APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1987, p. 577-583 Vol. 53, No. 3 0099-2240/87/030577-07$02.00/0 Copyright © 1987, American Society for Microbiology Purification and Some Properties of a Membrane-Bound Aminopeptidase A from Streptococcus cremoris FRED A. EXTERKATE* AND GERRIE J. C. M. DE VEER Netherlands Institute for Dairy Research, 6710 BA Ede, The Netherlands Received 25 August 1986/Accepted 24 November 1986 A membrane-bound L-a-glutamyl (aspartyl)-peptide hydrolase (aminopeptidase A) (EC 3.4.11.7) from Streptococcus cremoris HP has been purified to homogeneity. The free y-carboxyl group rather than the amino group of the N-terminal L-a-glutamyl (aspartyl) residue appeared to be essential for catalysis. No endopeptidase activity could be established with this enzyme. The native enzyme is a polymeric, most probably trimeric, metalloenzyme (relative molecular weight, approximately 130,000) which shows on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels apparent high relative molecular weight values due to (lipid?) material dissociable with butanol. The subunit (relative molecular weight, approximately 43,000) is catalyti- cally inactive. The enzyme is inactivated completely by dithiothreitol, chelating agents, and the bivalent metal ions Cu2'and Hg2+. Of the sulfhydryl-blocking reagents tested, only p-hydroxymercuribenzoate appeared to inhibit the enzyme. Activity lost by treatment with a chelating agent could be restored by Co2+ and Zn2+ . The importance of the occurrence of an aminopeptidase A in S. cremoris with respect to growth in milk is discussed. Lactic streptococci possess proteinase activity associated successively, 25, 35, 45, and 55% (wt/vol) with solid AS. with the cell wall (7, 15, 22). In Streptococcus cremoris, After each addition, the solution was stirred for 15 min at 4°C peptidases have been detected which are located near the and the precipitate was collected by centrifugation (15 min at outside surface of and inside the membrane (8, 13). These 48,000 x g and 4°C). The precipitates were dissolved in peptidases are assumed to act in concert with the cell wall distilled water and either dialyzed overnight against an proteinase(s) to hydrolyze milk proteins to transportable excess of distilled water (4°C) and then freeze-dried or components (viz., amino acids and small peptides) (7), since concentrated by YM-10 (Amicon Corp., Lexington, Mass.) the size limit for transport through the membrane seems to filtration at 4°C. Usually >95% of the initial gluAP activity be approached with four to six residues in lactic streptococci was recovered and found entirely in the 25 to 35% (wt/vol) (14, 21). Subsequently, intracellular peptidases complete the AS fraction. This fraction was used for further purification. hydrolysis of transported peptides. In this way essential It contained 40 to 50% of the initial amount of protein in the amino acids are produced from milk proteins which other- cell extract. wise would limit growth of the organism. Butanol treatment. The AS fraction was dissolved in 0.05 Reports on the occurrence of membrane-bound peptidases M NaH2PO4-NaOH (pH 7.2), and n-butanol was added to are scarce, and knowledge of these peptidases is still scanty give a final concentration of 10% (vol/vol). The solution was (11, 13, 18). This is why our interest became focused stirred for 60 min at 25°C. After centrifugation (15 min, particularly on these enzymes in S. cremoris. Results (8) 48,000 x g), the supernatant was dialyzed twice against an have been obtained which indicate that a glutamate excess of distilled water at 4°C over a total period of 30 h and aminopeptidase (gluAP) is a membrane-bound enzyme. Ac- then centrifuged again to remove the precipitate obtained tivities detectable with the chymotrypsin substrate N- during dialysis. The supernatant was freeze-dried and dis- glutaryl-L-phenylalanine-4-nitroanilide and formerly desig- solved in 0.05 M NaH2PO4-NaOH buffer (pH 7.2). This nated as "endopeptidases" P37 and P50 have been found to known depend on a membrane-bound component (8) which might preparation exhibited gluAP activity, but all other be a peptidase responsible for the introductory release of the peptidase activities were inactivated by the butanol treat- glutaryl moiety. The gluAP activity could be due to a specific ment step (8). L-a-glutamyl (aspartyl) aminopeptidase (aminopeptidase A), Gel ifitration. For preparative purposes gel filtration was by the action of which the cell can provide itself with the performed at 4°C on a Sephacryl S-300 column (40 by 780 essential amino acid glutamic acid. The same enzyme could mm) equilibrated with 0.05 M NaH2PO4-NaOH buffer (pH be responsible for the release of a N-terminal glutaryl 7.2) with 350 mg of protein. Proteins were eluted with this moiety. To elucidate its function, the enzyme was purified buffer at a flow rate of approximately 0.6 ml min-', and 5-ml and its specificity was determined. fractions were collected. The pooled gluAP fraction was desalted and concentrated by filtration on an Amicon YM-10 MATERIALS AND METHODS filter. Analytical PAGE and IEF. Polyacrylamide gel electropho- Starting material for enzyme purification. A cell extract resis (PAGE) (8 or 8.5% acrylamide; 0.05 M imidazole buffer (500 ml) (7) obtained from milk-grown cells of S. cremoris system, pH 7.0) and analytical isoelectrofocusing (IEF) (pH HP (15-liter culture) suspended in 0.05 M NaH2PO4-NaOH 4 to 6) were performed essentially according to the instruc- buffer (pH 7.2) was subjected to fractionation by ammonium tions of LKB-Produkter AB, Bromma, Sweden (application sulfate (AS) precipitation. The extract (4°C) was brought to, notes 306 and 205, by respectively), by using the LKB 2117 Multiphor. The low-pl calibration kit (pH 2.5 to 6.5) was * Corresponding author. used for reference. 577 578 EXTERKATE AND DEVEER APPL. ENVIRON. MICROBIOL. Preparative IEF. Preparative flatbed IEF in a granulated showed impurities as judged by SDS-PAGE. The enzyme gel was performed with the LKB 2117 Multiphor essentially (102 pug of protein) in 250 ,ul of HEPES (N-2-hydroxy- according to the instructions described by Winters et al. in ethylpiperazine-N'-2-ethanesulfonic acid) buffer (25 mM), LKB application note 198. A bed of Ultrodex contained pH 7.5, was preincubated for 15 min at temperatures ranging Ampholine (LKB) carrier ampholytes of the pH range 4 to 6. from 30 to 80 or at 37°C in the presence of bivalent cation (1 The gluAP preparation (3 ml) supplemented with 5% mM) or the indicated amount of reagent. After preincuba- (vol/vol) Ampholine was applied as a zone after a prerun of tion, enzyme activity was measured at 37°C and pH 7.5 by 30 min, and IEF was performed overnight at 11°C. The the addition of a glu-pNA solution (4 mM) in HEPES buffer separate zones were collected by sectioning the gel bed and (50 mM). transferred to small columns. Elution of the gel fractions was Protein quantification. Proteins were estimated by the performed first with distilled water (6 ml) (for pH determi- micromethod of Bradford (4), by using crystalline serum nation) and then with 0.1 M NaH2PO4-NaOH buffer, pH 7.2 albumin (fraction V; BDH, Poole, England) as the standard. (6 ml). The pooled eluates were dialyzed against 0.002 M Chemicals and substrates. All reagents mentioned in this NaH2PO4-NaOH buffer, pH 7.2. study were of guaranteed grade. The following substrates Preparative PAGE. Preparative PAGE was performed by were used: L-ot-Glu-L-Ala (Sigma Chemical Co., St. Louis, applying the same buffer system and conditions as used for Mo.); Gly-L-ao-Asp and Gly-L-Phe (Fluka AG, Buchs, Swit- analytical PAGE. After completion of the electrophoretic zerland); L-Ala-L-Glu, L-0x-Glu-Gly, L-GluN-Gly, L-ot-Glu-L- run, the gels were first frozen at -80°C before the catalyti- Glu, L-oL-Glu-L-Ala-L-Ala, L-Ala-L-Asp, L-o-Asp-L-Phe, L-a- cally active component was cut out. Transfer of the protein Asp-L-Leu, L-Phe-L-Asp, L-Lys-L-Glu-Gly, Gly-Gly-L-Glu- from the gel into a small volume of buffer was accomplished L-Ala-methyl ester, L-y-Glu-L-Phe, and L--y-Glu-pNA (p- by electroelution, with the ISCO model 1750 sample concen- nitroanilide) (Bachem AG, Bubendorf, Switzerland); trator (Instrumentation Specialties Co., Lincoln, Nebr.) (3). pyroglu-pNA (Serva, Heidelberg, Federal Republic of Ger- The electrode compartment buffer was 0.05 M NaH2PO4- many); Z-L-Phe-L-Tyr and pyroglu-L-Ala (Cyclo Chemical, NaOH buffer, pH 7.2. Small pieces of the cut gel were placed Los Angeles, Calif.); L-a-Glu-pNa and glutaryl-L-Phe-pNa in the sample cups together with 0.005 M NaH2PO4-NaOH (Merck AG, Darmstadt, Federal Republic of Germany); buffer, pH 7.2. Electrophoresis was carried out for 6 h at 4°C Z-Gly-L-Phe, Z-L-(x-Glu-L-Phe, and Z-L-o(-Glu-L-Tyr (Mann (100 V, 20 to 30 mA). Research Laboratories, New York, N.Y.); succinyl-L-Phe- The enzyme fraction was removed from the small wells pNA (Boehringer GmbH, Mannheim, Federal Republic of with a plastic pipette. This fraction was dialyzed against Germany); N-acetyl-L-Ile-L-Glu-Gly-Arg-pNA (Kabi distilled water and freeze-dried. Diagnostica, Stockholm, Sweden). L-Arg-L-Glu-L-Leu was a Determination of (subunit) molecular weight. Relative mo- hydrolysis product obtained by the action of chymosin on lecular weights were estimated by electrophoresis in sodium 1-casein (25).