Purification and Characterization of an Aminopeptidase from Lactococcus Lactis Subsp
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1989, p. 2308-2314 Vol. 55, No. 9 0099-2240/89/092308-07$02.00/0 Copyright © 1989, American Society for Microbiology Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris AM2 E. NEVIANI,'t C. Y. BOQUIEN,2 V. MONNET,1* L. PHAN THANH,3 AND J.-C. GRIPON' Station de Recherches Laitieres, Institut National de la Recherche Agronomique, 78350 Jouy-en-Josas,' Laboratoire de Ge'nie des Procedes Biotechnologiques Agro-alimentaires, Institut National de la Recherche Agronomique, 78850 Thiverval-Grignon,2 and Station de Pathologie Porcine et d'Immunologie, Institut National de la Recherche Agronomique, Nouzilly, 37380 Monnaie,3 France Received 24 February 1989/Accepted 8 June 1989 An aminopeptidase was purified from cell extracts of Lactococcus lactis subsp. cremoris AM2 by ion- exchange chromatography. After electrophoresis of the purified enzyme in the presence or absence of sodium dodecyl sulfate, one protein band was detected. The enzyme was a 300-kilodalton hexamer composed of identical subunits not linked by disulfide bridges. Activity was optimal at 40°C and pH 7 and was inhibited by classical thiol group inhibitors. The aminopeptidase hydrolyzed naphthylamide-substituted amino acids, as well as dipeptides and tripeptides. Longer protein chains such as the B chain of insulin were hydrolyzed, but at a much slower rate. The Michaelis constant (Km) and the maximal rate of hydrolysis (Vmax) were, respectively, 4.5 mM and 3,600 pkat/mg for the substrate L-histidyl-i-naphthylamide. Amino acid analysis showed that the enzyme contained low levels of hydrophobic residues. The partial N-terminal sequence of the first 19 residues of the mature enzyme was determined. Polyclonal antibodies were obtained from the purified enzyme, and after immunoblotting, there was no cross-reaction between these antibodies and other proteins in the crude extract. Lactococcus lactis subsp. cremoris is a common constit- Cultures of 5 liters were grown in a medium containing uent of mesophilic lactic starters used in the cheese industry. lactose at 40 g/liter, tryptone (Difco Laboratories, Detroit, Aside from its property of acidification, this species is Mich.) at 20 g/liter, and yeast extract at 7 g/liter. Growth was characterized by being very fastidious for amino nitrogen. It at 30°C, pH was controlled at 6.5 by the addition of 10 N is incapable of synthesizing most of the amino acids it sodium hydroxide, and mechanical agitation was at 100 rpm. requires and must thus find them in its growth medium. Milk, Growth was observed spectrophotometrically in a culture however, is relatively poor in free amino acids and small sample diluted 1/10 in 0.2% EDTA (pH 12), as well as by peptides that can be used directly by the cells. To attain determining the consumption of 10 N NaOH. At the end of optimal growth, therefore, lactic acid bacteria must hydro- exponential growth, the cells were centrifuged at 6,000 x g lyze milk proteins (19, 23). This is done via a complex for 10 min and washed three times in 0.05 M 3-glycerophos- proteolytic system which is only now beginning to be more phate buffer (pH 8.0). thoroughly understood. A proteinase located in the cell wall Determining peptidase activity. The activity of whole cells is the first enzyme active in the degradation of caseins, was maximal with the substrate His-Phe-,-NA (Bachem, releasing peptides which can be used as substrates for cell Bubendorf, Switzerland) (1), and protein extracts obtained wall, membrane-bound, or cytoplasmic peptidases (see ref- during purification were routinely tested with this substrate. erence 24 for a review). Other n-NA derivatives were also used, including His- We (1) have previously shown that L. lactis subsp. cre- Pro-1-NA, Gly-Pro-3-NA, Leu-1-NA, Ala-,-NA, Lys-p- moris AM2 has high peptidase activity toward the substrate NA, Pro-1-NA, Phe-1-NA, His-,B-NA, and Glu-,-NA L-histidyl-L-phenylalanine-,3-naphthylamide (His-Phe-,B-NA). (Bachem). Enzyme activity was measured by assaying the This activity was used to specifically observe the growth of quantity of naphthylamine produced from the substrates, L. lactis subsp. cremoris AM2 cocultivated with Leuconos- using the method of Goldbarg and Rutenburg (9), adapted by toc lactis CNRZ 1091, which lacks aminopeptidase activity Boquien et al. (1). The unit of activity used was the katal, the against this substrate (1). The present work reports the quantity of enzyme releasing 1 mol of naphthylamine per purification and characterization of this enzyme and indi- second. cates that it differs from other aminopeptidases described in The activity of the purified extract was also tested against lactic acid bacteria (3, 4, 8). several dipeptides and tripeptides: Leu-Tyr, Met-Pro, Met- Ala, Leu-Gly, Leu-Leu, Leu-Leu-Leu, Ala-Leu-Gly, Leu- MATERIALS AND METHODS Gly-Gly, carboxybenzoyl (CBZ)-Gly-Tyr (Sigma Chemical Bacterial strain and culture conditions. L. lactis subsp. Co., St. Louis, Mo.), CBZ-Phe-Phe (Cyclo Chemicals, Los cremoris AM2, also called CNRZ 380, was obtained from the Angeles, Calif.), and His-Phe, Phe-Gly-Gly, Met-Leu-Gly culture collection of the Centre de Recherches de Jouy- (Interchim, Montluqon, France). Enzyme extracts were in- en-Josas, France, and was stored at -18°C in litmus milk. cubated at 37°C and pH 7 in the presence of 0.4 mM substrate. A volume of ninhydrin solution (20 g of ninhydrin, * Corresponding author. 750 ml of methylcellosolve [ethylene glycol monomethyl t Present address: Instituto Sperimentale Lattiero-Caseario, ether], 250 ml of 4 M acetate buffer [pH 5.51], 7 ml of 20075 Lodi, Milan, Italy. titanium chloride) equal to the reaction volume was then 2308 VOL. 55, 1989 AMINOPEPTIDASE FROM LACTOCOCCUS LACTIS SUBSP. CREMORIS 2309 added. After the solution was heated at 100°C for 10 min and (ii) Second ion-exchange chromatography. The second ion- 3 volumes of 50% ethanol were added, the absorbance of the exchange chromatography was done as described in the first solution was determined at 440 nm for proline and at 570 nm step, but with a linear NaCl gradient of 0.4 to 0.5 M in 60 for the other amino acids. min. During purification, active fractions were stored at Preparation of subcellular fractions. Spheroplasts were - 18°C in the presence of stabilizers (10% glycerol, 100 mM obtained after suspending washed cells (30 g [wet weight]) in ammonium sulfate, 1 mM dithiothreitol). 250 ml of 0.03 M Tris buffer (pH 8) containing 30% sucrose Characterization of aminopeptidase. (i) Electrophoresis. and lysozyme at 0.3 mg/ml. After incubating for 3 h at 37°C The purified fraction was analyzed by polyacrylamide gel (7), the spheroplasts were recovered by centrifugation at electrophoresis (PAGE) in 10% acrylamide gels with and 10,000 x g for 20 min. The supernatant, called the cell wall without sodium dodecyl sulfate (SDS) (13), followed by extract, was recovered and filtered through a 0.22-,um- staining with Coomassie blue. SDS-PAGE was used to pore-size filter. determine molecular sizes with the following markers: phos- The spheroplasts were suspended in the same buffer phorylase b (94 kilodaltons [kDa]), bovine serum albumin (67 without sucrose. After the spheroplasts burst, the suspen- kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), sion was centrifuged at 20,000 x g for 30 min to separate the soybean trypsin inhibitor (20.1 kDa), and a-lactalbumin (14.4 cell extract from membranes and cell wall debris. kDa). Migration was in Tris-glycine-hydrochloride buffer The degree of lysis during the formation and bursting of (pH 8.8) for 2 h at 4°C and 30 V/cm. the spheroplasts was determined by measuring A600 and by After PAGE in the absence of SDS, enzyme activity was assaying lactate dehydrogenase (22), expressed as a percent- detected directly in the gel by the method of Miller and age of the total cellular lactate dehydrogenase activity deter- McKinnon (18). The gel was incubated at 25°C for 30 min mined after lysis with lysozyme in Tris buffer without with the following mixture: 1 mg of His-Phe-i-NA in 0.1 ml sucrose. of dimethylformamide and 10 mg of fast Garnet GBC (Sigma) Purification of enzyme. Nucleic acids in the cell extract in S ml of 0.2 M Tris hydrochloride (pH 7.5). Hydrolysis of were hydrolyzed by adding RNase and DNase (21). The the substrate was reflected by the appearance of a red color solution was then precipitated in the presence of 50 mM at the level of the enzyme band. MnSO4 and centrifuged at 20,000 x g for 30 min. (ii) Gel filtration. The molecular size of the active enzyme (i) First ion-exchange chromatography. Fast protein liquid was estimated by filtration through Ultrogel AcA 34 (IBF, chromatography was done with a Mono Q HR 10/10 column Villeneuve-la-Garenne, France) in a column (90 by 1 cm) (Pharmacia, Uppsala, Sweden). The column was equili- with 0.15 M NaCl in 20 mM Tris hydrochloride (pH 7.5) at a brated with 20 mM Tris hydrochloride (pH 7.5). The extract flow rate of 12 ml/h. The column was calibrated with (150 mg of proteins) was injected into the column and eluted thyroglobulin (670 kDa), ferritin (440 kDa), immunoglobulin with a linear NaCl gradient of 0 to 0.6 M in 1 h with a flow G (158 kDa), ovalbumin (42.7 kDa), myoglobin (17 kDa), and rate of 3 ml/min, and 3-ml fractions were collected. Fractions vitamin B12 (1.35 kDa). active against His-Phe-13-NA were recovered and dialyzed (iii) Amino acid analysis.