Applied and Environmental Microbiology, February 2003, p . 1276-1282, Vol . 69, No . 2

Identification and Characterization of Lactobacillus helveticus PepO2, an Endopeptidase with Post-Proline Specificity

Yo-Shen Chen,¹ Jeffrey E . Christensen,^2, Jeffery R . Broadbent,³ and James L . Steele^1*

Departments of Food Science,¹ Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706,² Department of Nutrition and Food Sciences, Utah State University, Logan, Utah 84322³

Received 8 April 2002/ Accepted 1 November 2002

The proteolytic systems of LAB can be functionally divided into three components: (i) cell envelope-associated proteinases which hydrolyze CNs to oligopeptides; (ii) peptide transport systems, of which the oligopeptide transport system is the most important in milk and cheese; and (iii) numerous intracellular peptidases (9, 17) . The intracellular peptidases of LAB include both endopeptidases and aminopeptidases . Endopeptidases, due to their ability to hydrolyze peptide bonds within a peptide, are of particular interest in targeting peptides for rapid hydrolysis . In Lactococcus lactis, the best-characterized LAB, the endopeptidases that have been identified include PepO, PepO2, PepF1, and PepF2 . All of these enzymes are metalloproteases, and PepO, PepF1, and PepF2 are encoded in operons (9, 17) . The physiological roles of these endopeptidases remain unclear; however, PepF appears to be important for protein turnover during nitrogen starvation (24) . To date, one metalloendopeptidase, designated PepO (8), and a thiol-dependent endopeptidase, designated PepE (13), have been characterized from Lactobacillus helveticus .

The ability of L . helveticus CNRZ32 to accelerate cheese ripening and reduce bitterness when it is used as an adjunct culture is well documented (2, 3, 21) . While numerous enzymes of the proteolytic system of L . helveticus have been identified (9), our understanding of the specific enzymes responsible for this strain's ability to reduce the bitterness in cheese is incomplete . The peptide ß-CN(f193-209), which is produced by the activity of chymosin on ß-CN, has been implicated in the development of bitterness in cheese (4, 20) . The purpose of this study and another study (10) was to identify and characterize an L . helveticus endopeptidase(s) involved in the hydrolysis of ß-CN(f193-209) . Additionally, the accumulation of _s1-CN(f1-9) has been associated with bitterness (4, 5, 15); therefore, the hydrolysis of this peptide was also examined .

Screening of L . helveticus CNRZ32 genomic library.
A previously constructed genomic library of L . helveticus CNRZ32 in E . coli DH5 (25) was screened for endopeptidase activity by using an amino-terminal blocked chromogenic substrate, N-acetyl-ß-CN(f203-209)--nitroanilide [N-acetyl-ß-CN(f203-209)-NA] (SynPep Co., Dublin, Calif.); this substrate is based on the C-terminal amino acid sequence of Bos taurus ß-CN . Pooled cultures (10 isolates/pool) were grown overnight in Luria-Bertani broth containing erythromycin . Cells were pelleted by centrifugation at 13,000 x g for 1 min at room temperature, washed, and suspended in 10 mM bis(2-hydroxyethyl)imino-Tris (pH 6.5; Sigma) . Cell extracts (CFEs) were obtained from E . coli cultures by vortexing samples with glass beads alternating with cooling on ice (1 min each); this procedure was repeated twice, and the cell debris was removed by centrifugation for 1 min at 13,000 x g . CFEs obtained from mid-log-phase cultures of L . helveticus CNRZ32 and E . coli DH5(pJDC9) were used as positive and negative controls, respectively . The presence of endopeptidase activity was determined by adding 100 µl of CFE to 395 µl of 10 mM Bis-Tris (pH 6.5) containing 1 mM N-acetyl-ß-CN(f203-209)-NA . The appearance of an intense yellow color (resulting from release of NA) within 15 min was considered an indication of endopeptidase activity . In the coupled reaction, 20 µl of CFE from E . coli DH5 containing aminopeptidase N activity (pJDC9::pepN) was used . All assays were performed in duplicate .

Plasmid isolation and cloning.
Plasmid isolation from E . coli was performed as described by Sambrook et al . (27) . Restriction enzymes and T4 DNA ligase were purchased from Gibco-BRL and were used as recommended by the manufacturer . Electroporation of E . coli was performed by using a Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) as recommended by the manufacturer .

DNA sequencing and sequence analysis.
All primers were synthesized by GIBCO-BRL Custom Primers (Grand Island, N.Y.) . PCR and DNA sequencing reactions were performed with a Perkin-Elmer model 480 thermal cycler (Perkin-Elmer Corp., Norwalk, Conn.) . DNA sequencing reactions were performed by using a Prism Ready Reaction DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Inc., Foster City, Calif.) . DNA templates were purified with a Qiagen Inc . (Hilden, Germany) PCR purification kit . Sequencing was initially performed with primers M13 and M13R (GIBCO-BRL) . As the known sequenced progressed, new primers were designed accordingly . Additional primers were designed by using the Affinity program supplied by Ransom Hill Bioscience, Inc . (Ramona, Calif.) . DNA sequences were determined by the Nucleic Acid and Protein Facility of the University of Wisconsin-Madison Biotechnology Center with an ABI model 370/3 automated sequencer . Sequences were analyzed by using the GCG sequence analysis package (Genetics Computer Group, Inc., Madison, Wis.) . Protein homology searches were performed by using the BLAST network service (1) . All reported DNA sequence data were confirmed by sequencing both DNA strands from at least two independent PCR products .

mRNA analysis.
Transcription of the pepO2 gene was investigated by using an 810-bp internal pepO2 fragment (nucleotides 607 to 1416) that was amplified and the PCR product end labeled with digoxigenin (Genius system; Boehringer Mannheim GmbH, Mannheim, Germany) for Northern hybridization . The primers used for probe amplification were YC-2290 (5'GATGCGATTGCACTCG) and YC-2000 (5'GATAGCGGCAGGGAAG) . Total RNA was isolated by using an RNeasy kit (Qiagen) . RNA molecular weight markers, solutions, and reagents used for Northern hybridization and chemiluminescent detection were purchased from Boehringer Mannheim . Northern hybridization was performed by using the procedure recommended by the manufacturer . Mapping of the 5' end of the pepO2 transcript was accomplished by using a kit for 5' end rapid amplification of cDNA (5'RACE) (version 2.0; GIBCO-BRL) . The gene-specific primers used for 5'RACE were YC-2340 (5'GTTTTCGGTTTGCTTTTG), YC-2600 (5'CGGCATCTCTTTTGGC), and YC-2840 (5'GGACGATCGGCAGGG) . First-strand cDNA synthesis was performed with primer YC-2340 . Nested amplification of first-strand cDNA was carried out with primer YC-2600 and the anchor primer supplied with the 5'RACE kit . Sequencing reactions were conducted with primer YC-2840 by using the nested amplification product as the template .

Synthesis of peptide substrates.
The peptides _S1-CN(f1-9) and ß-CN(f193-209) were synthesized at the Utah State University Biotechnology Center . The synthesized peptides were subsequently purified by collection of appropriate fractions after preparatory reverse-phase high-performance liquid chromatography (RP-HPLC) . The peptides were analyzed by mass spectrometry, and the identities were confirmed by Edman degradation with an Applied Biosystems model 477B protein sequencer . The peptides were lyophilized and stored at -80°C . Stock solutions were prepared in sterile double-distilled water (ddH₂O) and also stored at -80°C .

Peptide hydrolysis reactions.
Peptide hydrolysis reactions were performed essentially as described in the accompanying paper (10) . A 10-µl aliquot of CFE (0.95 to 1.05 mg of protein per ml) was diluted in 500 µl (total reaction volume) of 0.1 M Bis-Tris buffer (pH 6.5) . The reactions were initiated by adding the substrate, and the reaction mixtures were incubated at 37°C for a minimum of 30 min . The initial substrate concentration in the reaction samples was 0.2 µg/µl for both ß-CN(f193-209) and _s1-CN(f1-9) . Reactions were stopped by immediately freezing preparations at -20°C .

Peptide separation and identification.
Samples were injected into a 20-µl loop by using a Gilson model 231 sample injector equipped with a model 401 dilutor module containing a ddH₂O-acetonitrile wash solution (1:1; Gilson Medical Electronics, Paris, France) . The peptides were separated by using a Phenomenex Columbus C₁₈ column (250 by 2 mm; 5µ; 100 Å; Phenomenex Columbus, Torrance, Calif.) preceded by a Brownlee RP-18 precolumn . The mobile phase flow rate and gradient were controlled with a Hitachi L-6200A pump (Hitachi Instruments, San Jose, Calif.) . Mobile phases were continuously degassed by slow helium sparging . Peptides were detected with a Hitachi L-4500A diode array detector in the low-absorbance mode . Data was collected by using the Hitachi Chromatography Data Station software with a wavelength range of 200 to 300 nm, a 4-nm spectral bandwidth, and a 3,200-ms spectral interval .

Mobile phase A consisted of ddH₂O-MeCN (99:1) with 0.1% trifluoroacetic acid, and mobile phase B consisted of ddH₂O-MeCN (20:80) with 0.05% trifluoroacetic acid . Separation and elution of _S1-CN(f1-9) hydrolysis samples were accomplished with the following gradient: 1 to 16% mobile phase B from 0 to 20 min at a rate of 0.25 ml/min, 90% mobile phase B from 20 to 22 min at a rate of 0.25 ml/min, and 90 to 1% mobile phase B from 22 to 25 min at a rate of 0.25 ml/min . Separation and elution of ß-CN(f193-209) hydrolysis samples were accomplished with the following gradient: 4 to 60% mobile phase B from 0 to 40 min at a rate of 0.25 ml/min, 60 to 98% mobile phase B from 40 to 41 min at a rate of 0.25 ml/min, 98% mobile phase B from 41 to 45 min at a rate of 0.25 to 0.50 ml/min, and 98 to 4% mobile phase B from 45 to 47 min at a rate of 0.50 to 0.25 ml/min . The pump back pressure was 1400 lb/in² at zero time and remained below 1,600 lb/in² for the duration of the gradients . Samples being separated for fraction collection were monitored in real time . Fractions were collected manually, taking into account a predetermined time for the peptide to travel from the detector flow cell to the capture point .

The masses of RP-HPLC-separated peptide fractions were determined by using a triple quadrupole mass spectrometer (Micromass Quattro II) with electrospray ionization sources at the Utah State University Biotechnology Center . To identify the hydrolysis products, the masses were compared to calculated molecular masses of peptides and/or amino acids derived from ß-CN(f193-209) and _s1-CN(f1-9) .

Nucleotide sequence accession number.
The nucleotide sequence of pepO2 has been deposited in the GenBank database under accession no . AF321529 .

 
A genomic library of L . helveticus CNRZ32 in E . coli DH5 was screened for endopeptidase activities with acetyl-ß-CN(f203-209)-NA . Of the 1,880 isolates screened, 2 had activity in a coupled reaction with PepN . The restriction endonuclease profiles of these two isolates were visually indistinguishable (data not shown) . One plasmid, designated pSUW99, was selected and used for further analysis .

Sequencing of the endopeptidase clone.
Restriction mapping of pSUW99 revealed a 6.0-kb insert . Two 3.0-kb SstI fragments and two PstI fragments (2.0 and 4.0 kb) were obtained when the insert was digested with restriction endonucleases SstI and PstI, respectively . Subclones containing individual SstI fragments or PstI fragments in pJDC9 were examined for endopeptidase activity with acetyl-ß-CN(f203-209)-NA . Activity was detected only in strains containing one of the 3.0-kb SstI fragments, suggesting that the gene was present on this SstI fragment and contained a PstI site .

The complete nucleotide sequence of the 3.0-kb SstI fragment encoding endopeptidase activity was determined, and a 1,947-bp open reading frame (ORF) was identified (Fig . 1) . This ORF could encode a 649-amino-acid polypeptide with a deduced molecular mass of 71.4 kDa . Protein sequence homology searches with current BLAST databases revealed high levels of similarity between the deduced amino acid sequence and the amino acid sequences of other LAB PepO-type endopeptidases (6, 14, 22, 32; GenBank accession no . AF179267) . This protein exhibited 56% identity and 72% similarity to L . helveticus CNRZ32 endopeptidase PepO (8); therefore, the gene was designated pepO2 . L . helveticus PepO2 exhibited 42% identity and 59% similarity to L . lactis PepO (22, 32), 41% identity and 61% similarity to L . lactis PepO2, 38% identity and 57% similarity to Streptococcus thermophilus PepO (6), and 36% identity and 53% similarity to Lactobacillus rhamnosus PepO (11) . Significant levels of similarity to mammalian metallopeptidases, including endothelin-converting enzyme (45% similarity) and enkephalinase (neutral endopeptidase; 43% similarity), were also observed . The sequence motif His-Glu-Xxx-Xxx-His, which is characteristic of many zinc-dependent metalloproteases, was also identified in PepO2 between residues 497 and 501 (Fig . 1) . The start codon of the ORF is preceded by a putative ribosome binding site (AAGGAG; nucleotides -8 to -13) and by putative promoter -10 (TATGAT; nucleotides -32 to -37) and -35 (TTTTCA; nucleotides -56 to -61) sequences (28) . An inverted repeat (nucleotides 1967 to 1979 and 2000 to 2012) was observed in the 3' noncoding region and may function as a rho-independent transcriptional terminator with a G at 25°C of -21 kcal (31) . No signal sequence was detected by using a hydrophilicity plot constructed as described by Kyte and Doolittle (19) .

 
mRNA analysis.
Northern hybridization performed with total RNA from an exponential culture of L . helveticus CNRZ32 resulted in detection of a transcript that was 2.1 kb long (data not shown) . This size corresponds to the size of the pepO2 ORF and indicates that pepO2 is monocistronic . The transcriptional start site for the pepO2 promoter was mapped 26 bp upstream of the pepO2 start codon by 5'RACE (data not shown) .

Substrate specificity of PepO2.
To determine if the PepO2 substrate specificity is similar to that of previously described endopeptidases from L . helveticus CNRZ32, the ability of CFE from E . coli DH5 expressing PepO2 to hydrolyze N-benzoyl-Phe-Val-Arg-NA, N-benzoyl-Pro-Phe-Arg-NA, and N-benzoyl-Val-Gly-Arg-NA was examined . These substrates were utilized previously to identify and differentiate PepO and PepE in a genomic library of L . helveticus constructed in E . coli DH5 (8, 13) . Derivatives of E . coli DH5 expressing PepO hydrolyzed N-benzoyl-Pro-Phe-Arg-NA and N-benzoyl-Val-Gly-Arg-NA, while derivatives of E . coli DH5 expressing PepE hydrolyzed N-benzoyl-Phe-Val-Arg-NA and N-benzoyl-Pro-Phe-Arg-NA . Hydrolysis of these substrates by PepO2, with or without PepN, was not observed (data not shown) . Additionally, hydrolysis of acetyl-ß-CN(f203-209)-NA by CFEs of E . coli DH5 expressing L . helveticus PepO or PepE in coupled assays with PepN was not observed . These results indicated that PepO2 substrate specificity is distinct from PepO and PepE substrate specificity . CFEs from E . coli DH5 expressing L . helveticus PepC or PepX were also examined in a coupled reaction with PepO2 (in place of PepN); the results indicated that only the combined activity of PepN and PepO2 was capable of releasing NA from acetyl-ß-CN(f 203-209)-NA .

To examine hydrolysis of the model CN-derived bitter peptides ß-CN(f193-209) and _s1-CN(f1-9) by PepO2, RP-HPLC was performed to separate and collect peptide hydrolysis products . No significant hydrolysis of either substrate was detected with CFEs from E . coli DH5(pJDC9) . However, significant hydrolysis of both ß-CN(f193-209) and _s1-CN(f1-9) was detected with CFEs from E . coli DH5(pSUW99) (Fig . 2) . The predominant peptide fractions were collected and analyzed . PepO2 was found to hydrolyze ß-CN(f193-209) at the Pro-196-Val-197, Pro-200-Val-201, and Pro-206-Ile-207 bonds . Hydrolysis of _s1-CN(f1-9) was observed at the Pro-5-Ile-6 bond (Fig . 3) .

 
Bitterness in cheese is believed to be the result of accumulation of low-molecular-weight hydrophobic peptides, such as _s1-CN(f1-9) and ß-CN(f193-209) (4, 5, 20) . In another study, hydrolysis of _s1-CN(f1-9) and ß-CN(f193-209) by the L . helveticus CNRZ32 wild-type strain and several peptidase-deficient mutants was investigated (10) . The results of that study indicated that L . helveticus contains a previously undetected endopeptidase capable of hydrolyzing ß-CN(f193-209) under conditions simulating the conditions in ripening cheese . In this study, the endopeptidase was identified by screening a genomic library of L . helveticus for the ability to hydrolyze the chromogenic substrate N-acetyl-ß-CN(f203-209)-NA . The gene identified was determined to code for a protein with the highest level of identity (55%) to PepO from L . helveticus CNRZ32, a previously described metal-dependent endopeptidase (8) . Additionally, this endopeptidase exhibits 42% identity to PepO from L . lactis (22, 32), 41% identity to PepO2 from L . lactis, and 36% identity to PepO from L . rhamnosus (11) . Unlike the lactococcal PepO proteins, the L . helveticus PepO proteins include cysteine residues and are translated from a monocistronic message . The presence of duplicated PepO proteins in both lactococci and lactobacilli suggests that these enzymes may have important physiological functions . However, inactivation of L . helveticus PepO did not result in any observable change in the ability to grow in milk or amino acid-containing defined media (8) . Several attempts to construct a PepO2 deletion mutant derivative of L . helveticus CNRZ32 via two-step gene replacement were unsuccessful (data not shown), suggesting that this enzyme is required for viability . Additional research is required to establish the physiological roles of L . helveticus PepO and PepO2 .

The substrate specificity of PepO2 was assessed with chromogenic peptide substrates and two CN-derived peptides . The inability of PepO2 to hydrolyze the chromogenic substrates used to identify PepO and PepE from L . helveticus suggested that PepO2 has distinct substrate specificity . This suggestion was supported by the ability of PepO2 to hydrolyze acetyl-ß-CN(f203-209)-NA in conjunction with PepN, while no hydrolysis by either PepE or PepO was observed under the same conditions . The bonds hydrolyzed in _s1-CN(f1-9) and ß-CN(f193-209) by PepO2 were either Pro-Val or Pro-Ile bonds, indicating that PepO2 is a post-proline endopeptidase . Hydrolysis of the ß-CN(f193-209) Pro-204-Phe-205 and _s1-CN(f1-9) Pro-2-Lys-3 bonds was not observed, suggesting that PepO2 may have a preference for small uncharged amino acids on the carboxy side of the scissile bond . The hydrolysis of peptide bonds involving Pro is likely to be important in the hydrolysis of CN-derived peptides as Pro constitutes 16.7% of ß-CN and 8.5% of _s1-CN amino acid residues (29) . Additionally, CN-derived bitter peptides have been observed to contain relatively large amounts of Pro, and it has been proposed that the spatial structure resulting from the presence of Pro in a peptide is directly related to bitterness (20) . Therefore, the specificity of PepO2 for bonds containing Pro suggests that this enzyme may have a central role in the demonstrated ability of L . helveticus CNRZ32 to reduce bitterness in cheese .

In future studies we will examine if strains overexpressing PepO2 can reduce bitterness and increase flavor development in bacterial ripened cheeses (i.e., Cheddar and Gouda) . Additionally, the possible interaction between PepO2 and other components of the L . helveticus CNRZ32 proteolytic system, such as PepN, will be assessed by using combinations of strains overexpressing peptidases .

This project was funded by Dairy Management, Inc . through the Wisconsin Center for Dairy Research and the College of Agricultural and Life Sciences at the University of Wisconsin-Madison .

Present address: Clinical Research Department, Marshfield Medical Research Foundation, Marshfield, WI 54449 .

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