The aminopeptidase C (PepC) from Lactobacillus helveticus CNRZ32. A comparative study of PepC from lactic acid bacteria

 The aminopeptidase C (PepC) of Lactobacillus helveticus CNRZ32 was purified by anion exchange chromatography from cell free extracts of an E. coli DH5α clone overexpressing the Lactobacillus aminopeptidase. PepC was found to have a tetrameric
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  Eur Food Res Technol (2000) 212:89–94 Q Springer-Verlag 2000 ORIGINAL PAPER Pilar Fernández de Palencia 7 Félix López de FelipeTeresa Requena 7 Carmen Peláez The aminopeptidase C (PepC) from Lactobacillus helveticus CNRZ32. A comparative study of PepC from lactic acid bacteria Received: 25 February 2000P.F. de Palencia 7 F.L. de Felipe 7 T. Requena, C. Peláez ( Y )Department of Dairy Science and Technology,Instituto del Frío (CSIC), Ciudad Universitaria,28040, Madrid, Spaine-mail: cpelaez 6 if.csic.es Abstract The aminopeptidase C (PepC) of Lactobacil-lus helveticus CNRZ32 was purified by anion exchangechromatography from cell free extracts of an E. coli DH5 a clone overexpressing the Lactobacillus amino-peptidase. PepC was found to have a tetrameric struc-ture in its native form with subunits of 50kDa each, apH optimum of 6.5 and maximum activity at 45 7 C. Sulf-hydryl-blocking reagents inhibited the enzyme activitywhereas reducing or metal chelating reagents had anactivating effect on the PepC activity. The PepC hydro-lyzed a wide range of  p- nitroaniline derivatives, dipep-tides and several tripeptides which contained basic ami-no acids (Arg, Lys), Pro residues, or cheese flavourprecursor amino acids (Met, Leu, Phe) at the N-termi-nal position. The substrate specificity and residual ac-tivity of PepC from several lactic acid bacteria, includ-ing the PepC described above, were compared at condi-tions of pH and NaCl present in cheese. Keywords Aminopeptidase C 7 Lactobacillushelveticus CNRZ32 7 Lactic acid bacteria 7 Enzymaticspecificity Introduction Lactic acid bacteria (LAB) are widely used in the man-ufacture of a variety of fermented dairy products.These microorganisms are auxotrophic for some of theessential amino acids [1] so they have developed a pro-teolytic system able to hydrolyze the major milk pro-tein, casein, into amino acids, thereby allowing an opti-mum growth of the bacteria in milk [2]. These aminoacids are important not only for the microbial growthbut also as precursors of the volatile components char-acteristic of cheese flavour [3].In the last few years the interest in the role of non-starter LAB on cheese ripening has increased greatly[4]. Most of the studies have concentrated on the in-fluence that these microorganisms may have on proteo-lysis and flavour development, and several studies havereported the characterization of the proteolytic systemof a number of lactobacilli strains [5–8].The general aminopeptidases N (PepN) and C(PepC) are important in peptide hydrolysis. These en-zymes have been purified and characterized and thegenes cloned and sequenced [2]. The debittering activi-ty of the lactococcal PepN and its role in flavour devel-opment has been referred to by several authors [9–11].Information concerning the role of PepC in cheese fla-vour is scarce. The use as cheese starter of L. lactis sub-sp. cremoris TIL61 showing a tenfold increase in thegeneral aminopeptidase PepC from L. lactis subsp. cre-moris AM2 [12, 13] did not lead to an increased rate of proteolysis [14]. Nevertheless, this result cannot be gen-eralized for PepCs from all LAB strains because differ-ences in activity under cheese conditions, as well as dif-ferent substrate specificities, consequences of the en-zyme primary structure, may also have a very distinctimpact on flavour development. This has already beenshown for lactococcal PepN activity [15] but there wasnot been a comparative study among PepCs from dairyLAB.The thiol-dependent PepC has been purified andcharacterized in a number of lactic acid bacteria [12, 16,17]. PepC encoding genes have been cloned and se-quenced [18–21]. Recent studies regarding PepC havecharacterized its bleomycin hydrolase activity [22]. Lactobacillus helveticus CNRZ32 has been shown toimprove flavour development, reduce bitterness andaccelerate cheese ripening when used as an adjunct cul-ture [23, 24]. In the present work we report on the pu-rification and characterization of the PepC from Lacto-bacillus helveticus CNRZ32 overexpressed in an E. coli strain. We consider this information is of great rele-  90 Table1 Relative activity of Pep C from L. helveticus CNRZ32 towards various substratesSubstrateRelativeactivity a (%)SubstrateRelativeactivity b (%)SubstrateRelativeactivity b (%)Lys-  p NA100Arg-Phe100Ala-Pro-Gly5Leu-  p NA42Ala-Ala9Ala-Leu-Gly34Arg-  p NA238Ala-Met69Gly-Pro-Ala52Pro-  p NA4Ala-Phe34Leu-Gly-Phe36Ala-  p NA25Ala-Pro10Leu-Leu-Leu60Met-  p NA67Ala-Val21Pro-Gly-Gly4Val-  p NAND c Arg-Leu69Ala-Ala-Ala-Ala48Glu-  p NANDArg-Lys23CBZ d –Arg10Ala-Ala-  p NA2Asp-Gly26Gly-Pro-  p NANDGlu-Glu38Arg-Pro-  p NA14His-Ala94Glu-Pro-  p NANDHis-Phe83Ala-Pro-  p NA9Leu-Gly30Glu-Phe-  p NANDLeu-Leu87Leu-Met81Leu-Pro20Leu-Tyr45Lys-Ala55Met-Ala63Phe-Ala81Phe-Leu38Phe-Met99Phe-Pro9Pro-Leu28Pro-Phe22Val-Ala10Val-Asp20 a Activity towards Lys-  p NA was taken as 100% b Activity towards Arg-Phe was taken as 100% c ND, not detected d CBZ, carbobenzoxy vance in comparing substrate specificities and residualactivity under cheese conditions among all PepCs de-scribed in LAB. Materials and methods Microorganism and growth conditionsThe strain E. coli DH5 a harbouring the Lactobacillus helveticus CNRZ32  pepC gene cloned in the medium-copy number vectorpJDC9 [18, 25] was kindly provided from the laboratory of Dr.J.L. Steele (Department of Food Science, University of Wiscon-sin, USA). The E . coli strain was grown in Luria-Bertani (LB)broth [26] supplemented with erythromycin (1mg ml) for plasmidmaintenance.Enzyme Purification Preparation of the crude extract. LB broth (5l) containing 1mgml erythromycin were inoculated with 100ml of an overnight cul-ture of the E. coli DH5 a strain described above. After growth at37 7 C for 16h, cells were harvested by centrifugation (7500  g ,20min, 4 7 C) and washed with 50mmol l –1 sodium phosphate buf-fer, pH 7.0. The pellet was resuspended in 15ml of 20mmol l –1 Tris-HCl, pH 7.5, and the cell-free extract was obtained after dis-ruption with glass-beads as described previously [6].  Anion exchange chromatography. A 3-ml sample (14mg of pro-tein) of the cell-free extract was loaded onto a MonoQ HR 5/5column (Pharmacia Biotech, Uppsala, Sweden) equilibrated with20mmol l –1 Tris-HCl, pH 7.5. The enzyme was eluted in equili-brating buffer at a flow rate of 1ml min in a linear NaCl gradient(0mol l –1 to 1mol l –1 NaCl). The eluate with the highest amino-peptidase activity was loaded again onto the same column and thefraction containing the purified enzyme was stored at –80 7 C forfurther studies. Protein determination. Protein concentration was determined ac-cording to [27] using bovine serum albumin (Sigma) as stand-ard.Aminopeptidase activity and substrate specificityAminopeptidase activity was determined with Lysine-  p -nitroanil-ide (Lys-  p NA) as substrate (Sigma). For specificity studies, other  p -nitroaniline (  p NA) derivatives were also used (Table1), all ob-tained from Sigma. The reaction mixture contained 25 m l (5 m g)of enzyme solution, 425 m l of 50mmol l –1 sodium phosphate buf-fer, pH 7.0, and 50 m l substrate (1mmol l –1 final concentration).Enzyme activity was measured continuously over 10min at 37 7 Cby following the release of  p NA at 410nm using a Peltier CPS-40temperature controller in a Shimadzu UV-1601 spectrophotomet-er. One unit (U) of aminopeptidase activity was defined as theamount of enzyme required to release 1 m mol of  p NA per min. Amolar absorbance coefficient of 8800 M –1 cm –1 [28] was used tocalculate enzyme activity.The specificity of the aminopeptidase towards several pep-tides (Table1; all obtained from Sigma) was tested by the Cd-ninhydrin method [29]. A 20- m l sample of substrate (10mmol l –1 in water) and 70 m l of 50mmol l –1 sodium phosphate buffer, pH7.0, were added to 10 m l (2 m g) of the purified enzyme and incu-bated at 37 7 C for 20min. The reaction was stopped by addition of 0.9ml of ninhydrin reagent. The samples were heated for 5min at  9184 7 C and subsequently ice cooled. Absorbance was measured at507nm.Molecular mass determinationThe molecular mass of the enzyme was estimated by SDS-PAGEand by gel filtration. SDS-PAGE under reducing conditions wasperformed by using the Phast System (Pharmacia) according tothe instruction manual. Electrophoresis was carried out with12.5% polyacrylamide gel and SDS buffer strips (Pharmacia).Proteins were stained with Coomassie Blue R-250 (Merck). SDSmolecular weight markers (molecular mass 14–94kDa; LMW cal-ibration kit, Pharmacia) were used as reference proteins. For de-termination of molecular mass by gel filtration, 200 m l of purifiedenzyme were injected onto a Superose 12 HR 10/30 column(Pharmacia) and eluted with 0.1mol l –1 Tris-HCl, pH 7.5, at aflow rate of 0.3ml min –1 . The column was calibrated using a kitfor molecular mass markers for gel filtration (MW-GF-200 kit;Sigma).Effect of pH and temperature on the enzyme activityThe effect of pH on the aminopeptidase activity at 37 7 C was de-termined with an incubation mixture containing 25 m l of enzymesolution, 425 m l of buffer at the desired pH value (0.1mol l –1 ace-tate, pH 4.0 and 5.0; 0.1mol l –1 sodium phosphate, pH 6.0, 6.5 and7.0; 0.1mol l –1 Tris-HCl, pH 7.5 and 8.0; glycine-NaOH, pH 9 and10), and 50 m l 10mmol l –1 Lys-  p NA. To estimate pH stability, thepurified enzyme was incubated for 1h at 25 7 C in the appropriatebuffer.The influence of temperature on aminopeptidase activity wasdetermined by measuring the hydrolysis of Lys-  p NA in the tem-perature range 20–70 7 C (pH 7.0). To estimate thermostability, apreincubation of enzyme solution in 50mmol l –1 sodium phos-phate, pH 7.0, was carried out at different temperatures(20–65 7 C) for 30min. After cooling, residual activities were de-termined at 37 7 C by addition of the substrate.Effect of chemical reagents and metal ions on activityA mixture containing 25 m l of the purified enzyme, 25 m l of theindicated concentrations of chemical reagents (see Table2) ormetal ions (see Table3) and 400 m l of 50mmol l –1 sodium phos-phate buffer, pH 7.0, or 20mmol l –1 Tris-HCl, pH 7.0, respective-ly, were incubated for 30min at 25 7 C. For the determination of the residual aminopeptidase activity, 50 m l of 10mmol l –1 Lys-  p NA were added.Kinetic parametersThe kinetic of the purified aminopeptidase towards Arg-, Leu-and Lys-  p NA substrates were determined at concentrations rang-ing from 0.005mol l –1 to 9mmol l –1 . Lineweaver-Burk plots wereconstructed and the K  m and V  max were calculated from the slopeand intercept, respectively, of the regression lines. Results Enzyme purificationCell free extracts of the E. coli DH5 a strain harbouringthe recombinant plasmid described in experimentalprocedures showed a specific activity for Lys-  p NA tentimes greater than the basal activity of E. coli DH5 a  .This facilitated the purification of the enzyme to homo- Table2 Effect of chemical reagents on the aminopeptidase activ-ityChemical reagentConcentrationRelativeactivity a (%)E-6410 m g ml –1 5Iodoacetic acid1mmol l –1 0Iodoacetamide1mmol l –1 0 N - Ethylmaleimide1mmol l –1 0  p -Hydroxy benzoic acid1mmol l –1 0PMSF b 1mmol l –1 13Pefabloc0.1mg/mL77EDTA1mmol l –1 1435mmol l –1 1711–10-phenanthroline1mmol l –1 94Phosphoramidon4 m g ml –1 83Antipapain50 m g ml –1 151Bestatin40 m g ml –1 84Leupeptin0.5 m g ml –1 100Pestatin0.7 m g ml –1 85Aprotinin2 m g ml –1 76DTT1mmol l –1 1725mmol l –1 191 b  -mercaptoethanol1mmol l –1 89 a Activity is expressed as percentage relative to an untreated sam-ple b Phenylmethylsulfonylfluoride Table3 Effect of various metal ions on aminopeptidase activityMetalion b Aminopeptidase activity a (%)0.01mmol l –1 0.1mmol l –1 1mmol l –1 Mg 2 c 1701655Cu 2 c 005Fe 3 c 001Zn 2 c 9750Mn 2 c 100412Co 2 c 112613Ca 2 c 14512927Cd 2 c 703100.1mol l –1 0.25mol l –1 1mol l –1 Na c 17140.2 a Samples not treated by metal ions are taken as 100% b The associated anion was Cl – in all cases geneity in a single chromatographic step. Results of thepurification procedure are summarized in Table4. Thetotal aminopeptidase activity against Lys-  p NA was re-covered in the cell free extract. Then this fraction wassubjected twice to anion-exchange chromatography(Mono Q column) and the activity was eluted at 0.2moll –1 NaCl. After this step, electrophoretic purity of theenzyme was achieved (Fig.1). The specific activity wasenriched about twofold with a recovery of 6% of thetotal activity.Molecular massMolecular mass of the aminopeptidase was estimated tobe approximately 50kDa by SDS-PAGE under reduc-  92 Table4 Purification procedure for PepC from Lactobacillus helveticus CNRZ32PurificationstepTotalprotein(mg)Totalactivity(U) a Specificactivity(U/mg)Purifi-cation(-fold)Activityyield(%)Cell-free extract59.4741.31.0100Anion-exchange I4.421.24.83.828.6Anion-exchange II1. a Activity is expressed in units (U) as m mol of  p- nitroaniline released per minute under the assay conditions Fig.1a, b SDS-PAGE (12.5%) of the purified PepC from Lacto-bacillus helveticus CNRZ32:Lanes. a Molecular mass standardproteins (kDa). b Purified enzyme after the anion exchange chro-matography ing conditions (Fig.1). A molecular mass of 200kDawas determined by gel filtration, which implies a te-trameric structure of the enzyme in its native form.Effect of pH, temperature and NaCl on the enzymeactivityMaximum aminopeptidase activity was observed at pH6.5 and 45 7 C. The activity was reduced by 50% at pH5.4. After 1h of incubation at pH values between 6.0and 7.0, nearly 100% of the activity remained, while in-cubation at pH 5.0, 8.0 and 9.0 resulted in a decrease of approximately 75% in activity. The aminopeptidasewas quite thermostable. The enzyme retained 80–100%of the activity after 30min of incubation in a tempera-ture range of 20–35 7 C, and about 50% of the activity at45–55 7 C. Incubation of the purified enzyme in the pres-ence of 4% NaCl led to suppression of the activityEffect of chemical reagents and metal ions on theactivityThe effect of chemical reagents and metal ions is shownin Tables 2 and 3, respectively. Inhibitors of metalloen-zymes, such as 1–10-phenanthroline and phosphorami-don had no effect on enzyme activity, while EDTAstrongly activated the enzyme at concentrations of 1mmol l –1 and 5mmol l –1 . This suggests that this ami-nopeptidase is not a metalo-dependent enzyme. An ac-tivating effect was also observed with antipapain andwith the reducing agent DTT at concentrations of 1mmol l –1 and 5mmol l –1 . Enzyme activity was strong-ly inhibited by all the sulphydryl-groups blocking rea-gents assayed (E-64, iodoacetic acid, iodoacetamide, N  -ethylmaleimide and  p -hydroxy benzoic acid).The presence of certain divalent cations such as Mg 2 c , Co 2 c and Ca 2 c increased, while Cu 2 c , Fe 3 c ,Zn 2 c , Mn 2 c , Co 2 c and Cd 2 c inhibited the aminopep-tidase activity at the concentrations indicated in Ta-ble3.Substrate specificityThe purified PepC hydrolyzed  p NA derived aminoacids, dipeptides and several tripeptides. The relativeactivity of PepC towards these substrates is expressedin Table1. Positive charged amino acids (Lys-  p NA andArg-  p NA) were the preferred substrates of the enzyme,followed by hydrophobic ones such as Met- and Leu-  p NA.The enzyme also hydrolyzed a broad range of dipep-tides. The substrates cleaved at the highest rates wereArg-Phe, His-Ala, His-Phe, Leu-Leu, Leu-Met, Phe-Ala and Phe-Met. PepC also showed a high rate of cleavage against dipeptides containing sulphur aminoacids such as Ala-Met and Met-Ala. All tripeptidestested were hydrolyzed at different rates.Kinetics parametersThe affinity of the PepC was examined for the sub-strates Arg-, Leu- and Lys-  p NA, for which K  m valuesof 0.063, 0.068 and 0.619mmol l –1 , respectively, werefound. Although the enzyme showed a similar affinityfor the substrates Arg- and Leu-  p NA, the rate of hy-drolysis for Arg-  p NA was the highest (3.252 m molmin –1 per mg of protein) compared to Leu-  p NA(0.066 m mol min –1 per mg of protein) and Lys-  p NA(2.164 m mol min –1 per mg of protein).  93 Table5 Comparison of known PepC aminopeptidases from lactic acid bacteria L. delbrueckii subsp .bulgaricus B14 L. delbrueckii subsp .lactis DSM7290 L. helveticus 53/7 L. helveticus CNRZ32 L. casei subsp. casei IFPL 731 L. lactis subsp. cremoris AM2 S. thermophilus CNRZ 302Specificity a Lys  c c c c P c c Arg  P c ND  c P ND  c Pro  P P ND  c c P P Met  c c ND  c c c c Leu  c c P c c c c Phe  P c ND  c c c c Activity b (%)pH 5.47070ND501015124% NaClNDNDND0110NDND Lactobacillus delbrueckii subsp . bulgaricus B14 [16]. Lactobacil-lus delbrueckii subsp . lactis DSM7290 [21]. Lactobacillus helveti-cus 53/7 [20]. Lactobacillus helveticus CNRZ32 (this work). Lac-tobacillus casei subsp. casei IFPL731 [17]. Lactococcus lactis sub-sp. cremoris AM2 [12]. Streptococcus themophilus CNRZ 302[19] a Hydrolysis against  p- nitroanilide or b-naphthylamide aminoacylsubstrates, dipeptides and tripeptides, all containing the indicatedresidue at the N-terminal end b Expressed as percentage of maximal enzyme activity c :detected–: not detectedND: not determined Discussion This work describes the purification and characteriza-tion of a cysteine aminopeptidase (PepC) from L. hel-veticus CNRZ32. This information, together with theprevious characterization of other PepCs from LAB, al-low us to compare activities and determine the poten-tial impact on cheese ripening.The  pepC gene from L. helveticus CNRZ32 was pre-viously cloned in the plasmid pJDC9 and introduced in E. coli DH5 a [18, 25]. This E. coli strain overproducedten times the Lactobacillus enzyme, thus facilitating itspurification.The molecular mass of the PepC from L. helveticus CNRZ32, determined by SDS-PAGE under reducingconditions, was 50kDa, coincidental with the48.84kDa deduced from the  pep C gene previously de-scribed [25]. In the present work, the PepC molecularmass, determined by gel filtration, has been estimatedto be 200kDa. Therefore, the enzyme in its native stateconsists of four identical subunits of 50kDa. This te-trameric structure is a common characteristic sharedwith the other characterized PepC enzymes from Lac-tobacillus [16, 17, 21], while PepCs from Lactococcus and Streptococcus have been described as hexamers[12, 19].Thiol groups play an important role in the active siteof the enzyme, since aminopeptidase activity wasstrongly inhibited by treatment with all the inhibitors of sulphydryl-groups assayed. Moreover, it was not af-fected by the metal-chelating agents 1–10-phenanthrol-ine, phosphoramidon and EDTA. The purified enzymewas even activated by EDTA at concentrations of 1–5mmol l –1 . Similar feature has been reported for thePepC of L. delbrueckii [16]. A comparative study of substrate specificity and activity under cheese condi-tions (pH 5.4 and 4% NaCl) of known PepC from dairyLAB is presented in Table5. The PepC of L. helveticus CNRZ32 showed the highest specificity against  p NAderivatives of positively charged amino acids (Lys-  p NAand Arg-  p NA) placed at the N-terminal position. Theenzyme also hydrolyzed efficiently dipeptides with ahistidine residue at the N-terminal end. According toHabibi-Najafi and Byong [30], a basic amino acid at theN-terminal position appears to enhance significantlypeptide bitterness. Therefore, hydrolysis of positivecharged amino acids by the PepC from L. helveticus CNRZ32 may have an impact on cheese debittering.Only the aminopeptidases from L. delbrueckii subsp. lactis DSM 7290 and S. thermophilus CNRZ 302 areable to release both amino acids.Sulphur-containing amino acids, branched chain andaromatic amino acids have been demonstrated to beprecursors of aroma compounds in cheese [3]. Availa-ble data from all characterized PepC enzymes (Table5)indicate that they are capable of hydrolyzing  p NA- or b  -naphthylamide derivative amino acids, dipeptidesand tripeptides containing Met, Leu and Phe at the N-terminus.The PepC from L. helveticus CNRZ32 retainedabout 50% of the activity after incubation at pH 5.4(Table5). The PepC of L. delbrueckii also retainedhigh activity at this pH (70%), while L. casei IFPL731retained only about 10%, a value which is similar tothat found for L. lactis AM2 and S. thermophilus . Be-sides, the effect of NaCl is also variable among thestrains examined.The comparison of PepC activity from LAB strains,based on specificity of substrate, residual activity inconditions present during cheese ripening, etc., indi-cates a great deal of variability under these conditions.Technological impact of different varieties of PepCfrom LAB should be demonstrated in cheese makingtrials.
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