dichloroisocoumarin. Effect of inhibitors on dipeptidyl peptidase activity The hydrolysis of Gly-Pro p-nitroanilide by cell-free lysates of all strains of ¸actobacillus spp. and the cheese NSLAB examined was inhibited by inhibitors of serine protease action (Table 3). The mean activity reductions for all strains tested by 3,4-dichloroisocoumarin and PMSF were 70.7$14.9 and 79.1$20.9%, respectively. The inhibitory effects of the metalloprotease inhibitors 1,10-phenanthroline and EDTA (36.2$15.4 and 33.7$12.5%, respectively), were indicative of the presence of a second dipeptidyl peptidase activity or substrate hydrolysis by a metallo-type aminopeptidase. Hydrolysis by lysates of nine selected species (29 strains) was also inhibited by 0.1 M diprotin A (Table 3). The effectiveness of this specific inhibitor confirmed that in the strains examined, dipeptidyl peptidase activity was present in the lysates and involved in the hydrolysis of

Table 2. Effect of inhibitors on the leucyl aminopeptidase activity of ¸actobacillus spp. and non-starter lactic acid bacteria isolated from Cheddar cheese! Inhibition (%)" ¸actobacillus spp.

Strains

3,4-DCI

PMSF

1,10-Phe

EDTA

¸b. acidophilus ¸b. bifermentans ¸b. brevis ¸b. buchneri ¸b. casei ssp. casei ¸b. casei ssp. alactosus ¸b. casei ssp. pseudoplantarum (syn ¸b. paracasei ssp. paracasei) ¸b. collinoides ¸b. coryniformis ssp. coryniformis ¸b. coryniformis ssp. torquens ¸b. curvatus ¸b. delbrueckii ssp. bulgaricus ¸b. delbrueckii ssp. delbrueckii ¸b. farciminis ¸b. fermentum ¸b. gasseri ¸b. (syn ¼.) halotolerans ¸b. helveticus ¸b. kefir ¸b. parabuchneri ¸b. paracasei ssp. paracasei ¸b. paracasei ssp. tolerans ¸b. plantarum ¸b. rhamnosus ¸b. zeae (syn ¸b. rhamnosus) ¸b. (syn ¼.) viridescens

1748 2736, M7, 1749, AR8, M2 I10, K10, L7 161, ATCC334, G3, O1 2713 2743

32.3 26.7 13.3 12.7 38.0 42.1 17.9

20.1 8.6 7.1 7.6 22.2 18.4 3.0

93.8 82.5 86.4 91.2 81.2 86.8 97.8

88.6 74.7 89.7 84.4 73.4 92.1 98.1

2805, L8 NCIMB9711 2740 2739, AR9, C8, D7 1489 213 2330, C3, F4 1750, G9, D12 2233 F9 2712, I8, N6 2737, C13, L12 2748, L2, M12 151, F11, N6, L10, O3 2774 1752, E2, L9 243 NCIMB9537 D5

26.6 32.9 48.1 35.7 58.7 34.6 30.2 15.2 57.8 36.9 29.6 12.0 18.6 34.9 59.4 52.7 34.6 21.0 22.8

12.0 2.9 19.0 12.7 12.3 12.7 24.5 5.0 4.4 21.9 7.5 ND 3.5 17.9 37.7 26.1 6.6 ND 37.7

81.0 93.1 71.3 84.9 95.3 80.7 83.3 98.4 60.0 73.5 92.1 93.6 96.3 81.5 57.3 64.2 98.5 97.8 62.3

74.4 26.0 47.7 77.3 95.7 63.4 75.4 95.2 44.4 60.4 88.8 85.5 98.6 75.3 34.3 72.0 97.0 98.6 37.8

!Activity measurements were made in duplicate using Leu-p-nitroanilide as substrate. "Mean inhibition (%) of LAP activity for strains indicated. ND, no inhibitory effect detected.

259

Peptidases of Lactobacilli and NSLAB

the diagnostic substrate. Pepstatin, an aspartic-type inhibitor, and the thiol-type inhibitors, iodoacetamide and E-64, caused a reduction in activity of 45%, confirming that the principal dipeptidyl peptidase activity in NSLAB and ¸actobacillus spp. was effected by serine dipeptidyl peptidases. PAGE analysis of leucyl aminopeptidase activity The aminopeptidase profile of NSLAB and other ¸actobacillus spp. was characterized using PAGE electrophoresis; individual enzymes were located in situ by activity staining using amidomethylcoumarin amino acid derivatives. Three general leucyl aminopeptidase activ-

ities were detectable by this method; their apparent molecular masses were 95, 44 and 30 kDa (Fig. 1a). A fourth activity with a molecular mass of '180 kDa was probably a multimer of one of the other enzymes. Activity (band intensity) was maximal at or close to pH 7 and post-electrophoretic incubation with the AMC derivatives of alanine, arginine, lysine or phenylalanine did not reveal the presence of any other enzyme activities. The substrate specificities of the separated aminopeptidases differed. The 94 and 44 kDa enzymes degraded Ala-AMC, Arg-AMC, Leu-AMC and Phe-AMC; no activity was detected with the 30 kDa enzyme, in the preparations tested, against Arg-AMC whilst the '180 kDa enzyme did not appear to degrade Phe-AMC. Activity

Table 3. Effect of inhibitors on the dipeptidyl peptidase activity of ¸actobacillus spp.! Inhibition (%)" ¸actobacillus spp.

Strains

3,4-DCI

PMSF

1,10-Phe

EDTA

¸b. acidophilus ¸b. bifermentans ¸b. brevis ¸b. buchneri ¸b. casei ssp. casei ¸b. curvatus ¸b. delbrueckii ssp. bulgaricus ¸b. delbrueckii ssp. delbrueckii ¸b. delbrueckii ssp. lactis ¸b. farciminis ¸b. fermentum ¸b. gasseri ¸b. (syn. ¼.) halotolerans ¸b. helveticus ¸b. kefir ¸b. (syn ¼.) minor ¸b. parabuchneri ¸b. paracasei ssp. paracasei ¸b. paracasei ssp. tolerans ¸b. plantarum Lb. (syn ¼.) viridescens

1748 2736, M7 1749, AR8, M2 I10, K10, L7 161, ATCC334, G3, 2739, AR9, C8, D7 1489 213 1438 2330, C3, F4 1750, G9, O12 2233 2781, F9 2712, I8, N6 2737, C13, L12 2780 2748, L2, M12 151, 2743, F11, H6, L10, O3 2774 1752, E2, L9 D5

58.4 44.0 53.4 63.0 96.3 80.9 73.4 63.6 67.5 73.2 58.0 57.9 91.5 87.8 73.8 66.3 54.8 98.3 92.0 66.7 64.3

67.7 79.0 44.6 95.2 99.0 92.9 47.1 50.0 59.9 65.3 91.9 68.4 79.3 94.4 98.3 63.7 96.8 99.7 96.0 99.1 99.3

31.8 25.1 37.1 37.0 42.0 49.7 16.8 22.1 16.4 26.6 27.8 21.0 32.2 37.4 34.2 32.9 29.1 45.2 80.0 46.4 69.0

33.9 12.1 33.0 31.8 29.8 31.7 22.9 30.1 24.6 31.1 22.7 21.0 57.2 30.1 44.9 29.7 25.5 53.7 68.1 31.3 31.8

!Activity measurements were made in duplicate using Gly—Pro-p-nitroanilide as substrate. "Mean inhibition (%) of activity for number of strains indicated.

Fig. 1(a). Leucyl aminopeptidase activities of lactic acid bacteria. UV-visualised hydrolysis zones, after 5 min post-PAGE incubation of gel with Leu-AMC, effected by lysates from 1, ¸b. helveticus NCFB 2712; 2, ¸b. kefir NCFB 2737; 3, ¸b. rhamnosus NCFB 243; 4, ¸b. rhamnosus (syn ¸b. zeae) NCIMB 9537; 5, ¸b. maltaromicus (syn Carnobacterium piscicola) NCFB 2382. Fig. 1(b–d). Effect of inhibitors on leucyl aminopeptidase activities of NSLAB:- 1, ¸b. (syn ¼.) viridescens D5; 2, ¸b. casei G3; 3, ¸b. curvatus D7; 4, ¸b. fermentum G9; 5, ¸b. helveticus N6; 6, ¸b. (syn ¼.) halotolerans F9. The control gel (no inhibitor) Fig. 1(b), and the effects of 3,4-dichloroisocoumarin (c), and 1,10-phenanthroline (d) are shown. The positions of molecular mass markers are indicated.

260

A. G. Williams et al.

was, however, also detectable against Pro-AMC in the 30 kDa fraction of 10 of the preparations (213, 1438, 1489, 2712, 2740, AR8, E2, F9, L8, N6). Pre-incubation of the gels with protease inhibitors for 30 min prior to addition of Leu-AMC substrate revealed that the separated enzymes had different inhibitor susceptibilities (Fig. 1b—d). The 30 kDa component was a serine-type enzyme that was inhibited by 3,4-dichloroisocoumarin (Fig. 1c), whereas the 95 and 44 kDa aminopeptidases were metalloproteases which were inhibited by 1, 10-phenanthroline (Fig. 1d). The inhibition profile was consistent in all strains examined. Examination of lysates of over 20 species ('50 strains) of ¸actobacillus, 4 species (5 strains) of ¼eissella and 1 strain of Carnobacterium confirmed the widespread occurrence of multiple general (leucyl) aminopeptidase activities (Table 4). The presence of other enzymes cannot be excluded as those with low activity or different substrate specificity and those irreversibly inactivated during PAGE would not have been detected. This preliminary survey indicates that the occurrence of both serine- and metallo-type aminopeptidase enzymes is not uncommon in NSLAB and other dairy strains of lactic acid bacteria (LAB). PAGE analysis of dipeptidyl peptidase activity Two distinct enzymes with activity against Gly-Pro AMC were detected in situ in 10% (w/v) polyacrylamide gels after electrophoretic separation of lysates of ¸actobacillus spp. and other LAB (Figs 2a and 3a). Their apparent molecular masses were 88 and 125 kDa. A third zone of enzymic hydrolysis was detected with ¸b. delbrueckii subsp. bulgaricus 1489, ¸b. delbrueckii subsp. lactis 1438, ¸b. fermentum G9 and 012 and ¸b. kefir 2737, C13 and L12; the molecular mass of this activity was '180 kDa, indicating that it was a multimer of the 125 kDa enzyme, as the 88 kDa form was not detectable in two strains of ¸b. fermentum and ¸b. kefir (Table 5). There is also a possibility that a different form of dipeptidyl peptidase exists in some strains of lactobacilli, because although hydrolysis zones were not detectable after electrophoresis the crude lysates exhibited considerable dipeptidyl peptidase activity with Gly-Pro AMC as substrate. This latter group included strains of ¸b. brevis, ¸b. casei, ¸b. curvatus, ¸b. collinoides, ¸b. farciminis, ¸b. helveticus, ¸b. paracasei ssp. paracasei, ¸b. plantarum and ¼. halotolerans. A survey of the occurrence of the two principal forms of dipeptidyl peptidase confirmed that many strains (32%) possessed both the 88 and 125 kDa forms of the enzyme (Table 5). The 125 kDa enzyme was not detected in any of the strains of ¼eissella examined, but was shown to be present in 73% of the LAB strains examined, and was the only enzyme present in 42% of the isolates that exhibited some activity in gels. The 88 kDa form was the only dipeptidyl peptidase detected in 25% of the isolates, although it was shown to occur in just over half (57%) of the lysates (Table 5). The three forms of the enzyme were inhibited by the dipeptidyl peptidase type IV inhibitor, diprotin A (Fig. 3c), but not by the aminopeptidase inhibitor, amastatin (Fig. 3b), confirming that substrate hydrolysis had been effected by a dipeptidyl peptidase mechanism. Enzymic activity was inhibited by serine-type inhibitors (Figs 2b and 3d) but activity in gels was not affected by metalloen-

Table 4. Aminopeptidases detected in lysates of ¸actobacillus type strains and non-starter lactic acid bacteria isolates by activity staining with Leu-AMC after non-denaturing polyacrylamide gel electrophoresis Aminopeptidase (kDa) ¸actobacillus strain

30

¸b. acidophilus 1748 ¸b. bifermentans 2736 ¸b. bifermentans M7 ¸b. bifermentans J2 ¸b. brevis 1749, AR8 ¸b. brevis M2 ¸b. buchneri I10 ¸b. parabuchneri 2748 ¸b. parabuchneri L2, M12 ¸b. casei ssp. casei 161 ¸b. casei O1 ¸b. casei G3 ¸b. paracasei ssp. paracasei 151, AR5, F11, H6, L10, O3 ¸b. paracasei ssp. tolerans 2774 ¸b. collinoides 2805 ¸b. collinoides L8 ¸b. confusus 1586 (syn W. confusa) ¸b. coryniformis ssp. torquens 2740 ¸b. curvatus 2739, D7 ¸b. curvatus AR9, C8, K4 ¸b. delbrueckii ssp. delbrueckii 213 ¸b. delbrueckii ssp. lactis 1438 ¸b. delbrueckii ssp. bulgaricus 1489 ¸b. farciminis 2330 ¸b. farciminis F4 ¸b. fermentum 1750 ¸b. fermentum G9, O12 ¸b. gasseri 2233 ¸b. halotolerans F9 (syn ¼. halotolerans) ¸b. helveticus 11971, 2712, I8, N6 ¸b. kefir 2737 ¸b. kefir C13 ¸b. maltaromicus 2382 (syn. Carnobact. piscicola) ¸b. minor 2780 (syn. ¼. minor) ¸b. pentosus 363 ¸b. plantarum 1752, G2, J1 ¸b. plantarum E2 ¸b. plantarum AC1 ¸b. plantarum L9 ¸b. rhamnosus 243 ¸b. viridescens 1655 (syn ¼. viridescens) ¸b. viridescens D5 (syn ¼. viridescens)

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zyme and cysteine (thiol)-type enzyme inhibitors. The 88 kDa enzyme was more susceptible to inhibition by 3,4-dichloroisocoumarin (Fig. 2b), whereas the 125 kDa enzyme was inhibited to a greater extent by PMSF (Fig. 3d) indicating that they are different enzymes rather than multimers with a common basal monomer. At the

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261

Fig. 2. Dipeptidyl peptidase activities of lactic acid bacteria. UV-visualized hydrolysis zones after incubation with Gly-Pro-AMC, in the absence (a) or presence of 3,4-dichloroisocoumarin (b), of PAGE-resolved lysates of 1, ¸b. curvatus NCFB 2739; 2, ¸b. (syn. ¼.) minor NCFB 2780; 3, ¸b. parabuchneri NCFB 2748; 4, ¸b. delbrueckii subsp. lactis NCFB 1438; 5, ¸b. acidophilus NCFB 1748; 6, ¸b. gasseri NCFB 2233; 7, ¸b. maltaromicus (syn Carnobacterium piscicola) NCFB 2382; 8, ¸b. helveticus NCFB 2712; 9, ¸b. rhamnosus (syn. ¸b. zeae) NCIMB 9537. The positions of molecular mass markers are indicated.

Fig. 3. Effect of inhibitors on dipeptidyl peptidase activity of lysates of non-starter lactic acid bacteria resolved by PAGE with Gly-Pro-AMC as substrate. The following inhibitors were used: control (a); amastatin (b); diprotin A (c); PMSF (d). The NSLAB are 1, ¸b. fermentum G9; 2, ¸b. kefir L12; 3, ¸b. (syn. ¼.) viridescens D5 and 4, ¸b. casei G3. The positions of molecular mass markers are indicated.

concentrations used, inhibition by PMSF (1 mM) and 3,4-dichloroisocoumarin (0.2 mM) was not total and some residual activity and substrate hydrolysis was detectable under UV-illumination after incubation for 15—20 min at 30°C. DISCUSSION Lactic acid bacteria are nutritionally fastidious and require preformed amino acids for growth. The concentration of free amino acids in milk and cheese curd is insufficient to sustain growth and hence this group of microorganisms must have a well-developed proteolytic enzyme system to ensure their survival and development in milk-based products. The proteolytic system of the

starter ¸actococcus has been studied in detail and comprises a cell wall-associated proteinase, several intracellular peptidases and at least six aminopeptidases with differing specificities (Kunji et al., 1996). In comparison, the range and nature of the enzymes formed by ¸actobacillus, that constitute the dominant viable bacterial population in cheese during maturation, are less well characterized (Bockelmann, 1995). The peptidolytic enzymes formed by the NSLAB generate amino acids that are a prerequisite for the growth of the bacteria (Morishita et al., 1981; Hammes and Vogel, 1995) and are also precursors for the formation of cheese flavour components. Enzymes similar to many of the well-characterized lactococcal peptidolytic enzymes have been described in the lactobacilli (Bockelmann, 1995), although these studies have been performed on only a limited number of

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Table 5. Dipeptidyl peptidase activities detected in lysates of ¸actobacillus type strains and non-starter lactic acid bacteria isolates using activity staining with Gly-Pro-AMC as substrate after non-denaturing polyacrylamide gel electrophoresis Dipeptidyl peptidase (kDa) ¸actobacillus strain

88

125

¸b. acidophilus 1748 ¸b. bifermentans 2736 J2 ¸b. bifermentans M7 ¸b. buchneri I10, K10, L7 ¸b. parabuchneri 2748, L2, M12 ¸b. casei ssp. alactosus 2713 ¸b. casei ssp. casei G3 ¸b. paracasei ssp. paracasei 2743 (syn ¸b. casei ssp. pseudoplantarum) ¸b. paracasei ssp. tolerans 2774 ¸b. collinoides 2805 ¸b. confusus 1586 (syn W. confusa) ¸b. coryniformis ssp. coryniformis 9711 ¸b. coryniformis ssp. torquens 2740 ¸b. curvatus 2739 ¸b. delbrueckii ssp. delbrueckii 213 ¸b. delbrueckii ssp. bulgaricus 1489 ¸b. delbrueckii ssp. lactis 1438 ¸b. farciminis 2330 ¸b. fermentum 1750 ¸b. fermentum G9, O12 ¸b. gasseri 2233 ¸b. halotolerans 2781 (syn ¼. halotolerans) ¸b. helveticus 2712 ¸b. helveticus N6 ¸b. kefir 2737, C13 ¸b. kefir L12 ¸b. maltaromicus 2382 (syn Carnobacterium piscicola) ¸b. minor 2780 (syn ¼. minor) ¸b. pentosus 363 ¸b. rhamnosus 9357 (syn. ¸b. zeae) ¸b. viridescens 1655 (syn ¼. viridescens)

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Hydrolysis zones were not detectable in gels of the following resolved lysates, even though Gly—Pro-AMC was hydrolysed by the cell-free lysate before electrophoresis: ¸b. brevis NCFB1749, AR8, M2; ¸b. casei NCFB 161, ATCC334; ¸b. collinoides L8; ¸b. curvatus AR9, C8, D7; ¸b. farciminis C3, F4; ¸b. (syn ¼.) halotolerans F9; ¸b. helveticus I8; ¸b. paracasei ssp. paracasei NCFB151, F11, H6, L10, 03; ¸b. plantarum AC1, E2, G2, L9.

species. The similarity of the enzyme systems in the two genera is not unexpected as both types of lactic acid bacteria have evolved to survive in the same ecosystem. In view of the potential involvement of ¸actobacillus spp. in cheese ripening, this study was undertaken to characterize more fully the peptidolytic enzyme systems in

a wide range of NSLAB and other dairy lactic acid bacteria, that initially had been assigned to the genus ¸actobacillus. The hydrolysis of diagnostic substrates by the lysates was indicative of the presence of more than one type of aminopeptidase. Under the growth and assay conditions used, the activities of the prolyl- (proline iminopeptidase), proline- (aminopeptidase P), pyroglutamyl-(pyrrolidonecarboxylyl peptidase) and aspartyl (glutamyl) aminopeptidase-like enzymes were low in comparison to the leucyl aminopeptidase activity, and it may be that a single broad-specificity aminopeptidase was responsible for the degradation of all the substrates tested. An enzyme with a very broad substrate specificity, including prolyl aminopeptidase activity, has been purified, for example, from a dairy strain of ¸b. helveticus (Sasaki et al., 1996). However, there are also reports of the presence of the proline iminopeptidase (prolyl aminopeptidase) gene (pepI) in ¸b. helveticus, ¸b. delbrueckii subsp. bulgaricus and ¸b. delbrueckii subsp. lactis (Atlan et al., 1994; Klein et al., 1994b; Varmanen et al.,1996); the enzyme has also been purified from ¸b. casei and ¸b. helveticus (Miyakawa et al., 1994a; Habibi-Najafi and Lee, 1995). The detection of low enzyme activities with diagnostic aminopeptidase substrates in this and other studies (e.g. Sasaki et al., 1995a; Williams and Banks, 1997) is an indication that the lactobacilli, like the lactococci, possess several aminopeptidases with different specificities although this conclusion generally requires confirmatory biochemical or molecular biological evidence. However, enzyme characterization studies have identified the presence of several peptidolytic enzymes in ¸b. helveticus CNRZ32 (Kunji et al., 1996), ¸b. casei subsp. casei IFPL 731 (Fernandez de Palencia et al., 1997) and ¸b. delbrueckii subsp. bulgaricus B14 (Bockelmann, 1995), while distinct genes encoding two separate cysteine aminopeptidases have been identified in ¸b. delbrueckii subsp. lactis DSM 7290 (Klein et al., 1994a, 1997). The inhibitory effects of both metalloprotease and serine protease inhibitors on the aminopeptidase activity in the cell lysates indicated the presence of at least two distinct aminopeptidases in a wide range of lactic acid bacteria. At the concentrations used, the thiol inhibitors, E64 and iodoacetamide, had no significant effect on the total activity, although a cysteine aminopeptidase, pepC-like, has been characterized in ¸b. delbrueckii subsp. bulgaricus B14 (Wohlrab and Bockelmann, 1993) and homology to the pepC gene of ¸b. helveticus CNRZ32 occurs widely among the lactic acid bacteria (Fernandez et al., 1994). The failure to detect thiol aminopeptidase activity is undoubtedly related to the inhibitors used. E64 is an active site-specific inhibitor that does not affect other cysteine residues, while Wohlrab and Bockelmann (1993) have shown that 1 mM iodoacetate only slightly reduces the activity of the ¸b. delbrueckii subsp. bulgaricus enzyme (Wohlrab and Bockelmann, 1993); p-hydroxymercuribenzoic acid, however, strongly inhibited its activity. The presence of thiol aminopeptidase activity may therefore have been more reliably detected with higher concentrations or alternative inhibitors. Hydrolysis of the N-terminal pentapeptide (R-P-P-GF) of bradykinin was also inhibited by metalloprotease inhibitors. In the lactococci, an enzyme, aminopeptidase P, with a specificity for X-Pro-Pro N-terminal sequences

Peptidases of Lactobacilli and NSLAB

has been described (Mars and Monnet, 1995; McDonnell et al., 1997). The presence of this enzyme in other lactic acid bacteria has yet to be confirmed, and the activities observed here with crude lysates are equivocal, as the substrate degradation could have been effected by other proline-specific enzymes. However, there is little published evidence to indicate that aminopeptidases purified from lactobacilli are able to cleave N-terminal amino acids with an adjacent proline residue. A 32 kDa metalloenzyme from ¸b. delbrueckii subsp. bulgaricus (Wohlrab and Bockelmann, 1994) was able to hydrolyse some residues linked to proline, although Arg—Pro—Tyr was not hydrolysed. A serine aminopeptidase with broad specificity from ¸b. casei IFPL 731 degraded di- and tripeptides with proline adjacent to the N-terminal position (Fernandez-Espla et al., 1997a) and a serine dipeptidyl peptidase of another ¸b. bulgaricus strain (LBU147) exhibited activity against Arg—Pro—Pro (Miyakawa et al., 1991). A metallo-type prolidase (proline dipeptidase) has also been characterized in ¸b. casei subsp. casei IFPL731 (Fernandez-Espla et al., 1997b), but the substrate range of this enzyme was restricted to dipeptides. The presence of these proline-specific peptidases does not preclude the existence of a proline aminopeptidase (aminopeptidase P-like) in non-lactococcal species of lactic acid bacteria and further studies on the occurrence of this enzyme are warranted in view of the fact that aminopeptidase P appears to be an important debittering enzyme in cheese (McDonnell et al., 1997). The complexity of the peptidolytic systems of the NSLAB and other lactic acid bacteria was examined further using polyacrylamide gel electrophoresis and activity staining. It was also possible to establish some characteristics of the component enzymes without further purification stages. Three separate enzymes and a high molecular mass activity were detected using the fluorogenic substrate leucine amidomethylcoumarin. Further aminopeptidases that cannot be resolved by the protocol adopted may be present in the LAB. Nevertheless, these findings on the multiplicity of the aminopeptidase activity of lactobacilli are in general agreement with earlier studies that utilized electrophoretic resolution (El Soda and Desmazeaud, 1982; El Soda et al., 1982, 1983; Abo-Elnaga and Plapp, 1987; Hegazi and AboElnaga, 1987; Khalid et al., 1991), and provide information on the incidence and some characteristics of the enzymes in a considerably extended range of LAB. The 95 and 44 kDa enzymes are metalloenzymes that were inhibited by 1,10-phenanthroline. The 95 kDa leucyl aminopeptidase thus resembles PepN which has been characterized in a limited number of strains of ¸b. helveticus and the subspecies of ¸b. casei and ¸b. delbrueckii (Tsakalidou et al., 1993; Arora and Lee, 1994; Kunji et al., 1996; Fernandez de Palencia et al., 1997). It is a general aminopeptidase and, with the exception of Pro-AMC, exhibited activity against the other amino acid-AMC substrates tested. Its presence was detected in 26 different species of ¸actobacillus, Carnobacterium and ¼eissella; the enzyme occurs widely in the lactococci and other lactic acid bacteria, suggesting an important role for this enzyme in peptidolysis. The molecular mass of the other metallopeptidase (44 kDa) is similar to that reported for pepC and pepP (Kunji et al., 1996). However, aminopeptidase C is a thiol enzyme with a molecular mass of approximately 50 kDa in the three ¸actobacillus spp. studied (Wohlrab and Bockelmann, 1993;

263

Fernandez et al., 1994; Klein et al., 1994a). The occurrence of aminopeptidase P in lactobacilli has yet to be confirmed and although the activity detected was of the appropriate molecular mass, its substrate specificity was atypical and in addition the low level of aminopeptidase P-like activity in the crude hydrolysate would probably prevent its detection in the gel system used. However, a metallo-type aminopeptidase having a sub-unit molecular mass of 39 kDa has been characterized in ¸b. acidophilus (Machuga and Ives, 1984) and a metallotripeptidase is present in ¸b. delbrueckii ssp. bulgaricus (Bockelmann et al., 1997). In addition, a metal-dependent dipeptidase with a molecular mass of approximately 50 kDa has been purified from ¸b. delbrueckii, ¸b. casei, ¸b. sake and ¸b. helveticus (Wohlrab and Bockelmann, 1992; Vongerichten et al., 1994; Montel et al., 1995; Tan et al., 1995; Fernandez-Espla and Martin-Hernandez, 1997). There are also other reports of a similar metallotype dipeptidase in other strains of ¸b. helveticus (Kunji et al., 1996) and the activity detected in many of the strains examined may be this dipeptidase. The 30 kDa molecular mass aminopeptidase is a serine-type enzyme that was inhibited by 3,4-dichloroisocoumarin. This enzyme, therefore, differs from the 32 kDa metallo-aminopeptidase and 29 kDa metallotripeptidase of ¸b. delbrueckii subsp. bulgaricus (Wohlrab and Bockelmann, 1994; Bockelmann et al., 1995), the 30 kDa metallo-oligopeptidase of ¸b. paracasei (Tobiassen et al., 1997) and the 35 kDa thiol-aminopeptidase characterized in ¸b. sake (Sanz and Toldra, 1997). However, a serine prolyl aminopeptidase with a molecular mass of 33 kDa is present in some strains of ¸b. delbrueckii, ¸b. helveticus and ¸b. casei subsp. casei (Gilbert et al., 1994; Klein et al., 1994b; Habibi-Najafi and Lee, 1995; Varmanen et al., 1996), and a 33 kDa serine-dependent dipeptidase with prolinase (pepR) activity has been characterized in ¸b. helveticus CNRZ32 (Shao et al., 1997). The 30 kDa activity was present in most of the strains examined and in some of these, Pro-AMC was hydrolysed by the enzyme. It further resembled the proline iminopeptidase (prolyl aminopeptidase) of ¸b. casei in that it degraded Leu-AMC, but had no detectable activity on Arg-AMC (Habibi-Najafi and Lee, 1995). The electrophoretic and activity-staining procedure has thus enabled the tentative identification of some principal peptidase activities in the lactic acid bacteria. The frequency of detection among the isolates examined implies a general importance for these enzymes within the lactic acid bacteria. The method used was restricted to the detection of enzymes that were not inactivated during electrophoresis. A higher incidence of detection and the presence of other peptidolytic enzymes may result from the monitoring of enzymic protein expression or gene detection. X-prolyl dipeptidyl aminopeptidase (pepX) is a key proline-specific peptidase in dairy lactic acid bacteria. Dipeptidyl peptidase activity, detected using Gly-ProAMC as substrate, was present in lysates of all the strains of lactic acid bacteria examined. The frequent detection of this activity confirms earlier reports of the widespread occurrence of the enzyme in dairy lactic acid bacteria (Casey and Meyer, 1985; Williams and Banks, 1997). Inhibitor susceptibilities of Gly-Pro-AMC hydrolysis indicated that the principal activity was effected by a serine dipeptidyl peptidase type IV enzyme. The metalloprotease inhibitor susceptibility in crude lysates may also

264

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indicate the presence of another dipeptidyl peptidase form, or, alternatively, that some hydrolysis may have been mediated by aminopeptidase action. Lysates of lactic acid bacteria will hydrolyse Gly—Arg-AMC and Gly—Phe-AMC (Williams and Banks, 1997) although purified pepX has no effect on these dipeptidyl peptidase substrates (Khalid and Marth, 1990; Habibi-Najafi and Lee, 1994). Electrophoretic analysis of the dipeptidyl peptidase activity revealed two enzymes able to hydrolyse Gly—Pro-AMC. The presence of other forms was also indicated as in some strains a higher molecular mass component ('180 kDa) was detected, and in others the activity initially present in the crude lysate was not detectable after electrophoresis. In previous electrophoretic studies, a single activity has usually been identified, although the presence of two separate dipeptidyl activities in single ¸b. brevis and ¸b. delbrueckii subsp. bulgaricus strains was observed (Casey and Meyer, 1985; Sasaki et al., 1995b). In these latter studies, inter-species differences in the R values, and hence molecular masses, of & the activities were reported, whereas the data obtained in the present survey indicate a commonality in the 88 and 125 kDa dipeptidyl peptidases of the lactic acid bacteria. The two serine dipeptidyl peptidases were inhibited by diprotin A, but not by amastatin, and did not hydrolyse Pro-AMC or other single amino acid-AMC derivatives tested, confirming that dipeptide-AMC substrate degradation was not mediated by sequential aminopeptidase action. The 88 kDa enzyme thus resembles the enzyme described as X-prolyl-dipeptidyl aminopeptidase in the lactococci and a limited number of strains of ¸b. casei, ¸b. delbrueckii and ¸b. helveticus (Meyer and Jordi, 1987; Miyakawa et al., 1994b; Kunji et al., 1996). There are no previous reports of a 125 kDa enzyme with prolyl dipeptidyl peptidase activity, although the evidence presented indicates that the enzyme occurs widely in dairy lactic acid bacteria. Nine peptidolytic enzymes have been purified and characterized from dairy lactobacilli (Bockelmann, 1995; Kunji et al., 1996), but in many instances information is available only on an individual enzyme in a single strain. With the exception of the two industrially important strains ¸b. helveticus CNRZ32 and ¸b. delbrueckii subsp. bulgaricus B14 and the Spanish caprine cheese isolate ¸b. casei IFPL731, the complexity of the peptidolytic systems of most species of non-lactococcal lactic acid bacteria is not known. This study, using type cultures and strains of NSLAB isolated from cheese, has attempted to redress this imbalance. The implication is that the dairy lactobacilli, in common with cheese starter lactococci, have a complex of enzymes involved in peptide hydrolysis. The NSLAB have the enzymic capacity to contribute to peptide turnover and hence amino acid generation and flavour formation during cheese maturation. ACKNOWLEDGEMENTS The EC-AAIR Programme (Contract 93-1531) and The Scottish Office (Agriculture, Environment and Fisheries Department) provided financial support for these investigations. The technical support of Mrs A. Limond and Mr S. Hamilton is acknowledged.

REFERENCES Abo-Elnaga, I. G. and Plapp, R. (1987) Peptidases of ¸actobacillus casei and ¸. plantarum. Journal of Basic Microbiology 27, 123—130. Arora, G. and Lee, B. H. (1994) Purification and characterization of an aminopeptidase from ¸actobacillus casei subsp. rhamnosus S93. Biotechnology and Applied Biochemistry 19, 179—192. Atlan, D., Gilbert, C., Blanc, B. and Portalier, R. (1994) Cloning, sequencing and characterization of the pepIP gene encoding a proline iminopeptidase from ¸actobacillus delbrueckii subsp. bulgaricus CNRZ397. Microbiology 140, 527—535. Bockelmann, W. (1995). The proteolytic system of starter and non-starter bacteria: components and their importance for cheese ripening. International Dairy Journal 5, 977—994. Bockelmann, W., Beuck, H-P., Lick, S. and Heller, K. (1995) Purification and characterization of a new tripeptidase from ¸actobacillus delbrueckii ssp. bulgaricus B14. International Dairy Journal 5, 493—502. Bockelmann, W., Gollan, V. and Heller, K. J. (1997) Purification of a second tripeptidase from ¸actobacillus delbrueckii subsp. bulgaricus B14. Milchwissenschaft 52, 500—503. Bradford, M. (1976) A rapid sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Analytical Biochemistry 72, 248—254. Broome, M. C., Krause, D. A. and Hickey, M. W. (1990) The isolation and characterization of lactobacilli from Cheddar cheese. Australian Journal of Dairy ¹echnology 45, 60—66. Casey, M. G. and Meyer, J. (1985) Presence of X-prolyl-dipeptidyl peptidase in lactic acid bacteria. Journal of Dairy Science 68, 3212—3215. Dicks, L. M. T., Du Plessis, E. M., Dellaglio, F. and Lauer, E. (1996) Reclassification of ¸actobacillus casei subsp. casei ATCC393 and ¸actobacillus rhamnosus ATCC15820 as ¸actobacillus zeae nom. rev., designation of ATCC334 as the neotype of ¸. casei subsp. casei and rejection of the name ¸actobacillus paracasei. International Journal of Systematic Bacteriology 46, 337—340. Doi, E., Shibata, D. and Matoba, T. (1981) Modified colorimetric ninhydrin methods for peptidase assay. Analytical Biochemistry 118, 173—184. El Soda, M. and Desmazeaud, M. J. (1982) Les peptide-hydrolases des lactobacilles du groupe ¹hermobacterium. I. Mise en e´vidence de ces activite´s chez ¸actobacillus helveticus, ¸. acidophilus, ¸. lactis et ¸. bulgaricus. Canadian Journal of Microbiology 28, 1181—1188. El Soda, M., Zeyada, N., Desmazeaud, M. J., Mashaby, R. and Ismail, A. (1982) Les peptide-hydrolases des lactobacilles du groupe Betabacterium. Mise en e´vidence chez ¸actobacillus brevis, ¸. fermentum, ¸. buchneri et ¸. cellobiosus. Science des Aliments 2, 261—273. El Soda, M., Said, H., Desmazeaud, M. J., Mashaly, R. and Ismail, A. (1983) The intracellular peptide-hydrolase of ¸actobacillus plantarum. ¸e ¸ait 63, 1—14. Ferna´ndez, L., Bhowmik, T. and Steele, J. L. (1994) Characterization of ¸actobacillus helveticus CNRZ32 pepC gene. Applied and Environmental Microbiology 60, 333—336. Fernandez de Palencia, P., Pela´ez, C. and Martin-Herna´ndez, M. C. (1997) Characterization of the aminopeptidase system from ¸actobacillus casei subsp. casei IFPL731. Journal of Agricultural and Food Chemistry 45, 3778—3781. Fera´ndez-Espla´, M. D., Fox, P. F. and Martin-Herna´ndez, M. C. (1997a) Purification and characterization of a novel serine aminopeptidase from ¸actobacillus casei ssp. casei IFPL731. Journal of Agricultural and Food Chemistry 45, 1624—1628. Fera´ndez-Espla´, M. D. and Martin-Herna´ndez, M. C. (1997) Purification and characterization of a dipeptidase from ¸actobacillus casei ssp. casei IFPL731 isolated from goat cheese made from raw milk. Journal of Dairy Science 80, 1497—1504.

Peptidases of Lactobacilli and NSLAB Fera´ndez-Espla´, M. D., Martin-Herna´ndez, M. C. and Fox, P. F. (1997b) Purification and characterization of a prolidase from ¸actobacillus casei subsp. casei IFPL731. Applied and Environmental Microbiology 63, 314—316. Fox, P. F., Wallace, J. M., Morgan, S., Lynch, C. M., Niland, E. J. and Tobin, J. (1996) Acceleration of cheese ripening. Antonie van ¸eeuwenhoek 70, 271—297. Gilbert, C., Atlan, D., Blanc, B. and Portalier, R. (1994) Proline iminopeptidase from ¸actobacillus delbrueckii subsp. bulgaricus CNRZ397: purification and characterization. Microbiology 140, 537—542. Habibi-Najafi, M. B. and Lee, B. H. (1994) Purification and characterization of X-prolyl dipeptidyl peptidase from ¸actobacillus casei subsp. casei LLG. Applied Microbiology and Biotechnology 42, 280—286. Habibi-Najafi, M. B. and Lee, B. H. (1995) Purification and characterization of proline iminopeptidase from ¸actobacillus casei subsp. casei LLG. Journal of Dairy Science 78, 251—259. Hammes, W. P. and Vogel, R. F. (1995) The genus ¸actobacillus. In ¹he ¸actic Acid Bacteria, »ol. 2: ¹he Genera of ¸actic Acid Bacteria, eds. B. J. B., Woods, and W. H., Holzapfel, Blackie Academic and Professional, Glasgow, pp. 19—54. Hagazi, F. Z. and Abo-Elnaga, I. G. (1987) Proteolytic activity of crude cell-free extract of ¸actobacillus casei and ¸actobacillus plantarum. Die Nahrung 31, 225—232. Jordan, K. N. and Cogan, T. M. (1993) Identification and growth of non-starter lactic acid bacteria in Irish Cheddar cheese. Irish Journal of Agricultural and Food Research 32, 47—55. Khalid, N. M., El Soda, M. and Marth, E. H. (1991) Peptide hydrolases of ¸actobacillus helveticus and ¸actobacillus delbrueckii ssp. bulgaricus. Journal of Dairy Science 74, 29—45. Khalid, N. M. and Marth, E. H. (1990) Purification and partial characterization of a prolyl-dipeptidyl aminopeptidase from ¸actobacillus helveticus CNRZ 32. Applied and Environmental Microbiology 56, 381—388. Klein, J. R., Henrich, B. and Plapp, R. (1994a) Cloning and nucleotide sequence analysis of the ¸actobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC. FEMS Microbiology ¸etters 124, 291—300. Klein, J. R., Schmidt, U. and Plapp, R. (1994b) Cloning, heterologous expression and sequencing of a novel proline iminopeptidase gene, pepI, from ¸actobacillus delbrueckii subsp. lactis DSM7290. Microbiology 140, 1133—1139. Klein, J. R., Schick, J., Henrich, B. and Plapp, R. (1997) ¸actobacillus delbrueckii subsp. lactis DSM7290 pepG gene encodes a novel cysteine aminopeptidase. Microbiology 143, 527—537. Kunji, E. R. S., Mierau, I., Hagting, A., Poolman, B. and Konings, W. N. (1996) The proteolytic systems of lactic acid bacteria. In ¸actic Acid Bacteria: Genetics, Metabolism and Applications, eds G. Venema, J. H. J. Huis in’t Veld, and J. Jugenholz. Kluwer Academic Publishers, Dordrecht, pp. 91—125. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (¸ondon) 227, 680—685. Lane, C. N. and Fox, P. F. (1996) Contribution of starter and adjunct lactobacilli to proteolysis in Cheddar cheese during ripening. International Dairy Journal 6, 715—728. Law, J. and Haandrikman, A. (1997) Proteolytic enzymes of lactic acid bacteria. International Dairy Journal 7, 1—11. Lynch, C. M., McSweeney, P. L. H., Fox, P. F., Cogan, T. M. and Drinan, F. D. (1996) Manufacture of Cheddar cheese with and without adjunct lactobacilli under controlled microbiological conditions. International Dairy Journal 6, 851—867. Machuga, E. J. and Ives, D. H. (1984) Isolation and characterization of an aminopeptidase from ¸actobacillus acidophilus R-26. Biochimica et Biophysica Acta 789, 26—36.

265

Mars, I. and Monnet, V. (1995) An aminopeptidase P from ¸actococcus lactis with original specificity. Biochimica et Biophysica Acta 1243, 209—215. Martley, F. G. and Crow, V. L. (1993) Interactions between non-starter microorganisms during cheese manufacture and ripening. International Dairy Journal 3, 461—483. McDonald, J. K. and Barrett, A. J. (1986) Mammalian Proteases: A Glossary and Bibliography, »olume 2, Exopeptidases Academic Press, London, pp. 132—144. McDonnell, M., Fitzgerald, R., Fhaolain, I. N., Jennings, P. V. and O’Cuinn, G. (1997) Purification and characterization of aminopeptidase P from ¸actococcus lactis subsp. cremoris. Journal of Dairy Research 64, 399—407. McSweeney, P. L. H., Fox, P. F., Lucey, J. A., Jordan, K. N. and Cogan, T. M. (1993) Contribution of the indigenous microflora to the maturation of Cheddar cheese. International Dairy Journal 3, 613—634. Meyer, J. and Jordi, R. (1987) Purification and characterization of X-prolyl-dipeptidyl-aminopeptidase from ¸actobacillus lactis and Streptococcus thermophilus. Journal of Dairy Science 70, 738—745. Miyakawa, H., Kobayashi, S., Shimamura, S. and Tomita, M. (1991) Purification and characterization of an X-prolyldipeptidyl aminopeptidase from ¸actobacillus delbrueckii ssp. bulgaricus LBU-147. Journal of Dairy Science 74, 2375—2381. Miyakawa, H., Hashimoto, I., Nakamura, T., Ishibashi, N. and Shimamura, S. (1994a) Purification and characterization of a prolyl aminopeptidase from ¸actobacillus helveticus LHE-511. Milchwissenschaft 49, 615—619. Miyakawa, H., Hashimoto, I., Nakamura, T., Ishibashi, N. and Shimamura, N. (1994b) Purification and characterization of an X-prolyl dipeptidyl aminopeptidase from ¸actobacillus helveticus LHE-511. Milchwissenschaft 49, 670—674. Montel, M-C., Seronie, M-P., Talon, R. and He´braud, M. (1995) Purification and characterization of a dipeptidase from ¸actobacillus sake. Applied and Environmental Microbiology 61, 837—839. Morishita, T., Deguchi, Y., Yajima, M., Sakurai, T. and Yura, T. (1981) Multiple nutritional requirements of lactobacilli. Genetic lesions affecting amino acid biosynthetic pathways. Journal of Bacteriology 148, 64—71. Peterson, S. D. and Marshall, R. T. (1990) Nonstarter lactobacilli in Cheddar cheese: a review. Journal of Dairy Science 73, 1395—1410. Peterson, S. D., Marshall, R. T. and Heymann, H. (1990) Peptidase profiling of lactobacilli associated with Cheddar cheese and its application to identification and selection of strains for cheese ripening studies. Journal of Dairy Science 73, 1454—1464. Sanz, Y. and Toldra´, F. (1997) Purification and characterization of an aminopeptidase from ¸actobacillus sake. Journal of Agricultural and Food Chemistry 45, 1552—1558. Sasaki, M., Bosman, B. W. and Tan, P. S. T. (1995a) Comparison of proteolytic activities in various lactobacilli. Journal of Dairy Research 62, 601—610. Sasaki, M., Bosman, B. W. and Tan, P. S. T. (1995b) Immunological and electrophoretic study of the proteolytic enzymes from various ¸actococcus and ¸actobacillus strains. Journal of Dairy Research 62, 611—620. Sasaki, M., Bosman, B. W. and Tan, P. S. T. (1996) A new broad-substrate-specificity aminopeptidase from the dairy organism ¸actobacillus helveticus SBT 2171. Microbiology 142, 799—808. Schleifer, K. H. and Ludwig, W. (1995) Phylogeny of the genus ¸actobacillus and related genera. Systematic and Applied Microbiology 18, 461—467. Shao, W., Yu¨ksel, G. U®., Dudley, E. G., Parkin, K. L. and Steele, J. L. (1997) Biochemical and molecular characterization of pepR, a dipeptidase from ¸actobacillus helveticus CNRZ32. Applied and Environmental Microbiology 63, 3438—3443.

266

A. G. Williams et al.

Tan, P. S. T., Sasaki, M., Bosman, B. W. and Iwasaki, T. (1995) Purification and characterization of a dipeptidase from ¸actobacillus helveticus SBT2171. Applied and Environmental Microbiology 61, 3430—3435. Tobiassen, R. O., Sorhaug, T. and Stepaniak, L. (1997) Characterization of an intracellular oligopeptidase from ¸actobacillus paracasei. Applied and Environmental Microbiology 63, 1284—1287. Tsakalidou, E., Dalezios, I., Georgalaki, M. and Kalantzopoulos, G. (1993) A comparative study: aminopeptidase activities from ¸actobacillus delbrueckii spp. bulgaricus and Streptococcus thermophilus. Journal of Dairy Science 76, 2145—2151. Varmanen, P., Rantanen, T. and Palva, A. (1996) An operon from ¸actobacillus helveticus composed of a proline iminopeptidase gene (pepI) and two genes coding for putative members of the ABC transporter family of proteins. Microbiology 142, 3459—3468. Vongerichten, K. F., Klein, J. R., Matern, H. and Plapp, R. (1994) Cloning and nucleotide sequence analysis of PepV, a

carnosinase gene from ¸actobacillus delbrueckii subsp. lactis DSM 7290, and partial characterization of the enzyme. Microbiology 140, 2591—2600. Williams, A. G. and Banks, J. M. (1997) Proteolytic and other hydrolytic activities in non-starter lactic acid bacteria (NSLAB) isolated from Cheddar cheese manufactured in the United Kingdom. International Dairy Journal 7, 763—774. Wohlrab, Y. and Bockelmann, W. (1992) Purification and characterization of a dipeptidase from ¸actobacillus delbrueckii subsp. bulgaricus. International Dairy Journal 2, 345—361. Wohlrab, Y. and Bockelmann, W. (1993) Purification and characterization of a second aminopeptidase (pepC-like) from ¸actobacillus delbrueckii subsp. bulgaricus B14. International Dairy Journal 3, 685—701. Wohlrab, Y. and Bockelmann, W. (1994) Purification and characterization of a new aminopeptidase from ¸actobacillus delbrueckii subsp. bulgaricus B14. International Dairy Journal 4, 409—427.