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Purification and characterisation of an intracellularaminopeptidase from Brevibacterium linens ATCC 9174

Fp Rattray, Pf Fox

To cite this version:Fp Rattray, Pf Fox. Purification and characterisation of an intracellular aminopeptidase from Bre-vibacterium linens ATCC 9174. Le Lait, INRA Editions, 1997, 77 (1), pp.169-180. �hal-00929509�

Lait (1997) 77. 169-180© Elsevier/INRA

169

Original article

Purification and characterisation of an intracellularaminopeptidase from Brevibacterium linens ATCC 9174

FP Rattray, PF Fox

Department of Food Chemistry, University College, Cork. Ire/and

Summary - An intracellular aminopeptidase from Brevibacterium linens ATCC 9174 was purified4300-fold to homogeneity using ammonium sulphate fractionation, anion exchange chromatography,hydrophobie interaction chromatography and anion exchange chromatography. The pH and tem-perature optima were 8.5 and 35 "C, respectively. The purified aminopeptidase was stable over therange pH 8 to 10, and was thermally stable up to 20 "C at pH 8.5. The molecular mass of the enzymewas found to be 59 kOa by SOS-PAGE and 69 kOa by gel filtration, indicating that the nativeenzyme exists as a monomer. The aminopeptidase was strongly inhibited by the thiol blockingagent, p-hydroxymercuribenzoate, and by C02+ and Zn2+; activity was unaffected by metal chelators,reducing agents or phenylmethylsulphonyl fluoride. Km and kcat values for L-Ala-p-NA were3.3 mmol/L and 4.3 s-I, respectively, while the corresponding values for L-Gly-p-NA were 0.2mmol/L and 7.6 s-I, respectively. The aminopeptidase hydrolysed dipeptides with an alanine residueat the N-terminal but tripeptides were not hydrolysed. The sequence of the first 19 N-terminal aminoacids was NHrPro-Phe-Asp-Gly-Pro-Asp- Thr-Ala-Ala-I1e-I1e-Asp-Arg-Leu- ?-Asn-Ala-?- Thr.

aminopeptidase / Brevibacterium linens / cheese / purification

Résumé - Purification et caractérisation d'une aminopeptidase intracellulaire de Brevibac-terium linens ATCC 9174. Une aminopeptidase intracellulaire de Brevibacterium linens ATCC9174 a été purifiée 4300 fois par homogénéisation et fractionnement au sulphate d'ammonium,chromatographie d'échange d'anions, chromatographie par interactions hydrophobes et chro-matographie d'échange d'anions. Le pH et la température optimale étaient respectivement de 8,5 et35 oc. L'aminopeptidase était stable, de pH 8 à 10. De plus, elle était stable thermiquement à 20 "Cà pH 8,5. La masse moléculaire de l'enzyme, determinée par SOS-PAGE et filtration sur gel étaitrespectivement de 59 kOa et 69 kOa, ce qui indique que l'enzyme native existe sous forme de

Oral communication at the IDF Symposium 'Ripening and Quality of Cheeses', Besançon, France, February26-28, 1996.

170 FP Rattray, PF Fox

monomère. L'activité enzymatique est fortement inhibée par l'agent bloquant les groupementsthiols, le p-hydroxymercuribenzoate, et par les ions C02+ et Zn2+; l'activité enzymatique n'est pasinhibée par les chélateurs de métal, les agents réducteurs ou le fluorure de phenylmethylsulpho-nyle. Les valeurs Km et kcar pour le L-Ala-p-NA étaient respectivement de 3,3 mmollL et 4,3 ,1, tan-dis que les valeurs correspondantes pour L-G1y-p-NA étaient respectivement 0,2 mmol/L et 7,6 s-I.L'aminopeptidase hydrolysait les dipeptides possédant un résidu alanine en N-terminal mais lestripeptides n'étaient pas hydrolysés. L'ordre des 19 premiers acides aminés N-terminaux étaitNHrPro-Phe-Asp-G1y-Pro-Asp- Thr-Ala-Ala- I1e-Ile-Asp-Arg-Leu- ?-Asn-Ala-?- Thr.

aminopeptidase / Brevibacterium [il/ens / fromage / purification

INTRODUCTION

The surface microflora of various smear sur-face-ripened cheeses, such as Limburger,Gruyère, Münster, Brick, Appenzeller andTilsiter, includes yeasts, moulds and bacteria(Kelly, 1937; Langhus et al, 1945; EI-Erian,1969). During ripening, yeasts grow initially onthe surface of the cheese and raise the curd pH bya combination of lactate utilisation and ammoniaproduction. This increase in pH enables thegrowth of Brevibacterium linens, which even-tually dominates the surface microflora of thesecheeses. The growth of B linens on the surface isan essential prerequisite for the development ofthe characteristic flavour, colour, aroma and tex-ture of smear surface-ripened cheeses (Ades andCone, 1969; E1-Erian, 1969; Sharpe et al, 1977;Hemme et al, 1982).

The purification and characterisation of extra-cellular proteinases and aminopeptidases fromB linens has been described (Foissy, 1974,1978a--c; Hayashi and Law, 1989; Hayashi etal, 1990; J uhâsz and Skârka, 1990; Rattray etal, 1995, 1996). However, the level of informa-tion on the intracellular activities ofthis microor-ganism is quite limited. At least six differentpeptide hydrolases were identitied using a zymo-gram technique in the intracellular fraction ofB linens ATCC 9174 (Torgersen and Serhaug,1978). Using the same technique (Serhaug,1981), a total of 18 peptide hydrolases with dif-ferent substrate specificities were tentativelyidentified in six strains of B linens.

A parti aily purified aminopeptidase from anintracellular fraction of B linens HS was foundto have pH and temperature optima at 7.5 and30 "C, respectively; the aminopeptidase wasinhibited by metal chelators and p-hydroxy-mercuribenzoate (PHMB) (El-Soda et al, 1991).Intracellular peptidase activity (unspecified)from a cell-free extract of B linens was reported(Wong and Cone, 1964) to be optimally active atpH 7.9 and 45 "C, and was strongly inhibitedby Hg2+ and PHMB.

This paper reports the purification and char-acterisation of an intracellular aminopeptidasefrom B linens ATCC 9174.

MA TERIALS AND METHODS

Microorganism and growtlt conditions

Brevibacterium linens ATCC 9174 was obtainedfrom the culture collection of the Departmentof Microbiology, University College, Cork, Ire-land. The organism was precultured in 250 mLof medium [1.0% trypticase peptone (w/v), 0.3%Bacto peptone (w/v), 0.25% yeast extract (w/v),0.5% glucose (w/v), 0.25% K2HP04 (w/v) and0.02% MgS04.7H20 (w/v)] in a 500 mL flaskon an orbital shaker at 200 cycles pel' min at23 oc. After 48 h, 100 mL of preculture wereused to inoculate 10 L of the same medium in a10 L fermenter (B Braun Diessel Biotech GmbH,Melsungen, Germany). The temperature was

Intracellular aminopeptidase from B linens

maintained at 23 "C, with 10% 02 saturation;the pH was not regulated.

Preparation of celllysate

After cultivation for 65 h, ie, late log phase, cellswere harvested by centrifugation at 14500 g for25 min at 4 oc. The cells were then washed with20 mmol/L Tris-HCI, pH 8.0, and re-cen-trifuged. The cells (80 g) were suspended in500 mL of 20 mmollL Tris-HCI, pH 8.0, con-taining 10 mmol/L MgCI2 and 240 mg lysozyme(Sigma; from chicken egg white, 3 x crystal-lized), then incubated with gentle shaking for2.5 h at 37 oc. To the resulting celllysate, 10 mgof DNase 1 (Boehringer-Mannheim; grade II,from bovine pancreas) and 10 mg of RNase A(Sigma; type I-AS, from bovine pancreas) wereadded and incubated for 30 min at 37 oc. Thelysate was then centrifuged at 14 500 g for25 min at 4 "C, and the resulting supernatantused for aminopeptidase purification.

Measurement of aminopeptidase activity

Aminopeptidase activity was measured spec-trophotometrically using L-alanine-p-nitroanalide(L-Ala-p-NA) as substrate; 50 IIIof enzyme solu-tion were added to 400 IJL of 50 mmol/LTris-HO buffer, pH 8.5, and the reaction initiatedby adding 50 IJL substrate (10 mmol/L in water).The reaction mixture was incubated at 35 "C for30 min, after which 1 mL of 30% (v/v) aceticacid was added to terminate the reaction. Theabsorbance of the mixture was then measured at410 nm using a Cary lE UV-Visible Spec-trophotometer (Varian Australia PtYLtd, Victo-ria, Australia). A standard curve was generatedusing p-nitroaniline. One unit of enzymatic activ-ity was defined as the amount of enzyme thatliberates 1 nmol of p-NA per min at 35 "C,pH 8.5. Specifie activity was defined as units permg protein. Protein concentration was measuredby the method of Bradford (1976), using bovineserum albumin as standard.

17l

Enzyme purification

Prior to chromatographie separation, the Iysatewas fractionated using (NH4hS04' The Iysatewas adjusted to 45% saturation with (NH4hS04'then centrifuged at 14500 g for 25 min at 4 oc.After centrifugation, the 45% soluble fractionwas adjusted to 80% saturation, and re-cen-trifuged. The 80% insoluble pellet was dissolvedin 80 mL of 20 mmol/L Na phosphate buffer,pH 7.0, and dialysed overnight against 5 Lofthe same buffer.

The dialysed (NH4hS04 fraction was appliedto a DEAE-Sepharose Fast Flow anion exchangecolumn (5 x 20 cm gel bed); (Pharmacia LKBBiotechnology, Uppsala, Sweden) connected toa Bio-Rad Econo System (Bio-Rad Laborato-ries Ltd, Hertfordshire, United Kingdom). Thecolumn was washed initially with 600 mL of20 mmol/L Na phosphate buffer, pH 7.0, fol-lowed by a Iinear NaCI gradient (0 to 004 mol/L)in the same buffer (1400 mL); the NaCl con-centration was then maintained at 004 mollL fora further 500 mL, then followed by a linear gra-dient (004 to 0.5 mollL) for 360 mL, and main-tained at 0.5 mol/L for 420 mL. The flow ratewas 12 mL per min and 15 mL fractions werecollected. Active fractions were pooled and dial-ysed against 20 mmol/L Na phosphate buffer,pH 7.0. The dialysed solution was then adjustedto 2 mol/L (NH4hS04 and applied to aPhenyl-Sepharose High Performance hydropho-bic interaction column (1.6 x 14 cm gel bed);(Pharmacia LKB Biotechnology) connected toa Bio-Rad Econo System (Bio-Rad Laborato-ries Ltd). The column was washed initially with150 mL of 20 mmol/L Na phosphate buffer,pH 7.0, containing 2.0 mollL (NH4hS04' fol-lowed by a Iinear (NH4hS04 gradient from 2.0to 0 mol/L for 300 mL and then maintained ato mol/L (NH4hS04 for a further 100 mL. Theflow rate was 1 mL per min and the fraction sizewas 5 mL. Pooled active fractions were desaltedand concentrated using a Centricon-3 concen-trator (Amieon Ltd, Gloucestershire, UK), andthen diluted using 20 mmol/L Na phosphatebuffer, pH 7.0, and injected onto a Mono-Q

172 FP Rattray, PF Fox

HR5/5 column connected to FPLC System(Pharmacia LKB Biotechnology). The columnwas washed with 4 mL of 20 mmol/L Na phos-phate buffer, pH 7.0, followed by a linear NaCIgradient, 0 to 0.37 mol/L, in the same buffer(41 ml.), th en maintained at 0.37 mol/L for2 ml., followed by a linear NaCI gradient, 0.37to 0.4 mol/L, over 8 mL and finally by a gradi-ent of 0.4 to 0.5 mol/L over 10 mL. The flowrate was 1 mL per min, and 1 mL fractions werecollected.

Polyacrylamide gel electrophoresis

Purity and molecular weight were determinedby SOS-PAGE (Laernrnli, 1970) in a gel con-taining 10% (w/v) polyacrylamide. The gel wasstained for protein with 0.125% (w/v) Coomassiebrilliant blue R-250 in methanol-aceticacid-water (40:7:53, v/v) and destained inmethanol-acetic acid-water (25: 10:65). Molec-ular mass markers were: myosin (205 kOa), p-galactosidase (116 kDa), phosphorylase b(97 kOa), bovine serum albumin (66 kOa), oval-bumin (45 kOa), glyceraldehyde-3-phosphatedehydrogenase (36 kOa), carbonic anhydrase(29 kOa), trypsinogen (24 kOa), trypsin inhibitor(20 kOa) and œ-lactalburnin (14 kOa).

Estimation of molecular massby gel filtration

The molecular mass of the purified enzyme wasalso estimated by gel filtration on a TSK-GelG3000SW column (TosoHaas GmbH, Stuttgart,Gerrnany), using a 0.1 mol/L Na phosphatebuffer, pH 7.0, containing 0.1 M Na sulphate.Bovine thyroglobulin (666 kOa), bovine y-glob-ulin (156 kOa), bovine serum albumin (66 kOa),ovalbumin (45 kOa), p-Iactoglobulin (18 kOa)and œ-lactalburnin (14 kOa) were used as ref-erence proteins for standard curve calibration.

Temperature and pH optima

Optimum temperature was determined by assay-ing activity over the range 4 to 60 "C in

Tris-HCl, pH 8.5. The optimum pH was deter-mined at 35 -c using Na acetate (pH 5-5.5), Naphosphate (pH 6-7), Tris-HCl (pH 7.5-8.5),and Na carbonate (pH 9-11). Buffer concentra-tion in ail assays was 50 mmollL.

Temperature and pH stability

Temperature stability was determined by incu-bating 50 ul. enzyme solution in 50 mmol/LTris-HCI, pH 8.5, at 4,20,30,40 or 50 -c for upto 24 h. Residual activity was then measured atthe optimum temperature (35 "C), pH stabilitywas determined by incubating 30 ul. of enzymesolution with 70 ul, of buffer (Na acetate, pH S,Na phosphate, pH 6-7, Tris-HCl, pH 8-9, orNa carbonate, pH 10-11) for 24 h at 20 oc. ThepH was then re-adjusted to 8.5 by adding 350 ul,of 50 mmol/L Tris-HCI, and the residualaminopeptidase activity was measured asdescribed.

Effect of activators and inhibitors

To 25 ul, of dialysed purified enzyme solution,400 ul, of 50 mmol/L Tris-HCl, pH 8.5 wereadded, followed by 25 ul, of activator orinhibitor (10 mmol/L in water), and pre-incu-bated for 30 min at 25 oc. Residual activity wasthen assayed by the addition of 50 ul, substrate(10 mmol/L L-Ala-p-NA in water) and incuba-tion for 30 min at 35 oc. The final concentra-tion of activator or inhibitor was either 0.1 or1.0 mmol/L. For reactivation of p-hydroxymer-curibenzoate-treated enzyme (1.0 mmollL),1.0 mmol/L dithiothreitol was added, and theactivity then measured as described.

Kinetic studies

The Km and kcat of the purified aminopeptidasewere determined for L-Ala-p-NA or L-G1y-p-NAas substrate. The aminopeptidase was incubatedin 50 mmol/L Tris-HCI, pH 8.5, with substrateat final concentrations ranging from 0.2 to

Intracellular aminopeptidase from B linens

5.0 mmol/L for L-Ala-p-NA and 0.05 to1.6 mmol/L for L-Gly-p-NA. After incubationfor 15 min, the reaction was terminated and theactivity measured as described previously. Kmvalues were calculated from Lineweaver-Burkplots using an Enzfitter computer programme(BIOSOFf, Cambridge, UK).

Substrate specificity studies

Substrate specificity on a range of peptides wasdetermined by the addition of 50 ul. of peptidesubstrate (10 mmol/L in water) to 400 ul, of50 mmollL Tris-HCI, pH 8.5, followed by 50 ilLof enzyme solution. The reaction mixture wasincubated for 30 min at 35 oc. Liberated freeamino groups were reacted with 1 mL of cad-mium-ninhydrin reagent (0.8 g ninhydrin in 80mL ethanol and 10 mL acetic acid, to whieh wasadded 1 g CdCI2 in 1 mL of water) and heatingat 84 "C for 5 min (Doi et al, 1981). After cool-ing, the developed colour was measured at 507nm. Activity on various nitroanilide substrateswas also assayed, as described previously.

N-terminal amino acid sequence

The purified aminopeptidase was centrifugedthrough a ProS pin Sample Preparation Cartridge(Applied Biosystems Inc, Foster City, CA, USA)onto a ProBlott polyvinylidene difluoride(PVDF) membrane. The N-terminal amino acidsequence was determined at the National FoodBiotechnology Centre, University College, Cork,Ireland, by Edman degradation on an automatedpulsed liquid-phase protein-peptide sequencer(Applied Biosystems Inc, modeI477A). Liber-ated amino acids were detected as their phenyl-thiohydantoin derivatives using a 120A anal-yser (Applied Biosystems Inc).

RESULTS

Purification of aminopeptidase

Purification of the aminopeptidase was achievedusing four purification steps. Ammonium sul-

173

phate precipitation, in addition to concentration,removed a considerable amount of orange pig-mentation from the lysate. In the first chro-matographic step (DEAE-Sepharose Fast Flow),the aminopeptidase activity was eluted at approx-imately 0.4 mollL NaCI with a 25-fold increasein specifie activity (fig 1a). The enzyme waseluted from the hydrophobie interaction columnat 0.0 mol/L (NH4)2S04 (fig 1 b) and specifieactivity was increased 713-fold. In the final chro-matographic step (Mono-Q), activity was elutedat 0.37 mol/L NaCI (fig 1c), with a 4307-foldincrease in specifie activity. The results of thepurification protocol are summarised in table I.

SDS-PAGE and molecular mass

Purity of the isolated enzyme was confirmed bySDS-PAGE; a single band with a molecularmass of 59 kDa was observed (fig 2, lane 6).HPLC gel filtration indieated a single peak cor-responding to a molecular mass of 69 kDa, sug-gesting that the enzyme exists as a monomer inits native state.

Temperature and pH optima and stability

The optimum temperature of the aminopepti-dase was determined to be 35 "C (fig 3a), whilethe optimum pH was 8.5 under the assay condi-tions used (fig 3b). The aminopeptidase was sta-ble for 24 h at temperatures up to 20 "C (fig 4a),but at 30 "C, 25% activity was lost after 24 h.The aminopeptidase was unstable at 40 and50 "C; no activity remained after 8 h and 30 min,respectively. The enzyme was relatively unsta-ble at both acidic and alkaline pH; it was totallyinactivated after 24 h at pH 5-6 and pH Il(fig 4b); maximum stability was observed atpH 9.

Effect of activators and inhibitors

The effects of chemical reagents and divalentcations on aminopeptidase activity are sum-

174 FP Rattray, PF Fox

0(1 .~() 100 150 200 2.:'iO ]DO

TiJ1lc (min)

0.12 (b)

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D10 :~ 20S -

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marised in table II. The sulphydryl reducingagents, dithiothreitol (OTT), p-mercaptoethanoland cysteine, and the metal chelators, EOT A,ethylene gtycol-bistû-amino-ethyl ether)N,N,N',N'-tetraacetic acid (EGTA) andI,IO-phenanthroline, had no effect on activityat 0.1 or 1.0 mmol/L (table II). The serine pro-

(a)

'0 .10 40

Time tminj~o

Fig 1. Purification of the intracellular aminopeptidasefrom B linens ATCC 9174. a: OEAE-SepharoseFast Flow anion exchange chromatography; b:phenyl-Sepharose high performance hydrophobieinteraction chromatography; c: Mono-Q anionexchange chromatography. A28û om (--); activity(..... ); NaCI or (NH412S04 (- - -).Purification de l'aminopeptidase intracellulaire deB linens ATee 9174. a: chromatographie d'échanged'anions (DEAE-Sepharose Fast Flow) ; b : chro-matographie d'interactions hydrophobes (Phenyl-:Sepharose); c: chromatographie d'échange d'anions(Mono-Q}. A280llm (--); activité (..... ); NaCi ou(NH4hS04 (- - -y.

teinase inhibitor, phenylmethylsulfonyl tluoride(PMSF), and the alkylating agent, N-ethyl-maleimide, also had no effect on activity. lodoac-etate caused a slight reduction in activity at 1.0mmol/L, but the thiol-blocking agent, p-hydrox-ymercuribenzoate (PHMB), caused strong inhi-bition at 0.1 and 1.0 mmollL. The PHMB-treatedenzyme was almost fully reactivated (88%) by1.0 mmol/L OTT.

None of the divalent cations activated theaminopeptidase but reduced activity in the orderZn2+ >, Co2+ >, Cd2+ >, Ni2+ >, Hg2+ >, Cu2+ >,Mn2+ >, Ca2+ > and Mg2+; Fe2+ had no effect onactivity (table II).

Fig 2. SOS-PAGE of the intracellular aminopepti-dase from B linens ATCC 9174 after different purifi-cation steps. Lanes 1 and 7, molecular mass markers;lane 2, crude intracellular extract; lane 3, ammoniumsulphate precipitation; lane 4, OEAE-Sepharose FastFlow anion exchange chromatography; lane 5,Phenyl-Sepharose High Performance hydrophobieinteraction chromatography; lane 6, Mono-Q anionexchange chromatography.SDS-PAGE de l'aminopeptidase intracellulaire deB linens ATee 9174 après différentes étapes de purifi-cation. Voies 1 et 7, standards de masse moléculaire;voie 2, extrait intracellulaire brut; voie 3, précipita-tion sulphate d'ammonium; voie 4, chromatographied'échange d'anions (DEAE-Sepharose Fast Flow);voie 5, chromatographie d'interactions hydrophobes(Phenyl-Sepharose} ; voie 6, chromatograhied'échange d'anions (Mono-Q).

Intracellular aminopeptidase from B linens

HM)IllO

75~ ~ 7;;'.S: ':;."~ 50

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Substrate specijicity

The relative activity of the aminopeptidase onL-Ala-p-NA and L-Gly-p-NA was 100 and164%, respectively (table III). The aminopep-tidase had no activity on any of the remainingaminopeptidase, iminopeptidase or dipeptidylaminopeptidase substrates (table III). The rela-tive activity of the aminopeptidase on dipep-tides showed that only those with an alanineresidue in the N-terminal position were hydrol-ysed. Maximum activity was observed on DL-Ala-DL-Aia. No activity was detected on any ofthe tripeptides studied.

Temperature CC)

Ion (h)

~ 75:~~tl' 50

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rH

Fig 3. Effeet of temperature (a) and pH (b) on theaetivity of the intraeellular aminopeptidase fromB linens xrcc 9174.Effet de la température (a) et du pH (b} sur l'activitéde l'ominopeptidase intracellulaire de B linensATCC9174.

175

Kinetic studies

Km and kcat values for L-Ala-p-NA were 3.3mmol/L and 4.3 s-l, respectively, while the cor-responding values for L-Gly-p-NA were0.2 mmollL and 7.6 s-l, respectively.

Nsterminal amino acid sequence

The sequence of the first 19 N-terminal aminoacids was NHTPro-Phe-Asp-G1y-Pro-Asp-Thr-Ala-Ala-Ile-Ile-Asp-Arg-Leu- ?-Asn-Ala-?- Thr.Homogeneity of the enzyme preparation was

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Fig 4. Effeet of (a) temperature [4 (0), 20 (0), 30(0),40 (L\), or so-c (EE)] and (b) pH on the stabilityof the intraeellular aminopeptidase from B linensArec 9174.Effet (a) de la température [4 (0),20 (0),30 (0),40("'), ou 50 "C (EH) J et (h) du pH sur la stabilité det'ominopeptidase intracellulaire de B linensATCC9174.

176 FP Rattray, PF Fox

Table 1. Purification of an intracellu1ar aminopeptidase from B linens ATCC 9174.Purification d'une aminopeptidase intracellulaire de B linens ATCC 9174.

Purification step Total protein Total activity Specifie activity Yield Purification factor(mg) (units} (units/mg} (%)

Cell-free extract 3332 57418 17 100 1.0

Ammonium sulphate fractionation 743 30896 42 54 2.5

DEAE-Sepharose 27.6 11727 425 20 25

Phen yI-Sepharose 0.55 6662 12113 12 712

Mono-Q 0.05 3661 73220 6 4307

Table II. EFfect of chemical reagents and divalent cations on the activity of an intracellular aminopeptidaseFrom B linens ATCC 9174.Effet des réactifs chimiques et des cations bivalents sur l'activité d'une aminopeptidase intracellulaire de B linensATCC9174.

Reagent or cation a Aminopeptidase activity (%)

0.1 mmol/L 1.0 mmol/L

Dithiothreitolp-MercaptoethanolCysteineEDTAEGTAI,IO-PhenanthrolinePMSFN-EthylmaleimideIodoacetic acidPHMBFe2+Mg2+

Ca2+Mn2+

Cu2+Hg2+

Ni2+Cd2+C02+

Zn2+

1051011051041041031001039867

1041009698979575733723

105101106101101989999962596908578484141372315

a EDTA: ethylenediaminetetraacetic acid; EGTA: ethylene glycol-bis (J3-aminoethyl ether)-N,N,N' ,N' -tetraacetic acid; PMSF:phenylmethylsulfonyl fluoride; PHMB: p-hydroxymercuribenzoate.

Intracellular arninopeptidase from B linens 177

Table III. Relative activitiesê of intracellular aminopeptidase from B linens ATCC 9174 on various substrates.Activités relatives d'une aminopeptidase intracellulaire de B linens ATCC 9174 sur différents substrats.

Substrate Relative activity (%) Substrate Relative activity (%)

Ala-p-NAGly-p-NAVal-p-NAPhe-p-NAPro-p-NAGlu-p-NALeu-p-NALys-p-NAArg-p-NAGly-Phe-p-NAAla-Pro-p-NA

100164

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DL-Ala-DL-AlaD-Ala-D-AlaAla-AlaDL-Ala-DL-PheAla-LeuAla-PheTyr-LeuTyr-PheLeu-GlyLeu-LeuLeu-ProLeu-AlaPhe-AlaPhe-GlyGlu-ValHis-LeuPro-GlyAla-Ala-AlaLeu-Gly-GlyLeu-Gly-ProPhe-Gly-Gly

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'Rates ofL-Ala-p-NA or m-Ala-et-Ala hydrolysis were taken as 100%.a Les taux d'hydrolyse de L-Ala-p-NA ou DL-Ala-DL-Ala ont été fixés à 100 %.

confirmed by no significant background inter-ference during sequencing. The N-terminalamino acid sequence was compared againstSwiss-prot Release 31, PIR Release 43 and Gen-Bank Release 87 data banks (DNAST AR Inc,Madison, Wisconsin, USA) using theLipman-Pearson prote in alignment technique,but no significant homology was found.

DISCUSSION

An intracellular aminopeptidase from B linensATCC 9174 was purified to homogeneity usingfour purification steps, ie, ammonium sulphatefractionation, anion exchange, hydrophobie inter-action and anion exchange chromatography. The

purified aminopeptidase had pH and tempera-ture optima of 8.5 and 35 "C, respectively, whichare comparable to the values pH 7.5 and 30 "Cfound for a partially purified aminopeptidasefrom B linens HS (El-Soda et al, 1991). Themolecular mass of the aminopeptidase deter-mined by SDS-PAGE and HPLC gel filtrationwas 59 and 69 kDa, respectively, indicating thatthe enzyme exists as a monomer in its nativestate.

The aminopeptidase was strongly inhibited bythe thiol blocking agent, PHMB, with reactiva-tion by the reducing agent DIT, indicating thata thiol group is present at the active site of theenzyme. The partially purified aminopeptidasefrom B linens, HS, was likewise inhibited byPHMB, but also by chelating agents and by the

178 FP Rattray, PF Fox

serine proteinase inhibitor, PMSF (El-Soda etal, 1991). In contrast, the vast majority ofaminopeptidases from lactic acid bacteria aremetalloenzymes (Visser, 1993), with the notableexception of an aminopeptidase from Lacto-bacillus delbrueckii ssp bulgaricus B 14(Wohlrab and Bockelmann, 1993), which wasinhibited by thiol blocking agents. Of the metalsassessed, Zn2+ was the most inhibitory;aminopeptidases isolated from various lacticacid bacteria have also been shown to beextremely sensitive to Zn2+ (Bockelmann et al,1992; Midwinter and Pritchard, 1994; Rul et al,1994).

The aminopeptidase from B linens ATCC9174 had a narrow substrate specificity; ithydrolysed both aminopeptidase and dipepti-dase substrates, but not dipeptidylaminopepti-dase or tripeptidase substrates. Activity onaminopeptidase substrates was confined toL-Ala- and L-Gly-p-NA, with highest activityon the latter; no activity was recorded onL-Val-, L-Phe-, L-Glu-, L-Leu-, L-Lys- orL-Arg-p-NA. Evidently, the aminopeptidase isspecifie for srnall, non-polar amino acids. Thepartially purified aminopeptidase from B linensHS (El-Soda et al, 1991) was also active onL-Ala- and L-Gly-, with the highest activity onL-Gly-p-NA, and no activity on i.-Leu-,L-Lys-, L-Pro- or L-Arg-p-NA. The amino-peptidase hydrolysed peptides containing ala-nine at the N-terminal position. Il was twice asactive on n-Ala-n-Ala as on L-Ala-L-Ala, withthe highest activity on DL-Ala-DL-Ala. It wasnot possible to assay the aminopeptidase on gly-cyl-peptides such as Gly-Gly and Gly-Gly-Gly,due to high substrate blanks; this is an acknowl-edged limitation with the method of Doi et al(1981). .

This substrate specificity may indicate apotential role for the aminopeptidase in the for-mation of the peptidoglycan in the cell wall,which in the case of B linens is type A 1Y(Schleifer and Kandler, 1972). During the for-mation of peptidoglycan, chains of the acylatedamino sugars containing the peptide subunit areinitially produced (in the case of type A 1Y, the

peptide subunit is t-Ala-n-Glu-diaminopimelicacid-D-Ala-D-Ala). The next step in peptido-glycan formation is the chemical cross-lin king ofthese chains, which involves the amino groupof the D-asymmetric carbon of thediaminopimelic acid, forming a peptide bondwith the carboxyl group of the penultimateD-Ala of an adjacent peptide subunit. The energyfor the formation ofthis peptide bond is derivedfrom the hydrolysis of the D-Ala-D-Ala peptidebond in the peptide subunit. It may be that theaminopeptidase has a role in this hydrolysis reac-tion, which may indicate that it is more associ-ated with the cell envelope rather than with thecytoplasm of the bacterium.

Another possible in vivo significance of thisintracellular aminopeptidase may be in the util-isation of oligopeptides that may be transportedacross the cell envelope of the bacterium; sucholigopeptides can be utilised for energy andgrowth requirements. However, due to the lim-ited specificity of this arninopeptidase, it isunlikely that the microorganism depends entirelyon it for intracellular oligopeptide hydrolysis.It is highly probable that other intracellularaminopeptidases and dipeptidases are presentin this microorganism.

The influence ofthis aminopeptidase on theripening of smear surface-ripened cheese is prob-ably quite limited due to its intracellular loca-tion; however, upon autolysis of the cells, thereleased aminopeptidase may act on casein-derived peptides and therefore contribute to theripening process. B linens does lyse upon nutri-ent limitations (Boyaval et al, 1985); however,whether such conditions exist on the cheese sur-face is not known. Clearly, the extracellular pro-teinases and aminopeptidases produced by Blinens and other microorganisms in the surfacesmear will have a mu ch larger affect on theripening process than intracellular peptidases.

ACKNOWLEDGMENTS

We thank A Healy, National Food Biotechnol-ogy Centre, University College, Cork, Ireland

Intracellular aminopeptidase from B linens

for the N-terminal amino acid sequencing and WBockelmann, Bundesanstalt für Milchforschung,Institut für Mikrobiologie, Kiel, Germany forassistance with cell cultivation. The project wasfunded in part from EU structural funds fromthe Food Sub-Programme of the IndustryProgramme.

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