Streptococcus thermophilus - American Chemical Society

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New Insights into the Proteolytic System of Streptococcus thermophilus: Use of Isracidin To Characterize Cell-Associated Extracellular Peptidase Activities Zeeshan Hafeez,†,‡ Céline Cakir-Kiefer,†,‡ Jean-Michel Girardet,†,‡ Xavier Lecomte,†,‡ Cédric Paris,§ Wessam Galia,†,‡ Annie Dary,†,‡ and Laurent Miclo*,†,‡ Équipe “Protéolyse & Biofonctionnalités des Protéines et des Peptides” (PB2P), Unité de Recherche “Animal et Fonctionnalités des Produits Animaux” (UR AFPA), Université de Lorraine, Vandoeuvre-lès-Nancy, F-54506, France ‡ INRA, Unité de Recherche “Animal et Fonctionnalités des Produits Animaux” (UR AFPA), Unité Sous Contrat 340, Vandoeuvre-lès-Nancy, F-54506, France § Laboratoire d’Ingénierie des Biomolécules, École Nationale Supérieure d’Agronomie et des Industries Alimentaires (ENSAIA), Université de Lorraine, Vandoeuvre-lès-Nancy, F-54518, France

Downloaded by NANYANG TECHNOLOGICAL UNIV on August 29, 2015 | http://pubs.acs.org Publication Date (Web): August 24, 2015 | doi: 10.1021/acs.jafc.5b01647

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ABSTRACT: The influence on the hydrolysis of isracidin of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in addition to protease PrtS of Streptococcus thermophilus strains was investigated. S. thermophilus LMD-9 (PrtS+ phenotype) efficiently hydrolyzed the isracidin mainly through the PrtS activity, whereas strain CNRZ1066 (PrtS− phenotype) and two mutant strains LMD-9-ΔprtS and LMD-9-ΔprtS-ΔhtrA also displayed substrate hydrolysis, but different from that of the wild type strain LMD-9. Identification by mass spectrometry of breakdown products of isracidin revealed the existence of novel cell-associated extracellular carboxypeptidase and peptidyl dipeptidase activities in all PrtS− strains, besides known cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities. Both aminopeptidase and peptidyl dipeptidase activities were not able to cleave the isracidin at peptide bonds with proline residues. No hydrolysis of isracidin was detected in cell free filtrate for all the strains studied, indicating that no cell lysis had occurred. Taken together, these results suggested the presence of cell-associated extracellular peptidase activities in S. thermophilus strains that could be vital for the growth of PrtS− strains. KEYWORDS: S. thermophilus, cell-associated extracellular peptidases, bioactive peptide, isracidin

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INTRODUCTION The lactic acid bacterium Streptococcus thermophilus is greatly employed as starter culture component in yogurt fermentation and in the manufacture of a wide variety of cheeses. Besides its capacity of conferring organoleptic properties to the fermented milk products, it has been shown that S. thermophilus has the ability to generate bioactive peptides from various milk caseins through its cell-envelope associated proteolytic system.1 Unlike many lactic acid bacteria (LAB), S. thermophilus displays few amino acid auxotrophies (for example strain LGM18311 needs histidine and sulfur-containing amino acids).2 As milk contains relatively low amounts of free amino acids and peptides, the growth of S. thermophilus at high cell density depends on its ability to generate and incorporate peptides from milk proteins through its surface associated proteolytic enzymes as well as its peptide transport systems.3,4 The key element of this surface proteolytic system is the PrtS cell-wall associated protease, which hydrolyzes the milk caseins into oligopeptides and belongs to the subtilisin-like serine protease family, also known as the subtilase family.5,6 All the S. thermophilus strains do not possess PrtS, e.g., strain CNRZ1066 lacks prtS gene and, in contrast, strain LMD-9 displays strong proteolytic activity due to the presence and expression of prtS gene.7 However, it has been shown that, in the case of strain 4F44, PrtS does not exist only as a cell-wall associated protease but also is partially released into the growth medium.8 Besides PrtS, S. thermophilus © XXXX American Chemical Society

also harbors housekeeping surface protease HtrA (high temperature requirement A; E.C. 3.4.21.107) which is present in all the S. thermophilus strains sequenced until now (sequences available on the NCBI site at the address http:// www.ncbi.nlm.nih.gov/genome/?term= streptococcus+thermophilus). This protease is not considered to belong to the proteolytic system, which provides amino acids to S. thermophilus. Indeed, HtrA is a trypsin-like serine protease induced by heat shock and involved in basic cellular processes such as the degradation of misfolded exported proteins produced in response to environmental stresses.9 The peptides generally released by PrtS from milk proteins and transported into the cell are further hydrolyzed by a variety of intracellular exo- and endopeptidases4,10 well characterized in S. thermophilus at both biochemical and genetic levels (for review see Savijoki et al.).4 In addition to the intracellular peptidases, a recent study revealed the presence of cellassociated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities in both PrtS+ and PrtS− strains of S. thermophilus.11 Such cell-associated extracellular peptidases might liberate free amino acids or small peptides to accelerate Received: April 13, 2015 Revised: July 16, 2015 Accepted: July 20, 2015

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DOI: 10.1021/acs.jafc.5b01647 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Schematic representation of construction of (A) S. thermophilus LMD-9-ΔprtS strain and (B) S. thermophilus LMD-9-ΔprtS-ΔhtrA strain. P-PrtS: prtS promoter region. RBS-PrtS: prtS ribosome binding site. SPPrtS: PrtS signal peptide. Stop: sequence containing multiple stop codons. TerPrtS: prtS terminator region. P-HtrA: htrA promoter region. RBS-HtrA: htrA ribosome binding site. Ter-HtrA: htrA terminator region. emR: erythromycin resistance gene. specR: spectinomycin resistance gene. The inset reveals the PCR amplification of prtS gene. M: DNA molecular weight marker. Lanes 1, 2, 3, and 4 indicate PCR product obtained from the genomic DNA of S. thermophilus CNRZ1066, LMD-9-ΔprtS, LMD-9-ΔprtSΔhtrA, and LMD-9, respectively.

the growth of PrtS− strains, since these strains acidified their growth medium at a rate similar to PrtS+ strains when this medium contained a mixture of peptides, i.e., bactotryptone.7 Contrariwise, such cell-associated extracellular peptidase activities might be undesirable for the stability of milk-derived peptides carrying a given biological activity, eventually added to the fermented milk products or produced from milk proteins by the PrtS activity during the fermentation process. This hypothesis is reinforced by a current study in which Hafeez and colleagues11 revealed hydrolysis of several milk-derived bioactive peptides by cell-associated extracellular peptidases of S. thermophilus strains. Isracidin corresponds to the 1−23 fragment of bovine αs1casein (CN) and is released by chymosin digestion of this casein. It exhibits an antimicrobial activity against different bacteria such as Escherichia coli and staphylococci.12,13 It could be applied as a biopreservative in the dairy products due to its inherent antimicrobial properties.14 Besides this, isracidin has been widely employed as a substrate to determine cleavage specificities of the surface proteases of different LAB.15,16 Indeed, relatively large peptide size and presence of three proline residues make isracidin an ideal substrate for this purpose, as certain enzymes are unable to hydrolyze peptide bonds involving proline residue. Thereby, Fernandez-Espla and colleagues6 determined the cleavage preference of the purified PrtS of S. thermophilus CNRZ 385 on isracidin, showing that PrtS displayed a specificity that was intermediate between the specificities of PrtPI-type and PrtPIII-type cell-envelope proteases of Lactococcus lactis. Knowing that S. thermophilus strains also possess cell-associated extracellular peptidases and hydrolyze different bioactive peptides of smaller size,11 we have

chosen to use isracidin as substrate to study the proteolytic effect of PrtS and cell-associated extracellular peptidases (strain PrtS+ phenotype) as well as that of cell-associated extracellular peptidases alone (strain PrtS− phenotype), on this peptide. Therefore, first, we determined the contribution of PrtS activity of LMD-9 strain, among all of the other proteolytic activities present at the cell surface, in the hydrolysis of isracidin, and we investigated the subsequent evolution of the peptides formed by a kinetics approach. Second, in order to investigate the level of hydrolysis of isracidin by known cellassociated extracellular peptidase activities (aminopeptidase and X-prolyl dipeptidyl peptidase), two mutant strains, i.e., ΔprtS and ΔprtS-ΔhtrA, constructed from strain LMD-9, were incubated with the peptide. In addition, the level of hydrolysis of isracidin by the strain CNRZ1066 that does not naturally display any PrtS activity has been studied.

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MATERIALS AND METHODS

Bacterial Strains, Growth Conditions, and Construction of Mutants. S. thermophilus CNRZ1066 came from the CNRZ (Centre National de Recherches Zootechniques, INRA, Jouy-en-Josas, France) while S. thermophilus LMD-9 (ATCC BAA-491) came from ATCC (American Type Culture Collection, Manassas, VA, USA) collections. Their genomes have been entirely sequenced.17,18 S. thermophilus LMD-9-ΔprtS mutant used in this study was obtained in a previous study.19 Briefly, it was planned to replace the ORF of prtS gene by a fragment consisting of the ORF of prtH gene fused with the ORF of an erythromycin resistance (emR) gene. During this mutant construction, a clone presenting the LMD-9-ΔprtS sequence was obtained (Figure 1A). To construct S. thermophilus LMD-9-ΔprtS-ΔhtrA mutant strain, htrA gene (locus_tag: STER_RS09790; NCBI Accession Number: B


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to 1 mL of the substrate and incubated 1 h at 37 °C. The hydrolysis of Lys-p-NA was determined by measuring absorbance at 410 nm using BioSpectrometer Basic (Eppendorf France S.A.S., Le Pecq, France). Isracidin Preparation. αs1-CN from sodium caseinate was first purified by batch fractionation according to the method of Miclo et al.22 and then hydrolyzed with rennet from Mucor meihei, type II (Sigma-Aldrich), as described by Lahov and Regelson12 with some modifications to get pure isracidin. Crude isracidin was separated from the residual αs1-CN by solid phase extraction (SPE) using Plus C18 Sep-Pak cartridges (Waters, Milford, MA, USA) as follows: the cartridges were equilibrated with 5 mL of solvent A (5% acetonitrile and 0.1% trifluoroacetic acid in water) at a flow rate of approximately 1 mL/min. The αs1-CN hydrolysate (2 mL) prepared at 5 mg/mL in solvent A was loaded onto the cartridges. The salts were removed by washing the cartridges with 5 mL of solvent A while the crude isracidin was eluted with 5 mL of solvent B (30% acetonitrile and 0.1% trifluoroacetic acid in water). The liquid chromatography−mass spectrometry analysis (LC/ESI-MS/MS; see below) showed that the crude isracidin was a mixture of 1−23 and 3−23 peptides in a molar ratio 10:1 (data not shown). This crude isracidin was then injected into Uptisphere C18 column (length and internal diameter 250 × 10 mm, particle size 5 μm, porosity 10 nm; Interchim, Montluçon, France) connected to an Alliance high-performance liquid chromatography (HPLC) unit (Waters) to get pure 1−23 peptide. A linear gradient of 25−38% acetonitrile in water in the presence of 0.1% trifluoroacetic acid was applied at a flow rate of 3.0 mL/min and at 30 °C. Finally, isracidin was detected with a photodiode array detector 996 (Waters) at 215 nm, collected, and lyophilized. Hydrolysis of Isracidin. S. thermophilus strains were grown to the exponential phase (OD650nm of 1.00 ± 0.05) in LM17 medium with or without antibiotic(s). The cells were harvested by centrifugation at 3100g for 10 min at 20 °C and the resulting cell pellet was washed twice with PAB, followed by resuspension of the cells to obtain the initial OD650nm of 1.00 ± 0.05 in the same buffer. Crude and pure isracidin solutions prepared in the PAB were then added to the cell suspensions to a final concentration of 20 nmol/mL and incubated at 42 °C with shaking at 200 rpm. A sample (500 μL) was aliquoted immediately, and other samples of the same volume were collected after 10, 20, 45, 60, 120, 180, 240, and 360 min of incubation. The hydrolysis reaction was stopped by adding 15 μL of trifluoroacetic acid to each sample. The cells were removed by filtration through a 0.45μm filter (Phenomenex, Le Pecq, France), and the hydrolysis products were stored at −20 °C until the LC/ESI-MS/MS analysis. The experiments were done in duplicate. Stability of the isracidin in PAB was checked in the same conditions without bacteria. Identification of Peptides by LC/ESI-MS/MS. The peptide fragments resulting from isracidin hydrolysis were analyzed by reversed-phase HPLC on an Ultimate 3000 system (Thermo Fisher Scientific/Dionex) coupled to electrospray ionization tandem mass spectrometry (RP-HPLC/ESI-MS/MS) using a spectrometer equipped with a linear trap quadripole ion trap mass analyzer LQT-XL (Thermo Fisher Scientific). Peptides were separated using a C18 Alltima reversed-phase column (length and inner diameter 150 × 2.1 mm, 5-μm porosity; Grace/Alltech, Darmstadt, Germany) equipped with a C18 Alltima precolumn (7.5 × 2.1 mm, 5-μm porosity; Grace/Alltech) at 25 °C. The peptides were eluted using a linear gradient of 5−60% acetonitrile in water in the presence of 0.1% (v/v) trifluoroacetic acid for 60 min at a flow rate of 0.2 mL/min and quantified with a double photodiode array detector (Thermo Fischer Scientific/Dionex). Peptides were detected by photodiode array detector and by mass spectrometric detector allowing UV semiquantification at 214 nm and also accurate MS semiquantification due to specific screening of peptide ions, seen on single ion parent chromatograms (SIC). To perform the quantification by UV detection at 214 nm, the molar extinction coefficient of each identified peptide was calculated according to Kuipers and Gruppen.23 The mass spectrometry was conducted under the following conditions: ESI positive ionization mode was used; spray voltage was set at 4.5 kV; source gases were set (in arbitrary units per min) for sheath gas, auxiliary gas, and sweep gas at 20, 5, and 5, respectively;

NC_008532.1) located between positions 1854162 and 1855397 on the S. thermophilus LMD-9 chromosome was replaced by specR gene that encodes spectinomycin adenyltransferase. For this purpose, htrA (upper and down) and specR fragments were amplified form genomic DNA of S. thermophilus LMD-9-ΔprtS mutant strain and pSET4s plasmid, respectively, as described by Hafeez et al.11 The overlap extension polymerase chain reaction was carried out using the above three amplicons, and then S. thermophilus LMD-9-ΔprtS mutant strain was transformed with final amplicon (Figure 1B).11 The strains were conserved at −80 °C in sterile reconstituted skim milk (10%). They were grown overnight at 42 °C by inoculation (1%) in fresh sterile reconstituted skim milk (10%), and the precultures were then inoculated at 1% in LM17 medium (M1720 broth supplemented with 20 g/L lactose) and incubated at 42 °C. When required, erythromycin was added to the culture media at final concentration of 5 μg/mL as selection pressure for LMD-9-ΔprtS and both erythromycin and spectinomycin at concentrations of 2.5 and 150 μg/mL, respectively, to select LMD-9-ΔprtS-ΔhtrA mutant strain. Polymerase Chain Reaction Analysis for Detection of prtS Gene. The presence or the absence of PrtS was determined using forward and reverse primer pairs of prtS gene and genomic DNA of S. thermophilus LMD-9, as well as mutant strains, i.e., LMD-9-ΔprtS and LMD-9-ΔprtS-ΔhtrA, and of S. thermophilus CNRZ1066 as template. Chromosomal DNA of S. thermophilus strains were purified from 50 mL LM17 cultures after growth to an optical density at 650 nm (OD650nm) of about 1.0 as described by Fischer et al.21 The standard polymerase chain reaction (PCR) was conducted in a Mastercycler Pro thermocycler (Eppendorf, Hamburg, Germany) to detect the prtS gene. The forward (5′-CAATTCCATCTGTCTTCATTC-3′) and reverse (5′-CCGTTGTTAATTGTGTTGAGT-3′) primers used to amplify an internal fragment of the prtS gene were synthesized by Eurogentec (Serain, Belgium). The PCR reaction was performed in a final reaction volume of 20 μL consisting of 0.5 units of Taq DNA polymerase (Thermo Fisher Scientific, Villebon-sur-Yvette, France), 1× Taq buffer, 0.2 mM of each deoxyribonucleotide triphosphate (dNTP), 1.5 mM of MgCl2, 0.5 μM of each primer, and about 100 ng of purified DNA as template. PCR amplification was carried out under the following conditions: initial denaturation step at 95 °C for 3 min, 30 cycles consisting of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and an extension at 72 °C for 60 s followed by a single 10 min final elongation at 72 °C. As expected, after agarose gel electrophoresis, the single nucleotide band with the anticipated molecular mass of the prtS gene was detected only in S. thermophilus LMD-9 (Figure 1 inset). This thus confirmed that the prtS gene is absent from the genome of LMD-9-ΔprtS, LMD-9-ΔprtS-ΔhtrA, and CNRZ1066 strains. Evaluation of Cell Lysis. In order to verify cell integrity in the pH 6.5, 12.5 mM potassium phosphate/acetate buffer (PAB), the activity of intracellular peptidases, potentially liberated by a cell lysis during cell incubation in the medium, was measured using the lysine-paranitroanilide (Lys-p-NA) chromogenic substrate (Sigma-Aldrich France, Saint-Quentin Fallavier, France). The substrate was prepared at 2 mM as described elsewhere,8 whereas the samples were prepared as follows: the log phase (OD650nm = 1.00 ± 0.05) cells of each S. thermophilus strain grown in LM17 medium were centrifuged at 3100g for 10 min at 20 °C and resuspended in PAB to the initial OD650nm of about 1 preceded by two washings with PAB. A part of the resuspended cells was frozen at −20 °C while the other part was incubated at 42 °C with shaking at 200 rpm (revolutions per min). After 0, 2, and 6 h incubation, cells were removed by centrifugation at 12000g for 5 min at 20 °C and the supernatant obtained was filtered through a sterile 0.22-μm filter (Merck Millipore, Darmstadt, Germany) to obtain cell free filtrate. Frozen cell suspensions of S. thermophilus were thawed on ice and disrupted by sonication 3 × 1 min, power 5, and active cycle 50% with a Vibra-cell (Fisher Scientific SAS, Illkirch, France). The cell lysate was recovered by centrifugation at 13400g for 10 min at 4 °C and used as such as positive control of intracellular peptidase activity, as intracellular peptidases have the ability to hydrolyze Lys-p-NA substrate. 100 μL volume samples of both cell free filtrate and cell lysate (intracellular proteins) were added C


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Figure 2. Hydrolysis kinetics of isracidin (empty circle) by S. thermophilus LMD-9 with generation of fragments (1RPKHPIK7, filled triangle; 1 RPKHPIKH8, filled square; 1RPKHPIKHQ9, empty square; 1RPKHPIKHQGLPQ13, cross; 1RPKHPIKHQGLPQEVL16, empty triangle; 1RPKHPIKHQGLPQEVLN17, empty diamond; and 1RPKHPIKHQGLPQEVLNENLL21, filled circle) analyzed by using single parent ion chromatograms. Isracidin (20 nmol/mL) was incubated at 42 °C under shaking at 200 rpm with log phase cells of this bacterial strain in phosphate/acetate buffer 12.5 mM, pH 6.5. The inset indicates similar hydrolysis kinetics of isracidin after peak area integration at 214 nm corrected by the molar extinction coefficients according to Kuipers and Gruppen.23 A.U.: absorption unit. capillary temperature was set at 250 °C; capillary voltage at 26 V; tube lens, split lens, and front lens voltages at 90 V, −42 V, and −5.75 V, respectively. The ion optics parameters were optimized by automatic tuning using a standard solution of the dipeptide Val-Trp at 0.1 g/L infused in mobile phase (50% acetonitrile in water and in the presence of 0.1% trifluoroacetic acid) at a flow rate of 5 μL/min. Full scan MS spectra were repeatedly performed from 100 to 2000 m/z during the time of the run and, second, automatized MS/MS scans were realized in order to identify the peptides with certainty. The peptide masses were given with a precision of 0.01%.

Table 1. Identification by RP-HPLC/ESI-MS/MS of Peptide Fragments Generated upon 1-h Incubation of Isracidin with S. thermophilus LMD-9 in Phosphate/Acetate Buffer 12.5 mM, pH 6.5 mass (Da)

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RESULTS Hydrolysis of Isracidin by Surface Proteolytic System of S. thermophilus LMD-9. The ability of the surface proteolytic system of S. thermophilus LMD-9 to hydrolyze isracidin was evaluated. For this, the pure isracidin was incubated with nongrowing log phase cells of LMD-9 strain in PAB at pH 6.5 and 42 °C. It was determined by MS screening of isracidin corresponding to [M + 2H]2+ double charged parent ion that about 99% of the isracidin was already lost after 10 min incubation and that no intact peptide was detected after 20 min (Figure 2). This hydrolysis was attributed to the proteolytic activity of the LMD-9 strain and not to an eventual instability of the peptide since isracidin was not hydrolyzed when incubated without cells in the PAB under the same conditions. The degradation products were then analyzed by RP-HPLC/ ESI-MS/MS, and the peptides identified are listed in Table 1. It was observed that the cell-associated proteolytic system of S. thermophilus LMD-9 cleaved the isracidin at K7−H8, H8−Q9, Q9−G10, Q13−E14, L16−N17, N17−E18, and L21−R22 peptide bonds. Hydrolysis kinetics revealed that a large quantity (determined by peak area integration using single parent ion chromatogram) of peptide 1−17 was present just after a 10-min

peptide fragment

sequence

obsd

theor

1−7 1−8 1−9 1−13 1−16 1−17 1−21 17−23 18−23 22−23

RPKHPIK RPKHPIKH RPKHPIKHQ RPKHPIKHQGLPQ RPKHPIKHQGLPQEVL RPKHPIKHQGLPQEVLN RPKHPIKHQGLPQEVLNENLL NENLLRF ENLLRF RF

874.7 1012.0 1140.0 1535.0 1876.3 1991.0 2460.0 904.6 790.6 321.3

874.6 1011.7 1139.7 1534.9 1876.1 1990.1 2459.4 904.5 790.4 321.2

exposure of isracidin to the nonproliferating cells of LMD-9 strain (Figure 2) followed by smaller quantity of the peptide 1− 16. The quantity of both peptides decreased throughout 1 h of incubation, unlike the peptides 1−8, 1−9, and 1−13, the quantity of which increased slowly and consistently. On the other hand, the increase in the quantity of peptides 1−7 and 1− 21 was noted at much lower intensity during 1 h incubation (Figure 2). According to the results of Fernandez-Espla et al.,6 who reported the preferential cleavage sites of purified PrtS on the pure isracidin, and to those of Miclo et al.,1 who analyzed hydrolysis of the 1−23 region of the αs1-CN by cells of various strains of S. thermophilus, the cleavage sites observed in this study could be attributed to the protease PrtS. Indeed, these authors reported that the PrtS cleaved the bonds N17−E18 and L16−N17 most preferably followed by the bonds H8−Q9 and Q9−G10. The bonds Q13−E14 and L21−R22 are cleaved to a D


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Journal of Agricultural and Food Chemistry medium and lesser extent, respectively. Hydrolysis of the bond K7−H8 was reported only by Miclo et al.,1 who used bacterial cells and not purified PrtS. Only slight differences in the evolution of the quantity of the peptides (appeared or disappeared) were observed as a function of time when quantified using either single parent ion chromatograms or absorbance at 214 nm by taking into account the different molar extinction coefficients (Figure 2 inset). Hydrolysis of Isracidin by Cell-Associated Extracellular Peptidases. To assess the contribution of cell-associated extracellular peptidases in hydrolysis of isracidin, strains without cell surface PrtS activity (CNRZ1066, LMD-9-ΔprtS, and LMD-9-ΔprtS-ΔhtrA) were used. The purpose of deleting htrA gene was to remove all the surface protease activities of LMD-9 strain whether they make part of the proteolytic system or not. The isracidin was incubated individually for 6 h with the resting intact cells of these three strains. The hydrolysis of the peptide was less important than with the LMD-9 strain since 50% of isracidin was intact after 1 h of hydrolysis by CNRZ1066 and 78% by LMD-9 mutants. The released peptides were identified by RP-HPLC/ESI-MS/MS (Table 2) and seemed to be the result of different cell-associated extracellular peptidase activities and were grouped into five different categories. The Figure 3 presents an overview of the different categories of peptides generated according to the different exopeptidase activities highlighted for the three PrtS− strains. The category A included all those peptides, which resulted from isracidin cleavage at L21−R22, N19−L20, N17−E18, V15−L16, Q13−E14, Q9− G10, and K7−H8 peptide bonds (Figure 3). This thus indicated the consecutive removal of dipeptides from the C-terminal of the isracidin, indicating a peptidyl dipeptidase activity (for informations on this kind of activity see http://www.brendaenzymes.org). The presence of the C-terminal dipeptide 22RF23 was highlighted, but no other dipeptides were found by mass spectrometry analysis. On the other hand, peptidyl dipeptidase activity was not observed when a Pro residue was encountered at a potential cleavage site, as in the case of L11−P12, P5−I6, and R1−P2 peptide bonds. The peptides, whose release involved the cleavage of these bonds, as peptide 1−9 by example, were formed by previous action of other enzymes (see below). The type of cleavage leading to the release of peptides belonging to category A referred to a peptidyl dipeptidase activity and was observed for each of the three S. thermophilus strains exhibiting a PrtS− phenotype, i.e., CNRZ1066 as well as both LMD-9ΔprtS and LMD-9-ΔprtS-ΔhtrA. Furthermore, the release of the C-terminal Phe residue as well as of peptides whose number of residues was an even number (category B; Table 2) revealed a carboxypeptidase activity that might act in a recurrent manner. The existence of the peptides 1−14, 1−16, and 1−20 might also reveal a combined action of peptidyl dipeptidase and carboxypeptidase activities. As an example, the peptide 1−20 might be formed by the successive action of the peptidyl dipeptidase activity at peptide bond L21−R22 of isracidin (formation of peptide 1−21) followed by the action of carboxypeptidase activity at peptide bond L20−L21 (Figure 3). The peptides 1−14 and 1−16 seemed to be generated in a similar manner, whereas the release of carboxyl-terminal Phe residue might be produced likewise or by the action of the aminopeptidase activity mentioned below (see peptides belonging to category C) on the dipeptide 22RF23. Both peptidyl dipeptidase and carboxypeptidase activities were associated with the cell wall because no such activities were observed in cell free filtrate obtained even after 6 h incubation

Table 2. Isracidin Fragments Identified by RP-HPLC/ESIMS/MS from 0- to 6-h Exposure to S. thermophilus LMD-9ΔprtS, LMD-9-ΔprtS-ΔhtrA, and CNRZ1066 in Phosphate/ Acetate Buffer 12.5 mM, pH 6.5 mass (Da) peptide fragment 1−21 1−19 1−17 1−15 1−13 1−9 1−7 22−23 23−23 1−20 1−16 1−14 4−23 7−23a 8−23 9−23 10−23 11−23a 14−23 15−23 16−23 17−23 18−23 1−2 4−5 3−23 6−23 13−23a 3−21a 4−21 8−21 14−21 18−21

sequence Category A RPKHPIKHQGLPQEVLNENLL RPKHPIKHQGLPQEVLNEN RPKHPIKHQGLPQEVLN RPKHPIKHQGLPQEV RPKHPIKHQGLPQ RPKHPIKHQ RPKHPIK RF Category B F RPKHPIKHQGLPQEVLNENL RPKHPIKHQGLPQEVL RPKHPIKHQGLPQE Category C HPIKHQGLPQEVLNENLLRF KHQGLPQEVLNENLLRF HQGLPQEVLNENLLRF QGLPQEVLNENLLRF GLPQEVLNENLLRF LPQEVLNENLLRF EVLNENLLRF VLNENLLRF LNENLLRF NENLLRF ENLLRF Category D RP HP KHPIKHQGLPQEVLNENLLRF IKHQGLPQEVLNENLLRF QEVLNENLLRF Category E KHPIKHQGLPQEVLNENLL HPIKHQGLPQEVLNENLL HQGLPQEVLNENLL EVLNENLL ENLL

obsd

theor

2460.0 2233.7 1991.0 1763.0 1535.0 1140.0 874.7 321.3

2459.4 2233.2 1990.1 1763.0 1534.9 1139.7 874.6 321.2

165.0 2346.0 1876.3 1664.0

165.1 2346.3 1876.1 1663.9

2382.0 2304.2 1907.0 1768.6 1640.5 1583.9 1245.5 1116.5 1017.0 904.0 790.0

2381.3 2304.1 1906.0 1768.9 1640.9 1583.9 1245.7 1116.6 1017.6 904.5 790.4

271.0 252.0 2509.0 2147.0 1373.8

271.2 252.1 2509.4 2147.2 1373.7

2206.3 2078.0 1602.0 942.0 487.0

2206.2 2078.1 1602.8 942.5 487.3

a

Identified in the case of CNRZ1066 with crude isracidin (mixture of peptides 1−23 and 3−23 in a ratio 10:1) as substrate; the crude isracidin was not tested with the LMD-9 mutant strains.

of the bacterial cells in PAB (data not shown). This thus confirmed the absence of cell lysis which could have led to the release of intracellular peptidases in the incubation buffer. To our knowledge, peptidyl dipeptidase and carboxypeptidase activities associated with the cell wall have never been reported before in this bacterium. The second main group is the category C, which is composed of peptides generated by an aminopeptidase activity. Table 2 shows all the peptides identified by mass spectrometry that were the result of consecutive removal of N-terminal amino acids. Peptides ending with the Phe23 residue and the first residue of which corresponded to the 3rd to the 18th residue of isracidin were recovered except for peptides 2−23, 5−23, and 12−23. In the case of the latter three peptides, the E


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addition to 1−23, a low amount of fragment 3−23 (in a molar ratio 10:1 between the two peptides). Under these conditions, all the peptides belonging to category C (Figure 3) were found as hydrolysis of peptide 3−23 did not need the cleavage of the first bond of isracidin involving a Pro residue. Meanwhile, the existence of the dipeptides 1−2 and 4−5 (category D) showed that the isracidin was also cleaved by an X-prolyl dipeptidyl peptidase activity, which removed dipeptides containing proline residue and allowed aminopeptidase activity to continue downstream of these proline residues. However, the dipeptide 11−12 was not identified in our experimental conditions. The release of this peptide needed the cleavage of numerous bonds by the conjugated actions of (i) the aminopeptidase and X-prolyl dipeptidyl peptidase activities and (ii) peptidyl dipeptidase and/or carboxypeptidase activities on the polypeptide chain. The unknown relative rates and residue preferences of these activities do not allow to predict if the generation of this peptide is possible. The presence of certain peptides indicated that they were produced due to action of two or more peptidase activities. All the PrtS− strains of S. thermophilus, i.e., CNRZ1066, LMD-9ΔprtS, and LMD-9-ΔprtS-ΔhtrA, produced these peptides, and they are listed in category E (Figure 3). Kinetic Approach To Distinguish PrtS Activity from Exopeptidase Activities. Through three examples of selected peptides (1−13, 1−17, and 22−23), kinetics of isracidin degradation showed that all four S. thermophilus strains, i.e., CNRZ1066, LMD-9, LMD-9-ΔprtS, and LMD-9-ΔprtS-ΔhtrA, were able to cleave isracidin at N17−E18, Q13−E14, and L21−R22 peptide bonds, but differently (Figure 4). As shown in Figure 4A, the peptide 1−17 was produced in substantially high amount by LMD-9 strain and seemed likely to be the result of PrtS activity, whereas in the case of the PrtS− phenotype strains

Figure 3. Peptides identified by RP-HPLC/ESI-MS/MS released from 0- to 6-h contact of isracidin with PrtS− strains, i.e., S. thermophilus CNRZ1066, LMD-9-ΔprtS, and LMD-9-ΔprtS-ΔhtrA in phosphate/ acetate buffer 12.5 mM, pH 6.5. The peptides were obtained as a result of peptidyl dipeptidase activity (class A), carboxypeptidase activity (class B), aminopeptidase activity (class C), X-prolyl-dipeptidyl peptidase activity (class D), and a combination of previous activities (class E). Peptides with an asterisk symbol were identified with CNRZ1066 with crude isracidin (mixture of peptides 1−23 and 3−23 in a ratio 10:1) as substrate; the crude isracidin was not tested with LMD-9-ΔprtS and LMD-9-ΔprtS-ΔhtrA.

aminopeptidase activity was blocked due to the presence of a proline residue at the carboxyl side or P1′ position (according to the nomenclature of Schechter and Berger)24 of the bonds R1−P2, H4−P5, and L11−P12. Initially the aminopeptidase activity was detected upon incubation of S. thermophilus CNRZ1066 with the crude isracidin, which contains, in

Figure 4. Distinct release pattern of peptide fragments (A) 1−17, (B) 1−13, and (C) 22−23 by cell-associated extracellular proteolytic activities of S. thermophilus strains LMD-9 (filled diamond), LMD-9-ΔprtS (empty square), LMD-9-ΔprtS-ΔhtrA (empty triangle), and CNRZ1066 (empty circle) upon 6-h incubation with isracidin in phosphate/acetate buffer 12.5 mM, pH 6.5. F


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Journal of Agricultural and Food Chemistry such as CNRZ1066 and LMD-9 mutant strains, a minor quantity was produced as a result of action of peptidyl dipeptidase and/or carboxypeptidase activities. In contrast, the peptides 1−13 and 22−23 were generated in greater quantities by peptidase activities of CNRZ1066 strain than by PrtS and/or peptidase activities of the strain LMD-9 (Figures 4B and 4C). However, among the PrtS− phenotype, peptidase activities leading to generation of peptides 1−13 and 22−23 in strain CNRZ1066 appeared to be much higher than in both mutant strains of LMD-9.

Figure 5. Cleavage site preferences of S. thermophilus LMD-9 nonproliferating cells (Miclo et al.1 and this study) and of purified PrtS of S. thermophilus CNRZ 385 (Fernandez-Espla et al.6) on isracidin at different pH. In the case of work of Miclo et al.,1 the peptide bond 22−23 is cut preferably instead of bond 21−22 as αs1casein and not isracidin is the substrate. The relative cleavage rates are indicated by large (major site), medium (medium site), and dotted (minor site) arrows except for Miclo et al.,1 where the cleavage rates are not given.


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DISCUSSION Isracidin is a substrate widely used to determine cleavage specificities of the surface proteases of different LAB.15,16 In the present study, the impact of surface protease PrtS as well as exopeptidase activities of S. thermophilus on the degradation of isracidin was explored, as it has been shown that the 1−23 Nterminal region of αs1-CN is sensitive toward proteolytic attack of S. thermophilus (such as the LMD-9 strain) and other LAB strains that possess the main cell wall-associated protease.1,6,15,16 Isracidin was completely hydrolyzed after exposure to the intact cells of S. thermophilus LMD-9. It has been reported by Fernandez-Espla et al.6 that the purified PrtS of S. thermophilus CNRZ385 preferentially and primarily cleaved isracidin at N17− E18 and L16−N17 peptide bonds, and we have found similar results upon exposure of isracidin to the cells of S. thermophilus LMD-9. In another study the same results were obtained with the highly acidifying S. thermophilus strains PB302, PB385, 4F44, and ATE19PB8.1 Hence, it appeared that the proteolytic activity toward isracidin highlighted for S. thermophilus LMD-9 was overwhelmingly due to the activity of PrtS. Nevertheless some differences were noted, as the hydrolysis of peptide bond L21−R22 to a medium extent in this study whereas this bond appeared to be poorly hydrolyzed by PrtS of the strain CNRZ385.6 Moreover K7−H8, H8−Q9, and Q9−G10 peptide bonds were poorly hydrolyzed, in the present study, while they appeared to be preferred cleavage sites of PrtS in other studies,1,6 although the peptide bond K7−H8 was not hydrolyzed by PrtS of strain CNRZ385.6 The perceptible difference in the preference of some peptide bond hydrolysis might be explained by the difference in pH of incubation buffer. This observation is consistent with the previous findings that pH of the medium is one of the factors that alter the preference for bonds in the 1−23 fragment of αs1-CN, which can even result in a completely changed specificity.15,25 As shown in Figure 5, the cleavage preference of the PrtS protease for the peptide bonds N17−E18 and L16−N17 remains unchanged regardless of the pH tested. However, PrtS has pronounced cleavage preference toward K7−H8, H8−Q9, and Q9−G10 peptide bonds at pH 7.5 compared to pH 6.5, since no hydrolysis of K7−H8 peptide bond is observed at pH 8.0. Contrariwise, the weak hydrolysis of the peptide bond between the residues L21 and R22 is reported at pH 8.0 and 5.2. The peptide bond Q13−E14 is cleaved by PrtS in a similar way at all tested pH except 5.2 (Figure 5). In conclusion, the hydrolysis of a maximum of peptide bonds in the isracidin was observed at a pH (6.5−7.5) close to the initial pH of the milk fermentation process. This emphasizes that the sensitivity of the peptide toward PrtS can be modulated in the fermented products by changing the pH. Having a relatively large chain length, isracidin appeared to be a suitable substrate for further unveiling the potential

exopeptidase activities acting both at the N-terminal and Cterminal extremities. Moreover, the presence of three proline residues in the 1−12 N-terminal region of isracidin permitted us to highlight the X-prolyl dipeptidyl peptidase and aminopeptidase activities simultaneously as the latter is inhibited by proline residues. Two mutant strains of LMD-9 devoid of PrtS or both PrtS and HtrA, i.e., LMD-9-ΔprtS and LMD-9-ΔprtSΔhtrA, were used in this study. The LMD-9-ΔprtS strain permitted highlighting of peptidase activities associated with cell that were masked in the presence of PrtS in LMD-9 wild type strain, whereas the LMD-9-ΔprtS-ΔhtrA strain helped to conclude that observed hydrolysis was not due to the protease HtrA, another surface protease, which is involved in the hydrolysis of extracellular misfolded proteins. The strain CNRZ1066 represented naturally PrtS− phenotype and provided the means to evaluate the variability of the surface exopeptidase system by comparison with the LMD-9 mutant strains. When one or the other LMD-9 mutant strains was incubated with isracidin, hydrolysis of isracidin occurred in a similar way. The LMD-9 mutant strains displayed exopeptidase activities such as carboxypeptidase, peptidyl dipeptidase, aminopeptidase, and X-prolyl dipeptidyl peptidase that could act simultaneously or successively in an undefined order. Moreover, almost similar peptidase activities were shown by S. thermophilus CNRZ1066 after exposure to the isracidin. All these activities were cellassociated extracellular activities as no peptidase activity was observed in cell free filtrate. In this study, the identified carboxypeptidase activity was observed only on some of the peptides released after peptidyl dipeptidase activity as evidenced by the absence of the peptide 1−22. The poor carboxypeptidase and higher peptidyl dipeptidase activity could be the result of nonoptimal reaction conditions for carboxypeptidase, or of specificity of carboxypeptidase toward peptide bonds, or of relative velocity of the enzymatic activities or a combination of these hypotheses. It has been stated that the internal carboxypeptidase and dipeptidase of Lactobacillus casei NCDO 151 require different pH and temperature for their optimal activity.26 Macedo and workers27 also detected lower carboxypeptidase activity than the corresponding dipeptidase activity in the intracellular enzymatic extracts of Lactobacillus paracasei subsp. paracasei ESB230 and Lactococcus lactis subsp. lactis ESB117. However, to our knowledge, only one study highlighted the presence of intracellular carboxypeptidase activity in S. thermophilus.28 Moreover, Kang and colleagues29 reported the predicted presence of a membrane carboxypeptidase, and the gene of G


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Journal of Agricultural and Food Chemistry this peptidase is present in the genome of many strains of S. thermophilus available in databases. This carboxypeptidase corresponds to low molecular mass penicillin binding protein that cross-links disaccharide chains of peptidoglycan by peptide bridges. The presence of an arginine carboxypeptidase30 and of a dipeptidyl carboxypeptidase (peptidyl dipeptidase)31 associated with the cell wall has also been reported in Streptococcus mitis ATCC 15909 and Streptococcus gordonii FSS2, respectively. To our knowledge, no cell-associated extracellular peptidyl dipeptidase activity was ever reported in S. thermophilus. The peptide bonds L11−P12, P5−I6, or R1−P2 were not hydrolyzed by peptidyl dipeptidase activity, as no fragment corresponding to such a hydrolysis was generated (Figure 3). This might be correlated to the presence of proline residue. Indeed, Dasarathy et al.32 have reported that peptides containing a proline residue at ultimate or penultimate position are not hydrolyzed by the extracellular peptidyl dipeptidase-4 of Pseudomonas maltophilia. Contrarily, hydrolysis at P2−K3 and P5−I6 peptide bonds leading to the generation of the dipeptides 1−2 and 4−5 seemed more likely to be the result of X-prolyl dipeptidyl peptidase activity rather than that of peptidyl dipeptidase. With regard to aminopeptidase and X-prolyl dipeptidyl peptidase activities, recently, the presence of cell-associated extracellular aminopeptidase and X-prolyl dipeptidyl peptidase activities has been described in two S. thermophilus strains, i.e., LMD-9 and CNRZ1066.11 Similarly, aminopeptidase activity in the growth medium of S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Bifidobacteria has also been detected.33 Moreover, different cell wall-associated aminopeptidases such as those from Lactobacillus helveticus ITGL1,34 Lactobacillus lactis,35 and Lactococcus lactis subsp. cremoris36 have been purified and characterized. Besides, Streptococcus suis and S. gordonii FSS2 also possess extracellular and cell wall-associated arginine aminopeptidase activity.37,38 However, in the present study, the aminopeptidase activity on the isracidin was exhibited only after the action of the Xprolyl dipeptidyl peptidase, showing that the presence of proline residue as the second residue of the peptide precluded the aminopeptidase activity. Recently, the absence of hydrolysis of the β-casomorphin-7 (YPFPGPI) by cell-associated extracellular aminopeptidase of S. thermophilus has been reported due to the presence of proline residue at position P1′.11 Moreover, a cell-associated peptidase activity has been described after exposure of the antihypertensive dodecapeptide (FFVAPFPEVFGK) of bovine αs1-casein to Lactobacillus delbrueckii subsp. bulgaricus. Similarly, the cell-associated peptidase activity was not observed once a proline residue was encountered at position P1′.39 The presence of extracellular and cell-wall Xprolyl-dipeptidyl peptidase activity has already been described in Lactococcus lactis subsp. cremoris,40 Lactococcus lactis subsp. lactis IFPL359,41 and Streptococcus gordonii FSS2.42 This study provides new insights into the surface proteolytic system of S. thermophilus. Indeed, S. thermophilus possesses the well-known PrtS activity that is essentially pH-dependent, indispensable for milk fermentation and may also be involved in increasing the functional properties of fermented products by releasing bioactive peptides from milk proteins. Besides this activity, this bacterium also displays a broad range of exopeptidase activities associated with the cell. In the absence of PrtS, four distinct exopeptidase activities, i.e., carboxypeptidase, peptidyl dipeptidase, aminopeptidase, and X-prolyl dipeptidyl peptidase, were identified. The existence of cell-

associated extracellular carboxypeptidase and peptidyl dipeptidase activities in both PrtS+ and PrtS− phenotypes of S. thermophilus has been highlighted for the first time. This indicated that the surface proteolytic system of S. thermophilus is more complex than reported in previous studies. Now, it is clear that these cell-associated extracellular peptidase activities may play a role for the PrtS− phenotypes of S. thermophilus in the use of milk caseins to generate free amino acids, vital for bacterial growth. Nevertheless, this growth is slower because the proteolytic activity of these peptidases is weaker than that of PrtS as it was shown in this study where the presence of PrtS concealed peptidase activities. Second, the desirability of the S. thermophilus strains in certain cheeses could be associated with its cell-associated extracellular peptidase activities, as they may be involved in the degradation of bitter peptides comprising proline or hydrophobic residues (Leu, Phe), where the released amino acids ultimately act as flavor precursors. The detection of the S. thermophilus cell-associated extracellular peptidases should motivate further biochemical and genetic studies to understand completely their role in milk fermentation, which is still not clear.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +33 383684269. Fax: +33 383684274. E-mail: Laurent. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Chantal Poirson for her technical assistance. ABBREVIATIONS USED ATCC, American Type Culture Collection; EDTA, ethylenediaminetetraacetic acid; ESI, electrospray ionization; HPLC, high performance liquid chromatography; HtrA, high temperature requirement A protease; Lys-p-NA, lysine-para-nitroanilide; MS, mass spectrometry; PAB, potassium phosphate/ acetate buffer; PCR, polymerase chain reaction; PrtS, cell-wall associated protease of S. thermophilus; RP, reversed-phase; rpm, revolutions per min; SPE, solid phase extraction; TEA, trishydroxymethylaminomethane-acetate-EDTA

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REFERENCES

(1) Miclo, L.; Roux, E.; Genay, M.; Brusseaux, E.; Poirson, C.; Jameh, N.; Perrin, C.; Dary, A. Variability of hydrolysis of β-, αs1-, and αs2caseins by 10 strains of Streptococcus thermophilus and resulting bioactive peptides. J. Agric. Food Chem. 2012, 60, 554−565. (2) Pastink, M. I.; Teusink, B.; Hols, P.; Visser, S.; de Vos, W. M.; Hugenholtz, J. Genome-scale model of Streptococcus thermophilus LMG18311 for metabolic comparison of lactic acid bacteria. Appl. Environ. Microbiol. 2009, 75, 3627−3633. (3) Hols, P.; Hancy, F.; Fontaine, L.; Grossiord, B.; Prozzi, D.; Leblond-Bourget, N.; Decaris, B.; Bolotin, A.; Delorme, C.; Ehrlich, S. D.; Guedon, E.; Monnet, V.; Renault, P.; Kleerebezem, M. New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics. FEMS Microbiol. Rev. 2005, 29, 435−463. (4) Savijoki, K.; Ingmer, H.; Varmanen, P. Proteolytic systems of lactic acid bacteria. Appl. Microbiol. Biotechnol. 2006, 71, 394−406. (5) Shahbal, S.; Hemme, D.; Renault, P. Characterization of a cell envelope-associated proteinase activity from Streptococcus thermophilus H-Strains. Appl. Environ. Microbiol. 1993, 59, 177−182. H


Article


Journal of Agricultural and Food Chemistry (6) Fernandez-Espla, M. D.; Garault, P.; Monnet, V.; Rul, F. Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization. Appl. Environ. Microbiol. 2000, 66, 4772−4778. (7) Galia, W.; Perrin, C.; Genay, M.; Dary, A. Variability and molecular typing of Streptococcus thermophilus strains displaying different proteolytic and acidifying properties. Int. Dairy J. 2009, 19, 89−95. (8) Chang, O. K.; Perrin, C.; Galia, W.; Saulnier, F.; Miclo, L.; Roux, E.; Driou, A.; Humbert, G.; Dary, A. Release of the cell-envelope protease PrtS in the growth medium of Streptococcus thermophilus 4F44. Int. Dairy J. 2012, 23, 91−98. (9) Poquet, I.; Saint, V.; Seznec, E.; Simoes, N.; Bolotin, A.; Gruss, A. HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing. Mol. Microbiol. 2000, 35, 1042−1051. (10) Christensen, J. E.; Dudley, E. G.; Pederson, J. A.; Steele, J. L. Peptidases and amino acid catabolism in lactic acid bacteria. Antonie van Leeuwenhoek 1999, 76, 217−246. (11) Hafeez, Z.; Cakir-Kiefer, C.; Girardet, J.-M.; Jardin, J.; Perrin, C.; Dary, A.; Miclo, L. Hydrolysis of milk-derived bioactive peptides by cell-associated extracellular peptidases of Streptococcus thermophilus. Appl. Microbiol. Biotechnol. 2013, 97, 9787−9799. (12) Lahov, E.; Regelson, W. Antibacterial and immunostimulating casein-derived substances from milk: casecidin, isracidin peptides. Food Chem. Toxicol. 1996, 34, 131−145. (13) Hayes, M.; Ross, R. P.; Fitzgerald, G. F.; Hill, C.; Stanton, C. Casein-derived antimicrobial peptides generated by Lactobacillus acidophilus DPC6026. Appl. Environ. Microbiol. 2006, 72, 2260−2264. (14) Somkuti, G. A.; Paul, M. Enzymatic fragmentation of the antimicrobial peptides casocidin and isracidin by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. Appl. Microbiol. Biotechnol. 2010, 87, 235−242. (15) Exterkate, F. A.; Alting, A. C.; Slangen, C. J. Specificity of two genetically related cell-envelope proteinases of Lactococcus lactis subsp. cremoris towards αs1-casein-(1−23)-fragment. Biochem. J. 1991, 273, 135−139. (16) Sadat-Mekmene, L.; Genay, M.; Atlan, D.; Lortal, S.; Gagnaire, V. Original features of cell-envelope proteinases of Lactobacillus helveticus. A review. Int. J. Food Microbiol. 2011, 146, 1−13. (17) Bolotin, A.; Quinquis, B.; Renault, P.; Sorokin, A.; Ehrlich, S. D.; Kulakauskas, S.; Lapidus, A.; Goltsman, E.; Mazur, M.; Pusch, G. D.; Fonstein, M.; Overbeek, R.; Kyprides, N.; Purnelle, B.; Prozzi, D.; Ngui, K.; Masuy, D.; Hancy, F.; Burteau, S.; Boutry, M.; Delcour, J.; Goffeau, A.; Hols, P. Complete sequence and comparative genome analysis of the dairy bacterium Streptococcus thermophilus. Nat. Biotechnol. 2004, 22, 1554−1558. (18) Makarova, K.; Slesarev, A.; Wolf, Y.; Sorokin, A.; Mirkin, B.; Koonin, E.; Pavlov, A.; Pavlova, N.; Karamychev, V.; Polouchine, N.; Shakhova, V.; Grigoriev, I.; Lou, Y.; Rohksar, D.; Lucas, S.; Huang, K.; Goodstein, D. M.; Hawkins, T.; Plengvidhya, V.; Welker, D.; Hughes, J.; Goh, Y.; Benson, A.; Baldwin, K.; Lee, J. H.; Diaz-Muniz, I.; Dosti, B.; Smeianov, V.; Wechter, W.; Barabote, R.; Lorca, G.; Altermann, E.; Barrangou, R.; Ganesan, B.; Xie, Y.; Rawsthorne, H.; Tamir, D.; Parker, C.; Breidt, F.; Broadbent, J.; Hutkins, R.; O’Sullivan, D.; Steele, J.; Unlu, G.; Saier, M.; Klaenhammer, T.; Richardson, P.; Kozyavkin, S.; Weimer, B.; Mills, D. Comparative genomics of the lactic acid bacteria. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 15611−15616. (19) Lecomte, X.; Gagnaire, V.; Briard-Bion, V.; Jardin, J.; Lortal, S.; Dary, A.; Genay, M. The naturally competent strain Streptococcus thermophilus LMD-9 as a new tool to anchor heterologous proteins on the cell surface. Microb. Cell Fact. 2014, 13, 82. (20) Terzaghi, B. E.; Sandine, W. E. Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 1975, 29, 807− 813. (21) Fischer, G.; Decaris, B.; Leblond, P. Occurrence of deletions, associated with genetic instability in Streptomyces ambofaciens, is independent of the linearity of the chromosomal DNA. J. Bacteriol. 1997, 179, 4553−4558.

(22) Miclo, L.; Perrin, E.; Driou, A.; Papadopoulos, V.; Boujrad, N.; Vanderesse, R.; Boudier, J. F.; Desor, D.; Linden, G.; Gaillard, J.-L. Characterization of α-casozepine, a tryptic peptide from bovine αs1casein with bezodiazepine-like activity. FASEB J. 2001, 15, 1780−1782. (23) Kuipers, B. J. H.; Gruppen, H. Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis. J. Agric. Food Chem. 2007, 55, 5445−5451. (24) Schechter, I.; Berger, A. On the size of the active site in proteases. I. Papain. Biochem. Biophys. Res. Commun. 1967, 27, 157− 162. (25) Exterkate, F. A.; Alting, A. C. The conversion of the αs1-casein(1−23)-fragment by the free and bound form of the cell-envelope proteinase of Lactococcus lactis subsp. cremoris under conditions prevailing in cheese. Syst. Appl. Microbiol. 1993, 16, 1−8. (26) El Soda, B. M.; Desmazeaud, M. J.; Bergere, J.-L. Peptide hydrolases of Lactobacillus casei: isolation and genreal properties of various peptidase activities. J. Dairy Res. 1978, 45, 445−455. (27) Macedo, A. C.; Vieira, M.; Pocas, R.; Malcata, F. X. Peptide hydrolase system of lactic acid bacteria isolated from Serra da Estrela cheese. Int. Dairy J. 2000, 10, 769−774. (28) Deutsch, S. M.; Molle, D.; Gagnaire, V.; Piot, M.; Atlan, D.; Lortal, S. Hydrolysis of sequenced β-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides. Appl. Environ. Microbiol. 2000, 66, 5360−5367. (29) Kang, X.; Ling, N.; Sun, G.; Zhou, Q.; Zhang, L.; Sheng, Q. Complete genome sequence of Streptococcus thermophilus strain MNZLW-002. J. Bacteriol. 2012, 194, 4428−4429. (30) Linder, L. E.; Sund, M.-L.; Lonnies, H. Isolation and purification of cell wall-associated arginine carboxypeptidase from Streptococcus mitis ATCC 15909. Curr. Microbiol. 1994, 28, 91−96. (31) Harty, D. W. S.; Hunter, N. Carboxypeptidase activity common to viridans group streptococci cleaves angiotensin I to angiotensin II: an activity homologous to angiotensin-converting enzyme (ACE). Microbiology 2011, 157, 2143−2151. (32) Dasarathy, Y.; Stevens, J.; Fanburg, B. L.; Lanzillo, J. J. A peptidyl dipeptidase-4 from Pseudomonas maltophilia: purification and properties. Arch. Biochem. Biophys. 1989, 270, 255−266. (33) Shihata, A.; Shah, N. P. Proteolytic profiles of yogurt and probiotic bacteria. Int. Dairy J. 2000, 10, 401−408. (34) Blanc, B.; Laloi, P.; Atlan, D.; Gilbert, C.; Portalier, R. Two cellwall associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGLl. J. Gen. Microbiol. 1993, 139, 1441−1448. (35) Eggimann, B.; Bachmann, M. Purification and partial characterization of an aminopeptidase from Lactobacillus lactis. Appl. Environ. Microbiol. 1980, 40, 876−882. (36) Geis, A.; Bockelmann, W.; Teuber, M. Simultaneous extraction and purification of a cell wall-associated peptidase and β-casein specific protease from Streptococcus cremoris AC1. Appl. Microbiol. Biotechnol. 1985, 23, 79−84. (37) Goldstein, J. M.; Nelson, D.; Kordula, T.; Mayo, J. A.; Travis, J. Extracellular arginine aminopeptidase from Streptococcus gordonii FSS2. Infec. Immun. 2002, 70, 836−843. (38) Jobin, M.-C.; Grenier, D. Identification and characterization of four proteases produced by Streptococcus suis. FEMS Microbiol. Lett. 2003, 220, 113−119. (39) Paul, M.; Somkuti, G. A. Degradation of milk-based bioactive peptides by yogurt fermentation bacteria. Lett. Appl. Microbiol. 2009, 49, 345−450. (40) Kiefer-Partsch, B.; Bockelmann, W.; Geis, A.; Teuber, M. Purification of an X-prolyl-dipeptidyl aminopeptidase from the cell wall proteolytic system of Lactococcus lactis subsp. cremoris. Appl. Microbiol. Biotechnol. 1989, 31, 75−78. (41) Requena, T.; Pelaez, C.; Fox, P. F. Peptidase and proteinase activity of Lactococeus lactis, Lactobacillus casei and Lactobacillus plantarum. Z. Lebensm.-Unters. Forsch. 1993, 196, 351−355. I


Article



(42) Goldstein, J. M.; Banbula, A.; Kordula, T.; Mayo, J. A.; Travis, J. Novel extracellular X-prolyl dipeptidyl-peptidase (DPP) from Streptococcus gordonii FSS2: an emerging subfamily of viridans streptococcal X-prolyl DPPs. Infec. Immun. 2001, 69, 5494−5501.

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Streptococcus thermophilus - American Chemical Society

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