Genomic and Proteolytic Profiling of Lacticaseibacillus sp. PRA205: Insights into PepX- Mediated Bioactive Peptide Metabolism

Genomic and Proteolytic Profiling of Lacticaseibacillus sp. 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PRA205: Insights into PepX- Mediated Bioactive Peptide Metabolism Marianna Cristofolini, Alice Cattivelli, Alessandra Barbieri, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7544664/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted 9 You are reading this latest preprint version Abstract Species of the Lacticaseibacillus casei group are GRAS organisms extensively used in dairy fermentations to release bioactive peptides (BPs) with health-promoting properties. This study characterized the probiotic features, biosafety, and proteolytic system of the probiotic candidate Lacticaseibacillus sp. PRA205, a high producer of the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), originally isolated from Parmigiano Reggiano cheese, with a focus on the PepX enzyme. Whole-genome sequencing revealed a 3.2 Mb genome encoding 2979 predicted genes. Phylogenomics assigned PRA205, previously identified as Lacticaseibacillus casei , to a lineage closely related yet distinct from Lcb. parahuelsenbergensis . Genome annotation identified adhesion and stress-tolerance genes, along with three bacteriocin clusters, but no antibiotic resistance or virulence factors. Lacticaseibacillus sp. PRA205 exhibited a complete proteolytic system, consistent with its strong proteolytic phenotype. Notably, a single pepX gene, only weakly repressed under amino acid–rich conditions, encoded a serine protease of ~ 80 kDa that was partially purified and biochemically characterized. PepX degraded BPs, including the tripeptides VPP and IPP, and retained activity at low temperature and acidic pH, suggesting a dual role in BPs production and breakdown during dairy fermentation and storage. Overall, these findings elucidate the genetic basis of PRA205 proteolytic activity and support its safety and potential as probiotic culture for developing functional dairy foods enriched in BPs. Proteolytic system X-prolyl dipeptidyl aminopeptidase pepX gene Lacticaseibacillus probiotics bioactive peptides Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction In the dairy industry, microbial fermentation is one of the oldest and most effective methods to develop functional food [ 1 – 4 ]. In this context, lactic acid bacteria (LAB) serve as cell factories to produce biofunctional compounds, including milk protein-derived bioactive peptides (BPs) [ 5 – 7 ]. BPs are encrypted sequences within milk proteins that, once released, exhibit various biological activities such as antimicrobial, hypocholesterolemic, antihypertensive, antioxidant, anticancer, antithrombotic, osteogenic, immunomodulatory, and mineral binding activity effects [ 6 , 8 ]. These peptides typically range from 2 to 40 amino acids in length, with their activity influenced by sequence and composition [ 6 ]. Many are multifunctional, acting through multiple mechanisms and exerting more than one physiological effect [ 8 , 9 ]. In addition to their biological activity, short peptides released by LAB also affect cheese maturation, influencing texture and flavor of the final products [ 10 ]. The link between cheese bitterness and specific peptide fractions was recognized as early as 1932 [ 11 ]. Conversely, other peptides enhance salty and umami notes of cheese by interacting with compounds like reducing sugars and unsaturated fatty acids and forming flavor molecules such as sulfur compounds, methyl ketones, and aldehydes [ 12 ]. Key amino acids contributing to dairy flavor include branched-chain (valine, leucine, isoleucine), aromatic (tyrosine, tryptophan, phenylalanine), and sulfur-containing (methionine, cysteine) amino acids [ 13 , 14 ]. In LAB, a well-coordinated proteolytic system hydrolyzes milk proteins into peptides, including BPs, and essential amino acids. This system complements the autotrophies of LAB for several amino acids, enabling growth by utilizing external proteins as a nitrogen source [ 13 , 15 , 16 ]. This system includes cell surface proteinases (CEPs), responsible for the initial cleavage of caseins into oligopeptides (up to 17 amino acid residues); specific transport systems for the peptide internalization, such as oligopeptide permease (Opp), dipeptide permeases (DtpT, Dpp), and di/tri-peptide transporters; and a broad range of intracellular peptidases, including specific endopeptidases, aminopeptidases, tri- and dipeptidases, as well as peptidases involved in proline metabolism, which further degrade peptides into di/tri-peptides and/or free amino acids [ 17 – 19 ]. The considerable variability in proteolytic systems among LAB strains results in marked phenotypic differences in their ability to hydrolyze milk proteins and release BPs both at species and strain level [ 19 ]. Lacticaseibacillus sp. strain PRA205, isolated from Parmigiano Reggiano PDO cheese and previously identified as Lacticaseibacillus casei PRA205 [ 20 ], is a mesophilic, non-starter lactic acid bacterium with probiotic tolerance to environmental cues such as acidity, salinity, bile salts, and digestive enzymes [ 21 ] (Solieri et al. 2014). During milk fermentation it produced significant concentrations of anti-hypertensive tripeptides Valine-Proline-Proline (VPP) (32.88 mg/L) and Isoleucine-Proline-Proline (IPP) (7.52 mg/L) [ 22 ]. Consequently, fermented milk prepared with this strain exerts strong angiotensin-converting enzyme (ACE) inhibitory activity [ 22 , 23 ]. Moreover, when used as adjunct culture during yogurt fermentation with Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus , strain PRA205 maintained a viable cell count higher than 10⁸ CFU/g in yogurt at 4°C for 28 days, exceeding the minimum threshold required to confer probiotic health benefits [ 24 ]. In a previous work, we have characterized the cell envelope proteinase (CEP)-encoding prt gene in Lacticaseibacillus sp. PRA205, demonstrating that the enzyme PrtR1 is responsible for the release of several BPs through αs1- and β-casein hydrolysis [ 25 ]. However, despite the probiotic potential of strain PRA205, the entire proteolytic system is still unknown, and the intracellular peptidases involved in subsequent peptide processing remain uncharacterized. Among these, X-prolyl dipeptidyl aminopeptidase (PepX, EC 3.4.14.11) is a serine protease belonging to the MEROPS peptidase family S15 clan SC ( http://www.ebi.ac.uk/interpro/entry/IPR008252 ), which cleaves Xaa-Pro releasing N-terminal dipeptide from polypeptides [ 26 – 28 ]. In the model organism Lactococcus lactis , this aminopeptidase has a narrow specificity for proline (or alanine or glycine with, respectively, 10- and 100-fold lower efficiency) in the P1 position. In the positions P2 and P′1, PepX accepts any residue except proline [ 27 , 28 ]. While the positive role of PepX in cheese peptide debittering is well-known, its contribution to the release of BPs remains ambiguous. Several studies have linked PepX activity to notable biofunctions. For instance, PepX from Lactobacillus helveticus has been shown to hydrolyze caseins [ 29 , 30 ] and, together with PepC and PepO, it contributes to release VPP and IPP from a 28-amino-acid β-casein precursor peptide [ 31 ]. The PepX enzyme from Lactobacillus acidophilus has also been reported to hydrolyze highly proline rich gliadins and coeliac-toxic peptides that contain a high percentage of proline residues [ 32 – 34 ]. Moreover, PepX enzymes from five LAB species have been shown to generate ACE-inhibitory peptides from goat milk [ 35 ]. Deletion of the pepX gene resulted in the loss of hydrolytic activity on the typical substrate β-casomorphin-7 (β-CN 7) [ 36 , 37 ]. Conversely, other studies have negatively correlated the PepX activity with BPs release. In milk fermented by Lactococcus lactis and L. helveticus , high PepX activity did not enhance ACE-inhibitory capacity, suggesting that ACE-inhibitory peptides were primarily generated by early-stage proteases [ 38 ]. Similarly, in L. helveticus CNRZ32, deletion of pepX and pepN was associated with increased ACE-inhibitory activity, likely due to reduced degradation of proline-rich BPs [ 15 , 39 ]. The aim of the present study was to explore the genome of the probiotic candidate Lacticaseibacillus sp. PRA205 and to characterize its proteolytic system, with a focus on the X-prolyl dipeptidyl aminopeptidase PepX. Given the recent taxonomic revisions within the genus Lacticaseibacillus , including the introduction of four novel species [ 40 , 41 ], a comparative genomic approach was employed to accurately determine the phylogenetic position of strain PRA205, as well as to investigate its safety and probiotic traits. 2. Materials and Methods 2.1 Reagents and cultivation media Unless otherwise indicated, all media and anaerobic systems used in this study were purchased from Oxoid (Oxoid, Basingstoke, Hampshire, UK), while the chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), except for chemicals and solvents for mass spectrometry analysis that were supplied by Carlo Erba (Milan, Italy). The primers were provided by BMR Genomics (Padova, Italy). The molecular biology reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Amicon Ultra-4 centrifugal filter units (regenerated cellulose, nominal cut 30 kDa) were supplied by Millipore (Milan, Italy). 2.2 Bacteria Strain and Culture Conditions The Lacticaseibacillus sp. strain PRA205 used in this study was isolated from Parmigiano Reggiano PDO cheese [ 20 ] and was deposited in the Culture Collection of Department of Life Sciences (University of Modena and Reggio Emilia, Italy). The strain was cryo-preserved at -80°C in the Man, Rogosa and Sharpe (MRS) medium (pH 6.5) containing 25% (v/v) of glycerol. To activate the strain from its glycerol storage, it was propagated in 5 mL of MRS medium, supplemented with 1.5% (w/v) agar when required, and incubated at 37°C for 24 h, under anaerobic conditions. 2.3 Reference genomes In this study, genome sequences of Lacticaseibacillus casei group (LCG) species, including Lacticaseibacillus casei , Lacticaseibacillus paracasei , Lacticaseibacillus rhamnosus , Lacticaseibacillus zeae , and the four recently proposed species such as Lacticaseibacillus huelsenbergensis , Lacticaseibacillus zeae subsp. silagei , Lacticaseibacillus parahuelsenbergensis , and Lacticaseibacillus styriensis , were used for comparative purposes. In addition, two Lactobacillus species with a well-characterized proteolytic system, namely Lactobacillus helveticus and Lactobacillus delbrueckii subsp. lactis were used as reference genomes for the reconstruction of proteolytic system. All the genomes were obtained from the National Center for Biotechnology Information (NCBI, Bethesda, Rockville, ML, USA) genome database ( http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi , last accessed 12 March 2025). The genomes and the associated information are listed in Supplementary Table S1 . 2.4 Genomic Sequencing and annotation The genomic DNA was extracted carried out as previously reported [ 42 ]. BMR Genomics (Padova, Italy) performed both the library preparation and genome sequencing. Briefly, the purified DNA was processed using the Illumina DNAprep kit following the manufacturer’s instructions, and the resulting shotgun library was sequenced on the Illumina MiSeq Sequencing System, using the proprietary V3 reagent kit, producing 2 x 300 bp paired end reads. Reads pre-processing was carried out with Fastp v0.23.2 [ 43 ] to remove residual adapter sequences, short reads (length < 150) and low quality data (base quality < 20, average read quality < 25, read complexity threshold < 30). Possible contaminants were assessed by MetaPhlan v4.0.1 [ 44 ]. SPAdes v3.15.5 [ 45 ] with careful option was applied to perform de novo genome assembly. QUAST v5.0.2 [ 46 ] and BUSCO v5.4.3 [ 47 ], run on lactobacillales_odb10 (v2020-03-06) lineage dataset, were used for assessing assembly metrics and genomic completeness, respectively. The gene prediction and gene annotation were performed with Prokka v.1.14.6 [ 48 ] and eggNOG-mapper v2.1.7 [ 49 ]. Custom graphic map of the genome was obtained via Proksee v1.1.2 ( https://proksee.ca ; accessed 13 February 2025) [ 50 ] using the GenBank annotation file (gbk). BlastKOALA was used to calculate the relative abundances of genes in the KEGG categories as percentages of the genes assigned to each respective KEGG category versus the total genes number [ 51 ]. 2.5 Species identification and phylogenomics Draft genome sequence of PRA205 and seven Lacticaseibacillus complete genomes were uploaded to the JspeciesWS server ( https://www.ribocon.com/jspeciesws.html , accessed on 14 Mar 2025) to calculate ANI values with BLAST algorithm (ANIb) and MUMmer (Maximal Unique Match) alignment tool (ANIm), respectively [ 52 ]. FastANI, which is a k -mer and alignment-free method of ANI calculation [ 53 ], was implemented in EDGAR3.0 webtool [ 54 ] to cross-validate the ANI values. The calculations of average amino acid identity (AAI) and of the percentage of conserved proteins (POCP) between two genomes as proposed by [ 55 ] were also implemented in EDGAR3.0 pipeline [ 54 ]. For POCP analysis the following cutoff values were used: an e-value threshold of 1 e − 5 , a minimum sequence identity > 50%, and an alignment coverage of the query protein > 50%. GGDC 3.0 available in Type (Strain) Genome Server (TYGS) ( https://tygs.dsmz.de/ ) was used to calculate digital DNA-DNA hybridization (dDDH) values [ 56 ]. Phylogenomics were carried out with two approaches. Firstly, the core genes of 8 genomes were computed in EDGAR3.0 pipeline [ 54 ]. The alignments of each core gene set are generated using MUSCLE [ 57 ], and the alignments are concatenated to one huge alignment. This alignment was the input for the FastTree software ( http://www.microbesonline.org/fasttree/ ) to generate approximately-maximum-likelihood (ML) phylogenetic tree. The values at the branches of FastTree tree were local support values computed by FastTree using the Shimodaira-Hasegawa (SH) test. In the second phylogenetic analysis the PRA205 assembled genome was compared with 25 Lacticaseibacillus genomes (Supplementary Table S1 ) downloaded from National Center for Biotechnology Information (NCBI, Bethesda, Rockville, ML, USA). To perform such procedure, PhyloPhlAn v3.1.68 [ 58 ] was applied in fast mode against a customized PhyloPhlAn database containing only Lactobacillus associated markers (n = 339453). In addition, the following parameters were used to generate high-resolution strain-level phylogeny: --diversity low --trim greedy --min_num_entries 9 (~ 34.6% of the total number of genomes in order to keep more informative signals for distinguishing between very closely related organisms; 75481 markers were selected) --remove_fragmentary_entries --fast --force_nucleotides. The Maximum Likelihood (ML) tree was reconstructed from the PhyloPhlAn output using RAxML version 8.2.12 [ 59 ]. The GTRCAT model was applied to account for nucleotide substitution and rate heterogeneity. A rapid bootstrap analysis (-f a) with 1000 replicates was performed to assess branch support. To guarantee the result’s reproducibility, the parsimony random seed (-p) and the rapid bootstrap random seed (-x) were set to 1989 and 42, respectively. All the resulting phylogenetic trees were further refined and visually enhanced using the iTOL tool [ 60 ]. 2.6 Genome analyses The annotated genome (Genbank format) of PRA205 and 5 Lacticaseibacillus reference genomes (Supplementary Table S1 ) were submitted to EDGAR3.0 web-based tool to investigate core, dispensable, and accessory genes, as well as to identify KEGG Orthologs (KOs) and clusters Clusters of Orthologous Groups of proteins (COGs) functions across the different genomes [ 54 ]. Carbohydrate-active enzyme (CAZy) database implemented in the ProbioMinServer web-platform was used for functional assignment of CAZy enzymes [ 61 ]. The biosynthetic gene clusters (BGCs) encoding secondary metabolites were predicted using antiSMASH 4.0 [ 62 ] ( https://antismash.secondarymetabolites.org/#!/about , accessed on 20 June 2025). BAGEL5 web-server ( http://bagel5.molgenrug.nl , accessed on 20 May 2025) was used to identify potential bacteriocin gene clusters, as well as other bacterial ribosomally synthesized and post-translationally modified peptides, with all parameters set to default. Putative plasmids were identified using the PlasmidFinder v2.1 database ( https://cge.food.dtu.dk/services/PlasmidFinder/ ) according to the following screening criteria: 95% identity threshold and 60% minimum coverage [ 63 ]. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences and Cas protein encoding genes were identified using CRISPRCasFinder v2.2 [ 64 ]. The presence of genes of mobile elements was examined using BLASTX searches compared to the full mobileOG-db v1.1.3 database [ 65 ], with an identity > 90% and coverage > 90%. The in silico analyses for microbial safety assessments were implemented in ProbioMinServer [ 61 ], following the guidelines of the European Food Safety Authority (EFSA) [ 66 ]. Specifically, antibiotic resistance genes (ARGs) were identified using the Comprehensive Antibiotic Resistance Database (CARD) Variants v4.0.0 [ 67 ], ARMFinder [ 68 ], and ResFinder v4.3.2 [ 69 ]. BLASTN v2.8.1 + was used to detect virulence factors (VFs) by searching against the set B database from the Virulence Factor Database (VFDB) [ 70 ] and VirulenceFinder v2.0.3 [ 71 ]. BLASTP v2.8.1 + search was performed against the Pathogen Host Interaction v4.14 database to identify the probable pathogenic genes (PGs) [ 70 ] (Liu et al. 2022). The probiotic potential risk score (PPRS) was computed as defined by [ 72 ] to evaluate the risks associated with PRA205 probiotic strain. The score was classified as low-risk (≤ 4), medium-risk (4–6), and high-risk (≥ 6). 2.7 Identification of orthologs of the proteolytic system Protein sequences of experimentally verified proteolytic system members, i.e. CEPs and various peptidases, were derived from the non-redundant protein database Uniprot ( http://www.uniprot.org/ ; accessed on 17 May 2025; [ 73 ] (Bateman et al. 2021) (Supplementary Table S2 ). The amino acid sequences were used as query to perform a TBLASTN search against the nucleotide sequences of the selected species (Supplementary Table S1 ). Two sequences were considered homologous if their alignment had a minimum sequence identity of 30% and a query coverage of at least 70% [ 74 ] (Pearson 2013). The peptidase/protease candidates which were not present in genome annotation were subjected to HMMER v.3.2.1 using the hmmsearch function from HMMER v.3.4 [ 75 ]. The corresponding Hidden Markov Models (HMMs) of each protein family were obtained from the Pfam database [ 76 ]. 2.8 Milk fermentation Strain PRA205 was pre-cultured in 50 mL of MRS broth for 72 h at 37°C under anaerobic conditions. After centrifugation at 10,000× rpm for 20 min at 4°C, cells were washed with physiological solution (0.9% NaCl) and then inoculated in triplicates in scraw-cap flasks containing 45 mL UHT skimmed milk at the final concentration of 10 10 CFU/mL. After 72 h of incubation at 37°C under shaking conditions (10 rpm), fermented milk samples were left to settle for 10 min. The liquid phase was separated from the coagulated proteins through Whatman paper (grade 4). After centrifugation at 6,000x rpm for 20 min at 4°C, cells were resuspended in 6 mL of phosphate buffer pH 7.0 50 mM a final concentration of 10 10 CFU/mL and divided into two aliquots for RNA extraction and biochemical characterization of pepX enzyme, respectively. 2.9 Gene expression analysis RNA was extracted approximately from 2 x 10 8 cells out using the Zymo Direct-zol RNA MiniPrep kit (Cat. No. R2071, Zymo Research, Irvine, CA, USA) and applying few modifications to the manufacturer’s instructions. Briefly, after adding up to 700 µL of the Tri reagent, the mechanical lysis of cells was achieved using a Vortex Genie 2 instrument (Mo Bio Laboratories Carlsbad, CA, USA) by performing two rounds of 20 min at the highest speed alternating with 3 min on ice. The quantity of total RNA was measured spectrophotometrically using a Nanodrop Nd 1000 system (Nanodrop Technologies, Wilmington, DE, USA) and only samples with A260/280 absorbance ratio between 1.8 and 2.2 were considered for further analyses. The integrity of the total RNA was evaluated by denaturing gel electrophoresis on a 0.9% (w/v) agarose gel with formaldehyde (10 mL of 10× 3-morpholinepropane sulfonic acid [MOPS] running buffer) and 18 mL of 37% formaldehyde (12 mol/L) in pH 7.0 1× MOPS running buffer (0.4 mol/L MOPS, 1 mol/L sodium acetate, and 0.01 mol/L EDTA) after the RNA treatment at 65°C for 10 min. To remove any contamination of gDNA, 1 µg of the RNA sample was treated with dsDNase (Cat. No. EN0771, Thermo Fisher Scientific) (final volume 40 µL) and, thereafter, RNA was reverse transcribed to cDNA at 42°C for 60 min with random hexamers (Cat. No. SO142; Thermo Fisher Scientific) and oligo (dT)18 primers (Cat. No. SO131; Thermo Fisher Scientific) using the RevertAid RT Kit (Cat. No. EP0441; Thermo Fisher Scientific) according to the manufacturer’s instructions. The end-point RT-PCR amplification of pepX gene was carried out with a Dream Taq DNA polymerase (Cat. No. EP0712; Thermo Fisher Scientific). RT-PCR of the 16S rRNA gene was used as positive control and carried out as previously reported [ 77 ]. All RT-qPCR reactions were done in a 96-well plate using the PowerUp SYBR Green Master Mix (Cat. No. A25742; Thermo Fisher Scientific) on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). 16S rRNA was selected as housekeeping gene. Each reaction was prepared in a 20 µL mixture containing 10 µL of the Power SYBR Green master mix, 0.3 µM of each primer, and 5 µL of properly diluted cDNA (5 µg/µL). The thermal conditions were as follows: 50°C for 2 min, 95°C for 2 min, 40 cycles at 95°C for 15 s, and then at 60°C for 1 min with fluorescence measurement, and the melt curve stage including 95°C for 15 s, 60°C for 1 min, and increasing the temperature step to 95°C at a rate of 0.15°C/s. All the primers used in this study are listed in Supplementary Table S3 . The measurement of gene expression was tested in triplicate, and the mean of values for the target gene was analyzed and normalized to that of the 16S rRNA gene using the 2 −ΔΔCT method [ 78 ]. 2.10 Preparation of Lacticaseibacillus sp. PRA205 cytoplasmic extract Induced cells obtained from milk fermentation were harvested by centrifugation at 8000 rpm for 10 min at 4°C and washed 3 times with 100 mmol/L sodium phosphate buffer, pH 7.0. At the end of the washing procedure, the precipitate was re-suspended in 20 mmol/L Tris-Cl buffer pH 7.5 (1 mL of buffer per 10 10 total cells). Cell lysate was obtained by adding acid-washed glass beads (< 106 µm) (Cat. No. G4649; Sigma Aldrich) to the cell suspension in the proportion of 1:1 w/v and samples were vortexed at the maximum power with a Vortex-Genie 2 (Scientific Industries, Inc., Bohemia, NY, USA) for 4 min at 4°C. The extraction step was repeated four times with 2 min of resting in ice after each vortexing cycle. At the end of the last cycle, cell debris and glass beads were removed by centrifugation for 40 min at 10,000 rpm at 4°C. Finally, the supernatant, representing the cytoplasmic extract, was withdrawn, aliquoted and stored at -80°C until further analyses [ 79 ]. Proteins in the cytoplasmic extract were quantified by the Bradford method using bovine serum albumin (BSA) as standard [ 80 ]. The results were expressed in mg/L of BSA equivalents. 2.11 Determination of PepX activity in the cytoplasmic extract of Lacticaseibacillus sp. PRA205 In order to verify the presence of the X-propyl-dipeptidyl-aminopeptidase (PepX) in the Lcb. casei PRA205 cytoplasmic extract, the hydrolytic reaction was set up in a 96-well plate using the specific substrate glycyl-prolyl- p -nitroanilide (Gly-Pro- p NA) in the absence and presence of inhibitors [ 81 ]. The reaction mixture containing 235 µL of Tris-Cl buffer 50 mmol/L pH 7 and 5 µL of Gly-Pro- p NA substrate (previously dissolved in the same buffer in a concentration of 6.4 mmol/L) was pre-incubated at 37°C for 5 min, before the addition of 10 µL of cytoplasmic extract. The same reactions were also carried out in the presence of EDTA and PMSF at increasing concentrations of 0.1, 0.5, 1, 5 and 10 mmol/L. The corresponding control reactions were prepared by replacing the cytoplasmic extract with Tris-Cl buffer 50 mmol/L pH 7. The reaction was carried out for 2 h at 37°C and finally blocked by the addition of 50 µL of acetic acid (30%). At the end of the incubation, the amount of p NA released following the enzymatic hydrolysis was determined by spectrophotometric reading at 405 nm. The enzyme activity, defined as the amount of enzyme needed to release 1 µmol p-NA per minute (U = µmol/min x mL), was calculated with the following Eq. 1 . Equation 1 : $$\:U=\left[\:\frac{\left(\left(\frac{\varDelta\:\text{A}\text{b}\text{s}}{\text{t}}\right)*Vf\right)}{\left({\epsilon\:}\:\text{*}\text{V}\text{c}\right)}\:\right]$$ Where: ΔAbs is the absorbance change at 410 nm; t is the incubation time in minutes; Vf is the final reaction volume in mL; ε is the molar extinction coefficient of p-NA, 0,00945; and Vc is the sample volume in mL. Finally, the specific enzyme activity was calculated by relating the units of enzyme activity to the protein content expressed in mg/mL, using the formula U/mg = µmol/min x mg. 2.12 Partial purification of PepX from the cytoplasmic extract of Lacticaseibacillus sp. PRA205 PepX was partially purified by using a dimensional exclusion chromatographic column. During all stages of the purification process, both the protein content and the specific activity of PepX were monitored using the methods described above. First, a column for molecular exclusion chromatography packed with Sephadex G-100 resin, with a fractionation range between 5 and 150 kDa, was prepared and then conditioned with a Tris-HCl 50 mM buffer at pH 7.5 containing NaCl 0.1 M. The column was previously calibrated by eluting 4 standard components of known molecular weight (bromophenol blue, ferritin, bovine serum albumin and chymotrypsin). The cytoplasmic extract was first concentrated about 4 times by ultrafiltration with 30 kDa filters, centrifuging at 8360 rpm for 10 min at 4°C to obtain an optimum separation into small volumes. Elution was carried out under isocratic conditions using the same conditioning buffer, namely Tris-HCl 50 mM at pH 7,5 containing NaCl 0.1 M. All eluted fractions were subjected to spectrophotometric reading at 280 nm and 410 nm for the presence of proteins and specific reaction catalyzed by PepX using Gly-Pro-pNA as substrate. Subsequently, the fractions positive for enzyme activity were collected, joined and subjected to an ultrafiltration process as described above, to concentrate the sample and remove the sodium chloride contained in the elution buffer which would interfere with the subsequent analysis. 2.13 Effects of Temperature, pH, and Inhibitors on PepX Activity The effect of temperature on the PepX activity was determined as described above, modifying the incubation temperature while maintaining a constant pH of 7. The assay was carried out at four temperatures: 5°C, 35°C, 40°C, and 45°C. The effect of pH on enzyme activity was evaluated at the constant temperature at 37°C by modifying the pH of the reaction buffer. Tris-HCL buffer (50 mM) was utilized for reactions at pH 4–9, whereas sodium Tris-HCL buffer (50 mM) was used for the assay at pH 7. The effect of protease inhibitors was tested by supplementing the reaction mix with EDTA or PMSF at final concentrations of 0, 1, 2, 5, or 10 mM under standard conditions (pH 7, 37°C). 2.14 Peptide degradation by partial-purified PepX To evaluate the proteolytic activity of the partially purified PepX on various peptides, enzymatic reactions were carried out using the following well-known bioactive substrates: IPP, VPP, LPPT, APFPE, IPPL, and PPF. In each reaction mixture, 10 µL of partially purified PepX enzyme was incubated with 90 µL of peptide substrate, previously dissolved in 50 mM Tris-HCl buffer (pH 7), resulting in a final peptide concentration of 0.5 mM. Control reactions were also prepared by replacing the enzyme with 10 µL of potassium phosphate buffer (pH 7.5) containing 0.2 M NaCl, to assess potential spontaneous degradation of the peptides. The reaction mixtures were incubated at 37°C for 24 h. At the end of incubation, degradation products were analyzed using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, San Jose, CA, USA) mass spectrometer coupled to UHPLC system (UHPLC Ultimate 3000 separation module, Thermo Scientific, San Jose, CA, USA) equipped with a C18 column (Acquity UPLC HSS C18 reversed phase, 2.1 × 100 mm, 1.8 µm particle size, Waters, Milan, Italy). Chromatographic conditions and the mass spectrometer parameters were fully described in [ 82 ]. The relative quantities of the peptides IPP, VPP, LPPT, APFPE, IPPL, and PPF were estimated by integrating the area under the corresponding peaks in the extracted ion chromatograms. 3. Results and Discussion 3.1 Genome Sequencing Strain PRA205 was characterized at the genome level. The assembly of 1,124,896 reads resulted in 38 contigs, corresponding to a total of 3,195,478 bp and 3032 features. The GC content was 47.82% and a clear definition of the positive and negative strands was obtained (Fig. 1 ). The genome assembly metrics of strain PRA205 are summarized in Table 1 . The genome size of strain PRA205 was slightly larger than those of Lcb. casei and Lcb. rhamnosus and was more comparable to those of Lcb. zeae and four novel species recently isolated from silage, including Lcb. huelsenbergensis [ 40 ], Lcb. parahuelsenbergensis , Lcb. styriensis , and Lcb. zeae subsp. silagei [ 41 ] (Table 2 ). Table 1 Assembly and annotation of Lacticaseibacillus sp. PRA 205 genome. Assembly statistics Features Annotation statistics Features Contigs 38 Strand + 1323 Contigs (> 1000 bp) 33 Strand - 1709 Contigs (> 10.000 bp) 23 CDS 2977 Largest contig 684.487 tRNA 54 Total length 3.195.478 rRNA 6 N50 279.481 tmRNA (ssrA) 1 N90 43.054 ncRNA regions 20 L50 4 oriC/oriV 1 L90 14 GC (%) 47.82 Coverage (10X) 99.97 Avg. coverage depth 104 3.2 Species identification and phylogenomic analysis Strain PRA205 was previously identified as Lcb. casei (formerly Lactobacillus casei ) based on 16S rRNA gene sequencing [ 20 ]. Lcb. casei is closely related to other Lacticaseibacillus species, including Lcb. rhamnosus , Lacticaseibacillus paracasei , and the new described species Lcb. zeae [ 83 , 84 ], Lcb. huelsenbergensis [ 40 ], Lcb. parahuelsenbergensis , Lcb. styriensis , and Lcb. zeae subsp. silagei [ 41 ]. However, 16S rRNA barcoding is insufficient to distinguish phylogenetically related species sharing more than 99.7% sequence identity. To clarify the classification of PRA205, we computed ANIb and ANIm values of PRA205 genome with 11 phylogenetically related strains (Supplementary Tables S4 and S5 ). In two of these comparisons, ANI values exceed the 95% species threshold, making it difficult to assign PRA205 unambiguously to either Lcb. parahuelsenbergensis (ANIb/ANIm of 97.72%/98.07%) or Lcb. zeae subsp. zeae (ANIb/ANIm of 94.85%/95.33%). Similarly, dDDH values were 93.3% (confidence interval: 90.6–95.3%) with Lcb. parahuelsenbergensis DSM 116105 T ; 82% (confidence interval: 78.2–85.1%) with Lcb. zeae subsp. zeae DSM 20178 T ; and 69.0% (confidence interval: 65.1–72.7%) with Lcb. casei DSM 20011 T , respectively (Supplementary Table S6 ). Furthermore, FastANI and AAI analyses showed that strain PRA205 clustered with Lcb. parahuelsenbergensis DSM 116105 T (Fig. 2 a and 2 b), whereas POCP metrics supported the clustering of strain PRA205 with Lcb. zeae subsp. zeae DSM 20178 T (Fig. 2 c). Given that the proposed species boundaries are defined by an ANI value of 95–96% and a dDDH value of 70%, we can confidently exclude Lcb. casei as the species designation for strain PRA205. However, the data does not allow for a conclusive assignment of PRA205 to either Lcb. parahuelsenbergensis or Lcb. zeae subsp. zeae. To resolve the taxonomic position of strain PRA205, two different whole genome phylogeny construction methods were used. In the core genome based phylogenetic analysis performed with the EDGAR3.0 pipeline, a dataset of eight Lacticaseibacillus genomes was used, where strain PRA205 formed a separate branch close to Lcb. zeae subsp. zeae and Lcb. zeae subsp. silagei , suggesting close but distinct relatedness (Fig. 3 a). Analysis with PhyloPhlAn 3.0 included a larger dataset of 25 Lacticaseibacillus genomes. The resulting topology of the ML tree identified three clusters. The first cluster, referred to as Lcb. styriensis , consisted of Lcb. styriensis DSM 116297 T , Lcb. styriensis MRD1975, and Lcb. casei strain LC5 which should be re-attributed to Lcb. styriensis . The second cluster, referred to as Lcb. casei , included the only two genomes present in GenBank (last accessed March 2025), namely those of Lcb. casei strains DSM 20011 T and MGB0470. The third cluster, referred to as Lcb. huelsenbergensis , including Lcb. casei strains N and FBL6, in addition to the two strains previously described as Lcb. huelsenbergensis such as DSM 115425 T and DSM 115424 T [ 40 ]. Also in this case, strains FBL6 and N, previously annotated as Lcb. casei , could be re-attributed to the novel species Lcb. huelsenbergensis . According to [ 41 ], Lcb. parahuelsenbergensis DSM 116105 T branched alone. Finally, strain PRA205 was closely related but distinct from Lcb. parahuelsenbergensis , Lcb. styriensis , Lcb. huelsenbergensis , Lcb. zeae , and Lcb. casei . Based on these findings, strain PRA205 was strongly related to Lcb. parahuelsenbergensis but cannot be univocally attributed to this species. 3.3 Functional analysis and genome comparison Genome annotation of PRA205 predicted 2977 CDS, 53 tRNA, 6 rRNA, and 1 tmRNA (Table 1 ). The tRNAs, covering all 20 amino acids, were scattered across the contigs, with two clusters on contig 1 (772 bp, 8 tRNA) and contig 21 (∼2,293 bp,14 tRNA). The CDS counts in PRA205 and in Lcb. zeae subsp. zeae DSM 20178 T were higher than in other related species (Table 2 ). Out of 2977 CDS, 1544 (51.8%) were assigned to 23 different KEGG pathways. The most represented KOs were carbohydrate metabolism (282, 18.35%), protein families: genetic information processing (13.66%), and protein families: signaling and cellular processes (11.06%), respectively (Supplementary Table S7 ). Orthology relationships among PRA205 and five related strains were assessed using the EDGAR3.0 pipeline. The pan-genome included 3,930 CDS: 2,136 core genes (54.4%), 789 strain-specific genes (20.1%), and 1,005 dispensable genes (25.6%) (Fig. 4 a). The large core genome suggests a strong conservation of essential functions across all strains despite their attribution to different species. COG category distribution across demonstrated that most orthogroups were non annotated, followed by orthogroups classified as carbohydrate transport and metabolism in core, dispensable, and singletons genomes (Supplementary Table S8 ). Lcb. zeae subsp. zeae DSM 20178 T had the highest number of unique genes (Fig. 4 c), while PRA205 displayed 108 singletons, 105 of which are unclassified. The reaming three singletons are involved in unclassified – genetic information processing (1), environmental information processing (1), and glycan biosynthesis and metabolism (1) (Fig. 4 c). Mobile genetic elements (MGEs) contribute to genome plasticity and horizontal gene transfer (HGT) [ 85 ]. Analysis with MobileOG-db revealed that PRA205 displayed a similar MGE profile to Lcb. zeae subsp. zeae DSM 20178 T , and a greater MGEs number than Lcb. parahuelsenbergensis DSM 116105 T , Lcb. huelsenbergensis DSM 115425 T , Lcb. styriensis DSM 116297 T , and Lcb. zeae subsp. silagei DSM 116376 T . Most of 147 MGE-associated regions in the PRA205 genome were involved in integration/excision, replication/recombination/repair, and phage-related genes (Fig. 4 d). 3.4. Biosafety, prophage, and CRISPR-Cas assessments According to EFSA [ 66 ], the genome of the PRA205 strain was checked for the presence of AMR genes. All the results of in silico biosafety assessments were summarized in Supplementary Table S9 . Both AMRFinder and ResFinder analyses did not reveal AMR genes, indicating that strain PRA205 can be considered safe in relation to the potential dissemination of AMR genes. Neither virulence nor pathogenic genes were detected. PathogenFinder showed a probability of being a human pathogen of 0.092 (above 1), while the probiotic potential risk score (PPRS) was 2.00. These results are in accordance with the QPS status of Lacticaseibacillus sp. strain PRA205. No plasmid replication initiation proteins were detected in the genome of strain PRA205 using PlasmidFinder v2.1, suggesting the absence of plasmids. Similarly, no plasmid sequences were found in Lcb. zeae subsp. zeae DSM 20178 T , in Lcb. parahuelsenbergensis DSM 116105 T , or in the closely relative species Lcb. huelsenbergensis DSM 115425 T , Lcb. styriensis DSM 116297 T , and Lacticaseibacillus zeae subsp. silagei DSM 116376 T . At least one prophage sequence was detected in each of analyzed genomes (Supplementary Table S10 ). All these prophages belonged to the Siphoviridae family [ 86 ], which is the most prevalent prophage family infecting Lacticaseibacillus strains [ 87 , 88 ]. Specifically, strain PRA205 harbored three prophage regions, including one transposable element, whereas Lcb. parahuelsenbergensis DSM 116105 T had only one. Analysis of CRISPR-Cas systems showed that PRA205 genome displayed two putative cas clusters (13.2 kb and 3.5 kb) but none CRISPR arrays (Supplementary Tables S12 and S13 ). The absence of complete CRISPR-Cas systems may be due to assembly limitations from short-read Illumina sequencing, which struggles to resolve repetitive regions. Conversely, Lcb. parahuelsenbergensis DSM 116105 T and Lcb. huelsenbergensis DSM 115425 T had one CRISPR array each, but no cas genes. Lcb. styriensis lacked both CRISPR elements and cas genes. Complete CRISPR-Cas systems were found only in Lcb. zeae subsp. silagei and Lcb. zeae subsp. zeae (Table 2 ; Supplementary Tables S11 and S12 ). CRISPR-Cas systems can be lost under phage infection pressure [ 89 ]. The high abundance of phage-related MGEs in PRA205 could be linked to the absence of a functional CRISPR-Cas defense, supporting the hypothesis that MGEs play a dominant role in shaping its genome evolution. Table 2 Overview of genomic features, Glycosil transferase (GT) genes, and CRISPR-Cas systems in the genomes of strain PRA205 and its close relatives. Abbreviation s: Lh, Lcb. huelsenbergensis DSM 115425 T ; Lph, Lcb. parahelsenbergenesis DSM 116105 T ; Ls, Lcb. styriensis DSM 116297 T ; Lzs, Lcb. zeae subsp. silagei DSM 116376 T ; Lzz, Lcb. zeae subsp. zeae DSM 20178 T ; GT: Glycosyl transferase; na, not applicable. Features PRA 205 Lzz Ls Lh Lph Lzs Genome Size (Mb) 3.2 2.98 2.99 2.95 2.91 2.91 GC (%) 47.82 47.74 47.93 47.94 48.02 48.03 CDS 2977 2961 2906 2798 2792 2777 rRNA 6 6 15 15 15 15 tRNA 54 53 60 59 59 59 GT GT4 19 15 15 15 14 14 GT2 7 7 8 7 9 7 GT1 6 6 5 9 7 3 GT51 3 4 2 3 3 3 GT5 2 2 2 2 2 2 GT28 1 0 1 1 1 1 GT35 1 1 1 1 1 1 GT9 1 1 0 0 1 1 GT8 1 1 0 1 1 1 GH GH38 - - + - - - araA + - - + + + CRISPR region N° 0 6 0 1 1 8 cas gene N° 2 3 0 0 0 2 Type both CAS putative CAS-TypeIC, CAS putative, CAS putative na Na na CAS-TypeIC, CAS-TypeIIA_1 3.5 Search for functional traits-associated genes To identify genes potentially linked to probiotic traits, we first analyzed the genome of strain PRA205 for biosynthetic gene clusters (BGCs), putative bacteriocins, and carbohydrate-active enzymes (CAZymes). antiSMASH 8.0 predicted five BGCs in PRA205 genome, namely three ribosomally encoded and post-translationally modified peptides (RiPP)-like gene clusters, one linear azol(in)e-containing peptides gene clusters, and one terpene precursor (Supplementary Table S12 ). The three RiPP-like clusters resembled the Escherichia coli BGC encoding microcin L (similarity 0.44), the Lactobacillus gasseri BGC encoding gassericin T/E (similarity 0.53), and the Enterococcus faecium BGC encoding enterocin A (0.56 similarity), respectively. Among closely related strains, Lcb. parahuelsenbergensis exhibited the most similar BGCs profile to PRA205 (Supplementary Table S12 ). BAGEL5 analysis identified four areas of interest (AOIs), three of which overlapped with those predicted by antiSMASH results. The fourth AOI (PFCGCLEC_1.10.AOI_01) contained several genes involved in class IIc bacteriocin production (Fig. 5 ), supporting a potential antimicrobial role of PRA205. In particular, the cluster included two bacteriocins: the bacteriocin at orf00033 was identified as a ComC/BlpC family leader-containing pheromone/ bacteriocin (WP_138130143.1) from Lcb. zeae , and the adjacent downstream ORF encoded Enterocin X beta chain, identified as a bacteriocin leader domain-containing protein commonly reported in many Lacticaseibacillus strains [ 90 ]. The search for CAZymes revealed that 54.40% of the carbohydrate metabolism-related genes were annotated as glycosyltransferases (GTs) and 41.33% as glycoside hydrolases (GHs) in PRA205 genome (Table 2 ). A similar distribution was found in Lcb. zeae subsp. zeae DSM 20178 T (GT: 52.11%, GH: 42.25%), Lcb. parahuelsenbergensis (GT: 54.17%, GH: 40.28%), and Lcb. huelsenbergensis (GT: 54.29%, GH: 41.43%). In contrast, Lcb. styriensis and Lcb. zeae subsp. silagei showed slightly lower GT levels (48.61% and 47.89%, respectively) (Table 2 ). GHs have a critical role in the metabolism of complex carbohydrates as they are mainly used to hydrolyze glycosidic bonds or carbohydrates and glycosidic parts between carbohydrates [ 91 ]. Among GHs, glycoside hydrolases of family 38 are Class II α-mannosidases involved in the hydrolysis of terminal, non-reducing α-D-mannose residues in α-D-mannosides. Lcb. styriensis was the only Lacticaseibacillus species possessing a gene encoding α-mannosidase and therefore positive for α-mannosidase activity [ 41 ]. This gene was missing in strain PRA205 (Table 2 ). GTs catalyze glycosidic bond formation between phospho-activated sugars and various acceptors. These enzymes are involved in stress response, biofilm formation, and the biosynthesis of exopolysaccharides (EPS), which contribute to probiotic traits such as colonization and persistence [ 92 ]. PRA205 contained 41 GT genes from 9 families, 19 of which belonged to family 4 (Table 2 ). GT families 2 and 4 were most prevalent across all strains (Table 2 ). Notably, the GT8 gene, proposed as a molecular marker to distinguish Lcb. casei from Lcb. zeae [ 93 ], was present in PRA205 (96.44% identity), Lcb. zeae subsp. zeae , Lcb. zeae subsp. silagei , Lcb. parahuelsenbergensis , and Lcb. huelsenbergensis but absent in Lcb. styriensis and Lcb. casei DSM 20011 T . Analysis of pairwise sequence similarity revealed that GT8 nucleotide sequences are poorly conserved among these strains (Supplementary Table 13 ), suggesting that this gene may serve as a target for PRA205-specific primer design. The araA gene, which encodes L-arabinose isomerase, was detected in PRA205 (Table 2 ). This gene is also present in Lcb. parahuelsenbergensis , Lcb. zeae subsp. silagei , and Lcb. styriensis but absent in Lcb. zeae subsp. zeae DSM 20178 T [ 40 , 41 ]. Its presence suggests that PRA205 may metabolize arabinose, a potentially valuable probiotic trait [ 94 ]. Among the probiotic-associated traits, acid tolerance and adhesion capacity are of relevance. In our previous study, strain PRA205 was shown to withstand low pH conditions, exhibiting strong auto-aggregation and high cell surface hydrophobicity, two properties which are predictive of adhesion capability [ 21 ]. Consistent with these phenotypes, PRA205 genome contained multiple genes involved in acid stress response, including those encoding the F-type proton pump ( atpA – atpH ), Na+:H + antiporter ( npaA ), and ornithine decarboxylase ( odcI ) (Table 3 ). These systems contribute to intracellular pH homeostasis by coupling ATP hydrolysis to proton extrusion [ 95 ]. Notably, PRA205 lacks genes of the arginine deiminase (ADI) pathway as well as tyrosine-, lysine-, and histidine-decarboxylases. This indicates that, although genetically adapted to cope with acidic stress, the strain does not produce harmful biogenic amines, with the possible exception of putrescine (Table 3 ). The latter is generated via ornithine decarboxylation, a process generally coupled with amino acid transport by antiporter proteins [ 95 ]. This pathway contributes to cytosolic alkalinization and proton motive force generation, which can be exploited for stress resistance and ATP production. Consistently, PRA205 harbors both the ornithine decarboxylase encoding gene odcI and potE , encoding a substrate/product exchanger (Table 3 ) [ 96 ]. Although PRA205 also tolerates bile acids [ 21 ], another stress encountered in gastrointestinal transit, its genome lacks the cholylglycine hydrolase gene ( bsh ). This finding supports the view that bile salt tolerance is a polygenic trait determined by genes other than bsh [ 42 ]. Lastly, the adhesive phenotype of PRA205 is supported by several genes implicated in binding to intestinal epithelial cells, mucin, and fibronectin (Table 3 ) [ 97 , 98 ]. Table 3 Probiotic-related genes found in Lacticaseibacillus sp. PRA205. Phenotype Gene ID Name Gene description Position (Contig; coordinates) Acid tolerance PFCGCLEC_02899 atpC F-type H+-transporting ATPase epsilon chain Contig_19; 5940..6371 PFCGCLEC_02900 atpD F-type H+-transporting ATPase subunit beta Contig_19; 6386..7852 PFCGCLEC_02901 atpG F-type H+-transporting ATPase gamma chain Contig_19; 8028..8951 PFCGCLEC_02902 atpA F-type H+-transporting ATPase subunit alpha Contig_19; 8963..10492 PFCGCLEC_02903 atpH F-type H+-transporting ATPase subunit delta Contig_19; 10516..11061 PFCGCLEC_02904 atpF F-type H+-transporting ATPase subunit b Contig_19; 11048..11536 PFCGCLEC_02905 atpE F-type H+-transporting ATPase subunit c Contig_19; 11571..11783 PFCGCLEC_02906 atpB F-type H+-transporting ATPase subunit a Contig_19; 11806..12516 PFCGCLEC_02705 napA Na(+)/H(+) antiporter (CPA2 family) Contig_14; 30646..31800 PFCGCLEC_02301 odcI Inducible ornithine decarboxylase Contig_09; 39301..41391 PFCGCLEC_02602 potE Ornithine-Putrescine antiporter Contig_12; 23228..24583 Adhesion PFCGCLEC_02966 ltasS Lipoteichoic acid synthase Contig_21; 12038..14134 PFCGCLEC_01247 tuf Elongation factor Tu Contig_03; 244598..245788 PFCGCLEC_02369 tsf Elongation factor Ts Contig_09; 106024..106905 PFCGCLEC_00024 fusA Elongation factor G Contig_01; 28760..30862 PFCGCLEC_02853 dltA D-alanine–D-alanyl carrier protein ligase Contig_17; 33208..34728 PFCGCLEC_02962 cpoA Alpha-galactosylglucosyldiacylglycerol synthase Contig_21; 7736..8764 PFCGCLEC_01140 fbpA Fibronectin-binding protein Contig_03; 126400..128100 PFCGCLEC_00218 - Hypothetical protein with MucBP domain Contig_01; 221391..222581 PFCGCLEC_00997 - Hypothetical protein with MucBP domain Contig_02; 377598..379850 PFCGCLEC_02256 - Hypothetical protein with MucBP domain Contig_08; 142726..143214 PFCGCLEC_00943 oppA Oligopeptide-binding protein OppA Contig_02; 332806..334416 PFCGCLEC_00350 groL chaperonin GroEL Contig_01; 356658..358295 3.6 Reconstruction of the proteolytic system Among the functional traits of strain PRA205, the ability to release antihypertensive peptides from caseins has been well documented [ 22 , 23 ]. To elucidate the genetic basis of this phenotype, we analyzed the distribution protease and peptidase-encoding genes in PRA205 and 12 related Lacticaseibacillus species (Table 4 ). Table 4 Distribution of proteinase and peptidases in the proteolytic system of Lacticaseibacillus species. The number of identified genes is indicated. MEROPS families are indicated. Color shading shows absence of a gene (white), a single gene (yellow) or multiple genes (green). The Locus_Tag codes of the genes can be found in Supplementary Table S14 . Abbreviations: Lca: Lcb. casei DSM 20011 T ; Lpa: Lcb. paracasei BL23; Lc: Lcb. chiayiensis FBL7; LrT: Lcb. rhamnosus DSM 20021 T ; Lr GG: Lcb. rhamnosus GG; Lzz: Lcb. zeae subsp. zeae DSM 20178 T ; PRA, Lacticaseibacillus sp. strain PRA205; Lzs: Lcb. zeae subsp. silagei 116376T; Lh: Lcb. huelsenbergensis DSM 115425 T ; Lph; Lcb. parahuelsenbergensis DSM 116105 T ; Ls: Lcb. styriensis DSM 116297 T ; Lhel: L. helveticus DSM 20075 T and Ld: L. delbrueckii subsp. lactis DSM 20072 T . Peptidase Family Substrate / Annotation Lca Lpa Lc LrT LrGG Lzz PRA Lzs Lh Lph Ls Lhel Ld Proteinase Cell-wall bound proteinase PrtP S8-A 1 1 0 1 1 2 1 1 1 1 1 1 0 PrtR 0 2 1 1 1 1 1 1 1 1 1 0 0 PrtH 0 0 0 0 0 0 0 0 0 0 0 0 0 PrtB 0 0 0 0 0 0 0 0 0 0 0 0 1 PrtM 1 1 0 1 1 2 1 1 1 1 1 1 1 Peptidases Endopeptidase PepE/PepG C1-B (X)m|(X)n 1 1 1 1 1 1 1 1 1 1 1 3 2 PepF M3-B (X)m|(X)n 2 2 2 2 2 2 2 2 2 2 2 1 1 PepO M13 (X)m|(X)n 2 2 1 2 2 2 2 2 2 2 2 3 1 Aminopeptidase PepA M1 Glu/Asp|(X)n 0 1 1 1 1 1 1 1 1 1 1 1 1 PepC C1-B X|(X)n 1 1 1 1 1 1 1 1 1 1 1 1 1 PepM M24-A Met|(X)n 1 1 1 1 1 1 1 1 1 1 1 1 1 PepN M1 X|(X)n 1 1 1 1 1 1 1 1 1 1 1 1 1 PepS M29 1 1 0 1 1 1 1 1 1 1 1 0 0 Dipeptidase PepD C69 X|X 3 4 3 3 3 4 4 4 4 4 4 5 3 PepV M20-A X|X 1 2 1 2 2 2 2 1 1 1 1 1 1 Tripeptidase PepT M20-B X|X-X 0 1 1 1 1 1 1 1 1 1 1 1 2 Proline peptidase PepI S33 Pro|X-(X)n 1 1 1 1 1 1 1 1 1 1 1 1 1 PepR S33 Pro|X 1 1 1 1 1 1 1 1 1 1 1 1 1 PepL S33 Leu|(X)n 1 1 1 1 1 1 1 1 1 1 1 0 1 PepP M24-B X|Pro-(X)n 1 1 1 1 1 1 1 1 1 1 1 1 1 PepQ M24-B X|Pro 1 1 1 1 1 1 1 1 1 1 1 1 2 PepX S15 X-Pro|(X)n 1 1 1 1 1 1 1 1 1 1 1 1 1 Consistent with previous findings in L. helveticus and L. delbrueckii subsp. lactis [ 18 ], protease and peptidase genes were conserved across all genomes analyzed. Core peptidases—PepC, PepN, PepM, and proline-specific peptidases PepR, PepI, PepX, and PepQ—were present in single copies in all species. Gene encoding endopeptidases (PepO, PepF) and dipeptidases (PepV, PepD) were found in multiple copies in some genomes. Strain PRA205 exhibited a complete and enriched proteolytic profile, with two copies of PepO , PepF , and PepV and four of PepD . A similar profile was found in Lcb. zeae subsp. zeae DSM 20178 T . In contrast, Lcb. parahuelsenbergensis , Lcb. huelsenbergensis , L. styriensis , and Lcb. zeae subsp. silagei had only one PepV-encoding gene. Like the thermophilic species L. helveticus, Lacticaseibacillus sp. PRA205 was well-equipped to degrade proline-rich proteins like caseins as two catabolic pathways were identified in its genome: (i) PepX/PepQ, in which PepX cleaves X-Pro dipeptides, followed by PepQ-mediated hydrolysis; and (ii) PepI/PepN, where PepP releases Pro-Y-Z tripeptides, and PepI cleaves the N-terminal proline [ 99 ]. In contrast, mesophilic LAB such as L. lactis lack PepI and rely solely on the PepX/PepQ route [ 99 ]. In PRA205 genome, two candidate genes encoding for a X-prolyl dipeptidyl aminopeptidase (EC 3.4.14.11) were identified: pepX_1 (PFCGCLEC_02304; contig 9) and pepX_2 (PFCGCLEC_02630; contig 12). Alignment of deduced amino acid sequences with the prototype L. lactis PepX (A0A0V8AJV2) revealed that pepX_1 contained all domains characteristic of the S15 family, namely the PepX N-terminal domain (PF09168), the catalytic S15 domain (PF02129), and the C-terminal non-catalytic domain (PF08530) (Fig. 6 ). In contrast, pepX_2 lacked the N-terminal domain and resembled a CocE/Serine esterase (Q45289), indicating a misannotation. Furthermore, synteny analysis identified glnR and glnA genes 5’-downstream of pepX_1 gene according to the operon structure found in the close relative Lcb. rhamnosus [ 100 ]. These results confirm that PRA205 harbors a functional PepX-encoding gene ( pepX_1 ), supporting its proteolytic activity and the functional ability to degrade caseins, releasing BPs. 3.7 Expression analysis of the pepX gene Peptide-rich media downregulate proteolytic genes in Lacticaseibacillus spp., mediated mainly by the CodY regulator, which responds to intracellular BCAA levels by binding conserved motifs in promoters of nitrogen metabolism genes (e.g., prt, pepN, pepC, opp-pepO1 ) [ 25 , 102 , 103 ]. In L. lactis , CodY represses pepX in the presence of peptides, while in L. delbrueckii subsp. bulgaricus its regulation is CodY-independent [ 102 , 104 ]. In strain PRA205, prt was completely silenced in MRS medium but expressed in a milk-like medium [ 25 ]. To investigate the regulation of pepX expression under these two conditions, both RT-PCR and RT-qPCR were performed. RT-PCR showed pepX transcription in both media, with stronger signals observed in the milk-like medium (Fig. 7 a). Consistently, RT-qPCR confirmed that pepX_1 was constitutively expressed in MRS medium but significantly upregulated in the milk-like medium, indicating induction under amino acid-limited conditions (Fig. 7 b). Taken together, these results suggest that, unlike prt , pepX in PRA205 is not fully repressed in peptide-rich environments, pointing to a regulatory mechanism that is at least partially independent of CodY regulator. 3.8 PepX partial purification To link genomic data to enzymatic function, the prolyl-dipeptidyl aminopeptidase activity in Lacticaseibacillus sp. PRA205 was assessed on cytoplasmic extract obtained from cells grown on milk by using the specific substrate Gly-Pro-pNA. The specific prolyl-dipeptidyl aminopeptidase activity was found to be 2.78 ± 0.3 U/mg of proteins. Large variation has been found for the prolyl-dipeptidyl aminopeptidase activity of Lacticaseibacilluss spp. strains. Depending on the strain, the prolyl-dipeptidyl aminopeptidase activity ranged between 2.4 to 50 U/mg of proteins [ 81 , 105 ]. Purification of PepX from the cytoplasmic extract of Lacticaseibacillus sp. PRA205 was carried out as reported in [ 105 ]. Gel filtration chromatography (Supplementary Fig. S2 ) revealed that PepX from strain PRA205 exists predominantly as a monomer with an estimated molecular mass of approximately 80 kDa. In contrast, PepX had been previously purified as a dimer of about 170–200 kDa in L. acidophilus , L. delbrueckii , L. curvatus , L. helveticus , and L. lactis [ 17 , 106 , 107 ]. However, in several strains of L. helveticus , L. delbrueckii as well as Lcb. casei PepX was found active in a monomeric form of about 70–90 kDa [ 17 , 27 ]. The purification details of PepX from Lacticaseibacillus sp. PRA205 are summarized in the Supplementary Table S15 . 3.9 PepX characterization To define the biochemical characteristics of the purified enzyme, the effect of pH, temperature, and inhibitors was assessed by using the specific substrate Gly-Pro-pNA. The enzyme showed high activity over a wide pH range, from 6.0 to 8.0 with optimum activity at pH 7 (Fig. 8 a). Values of pH optima for PepX between 6.0 and 8.0 have been already published [ 105 – 107 ]. A significant reduction of about 64% of PepX activity was observed at pH value of 9. Interesting, at the pH value of 4.5 (as observed in fermented dairy products) the enzyme retained about the 40% of their original activity. The temperature dependence of Lacticaseibacillus sp. PRA205 PepX activity was assessed at 5 and 42°C, which are the typical temperature of yoghurt fermentation and fermented dairy food storage, respectively (Fig. 8 b). Data showed that, at the typical yoghurt fermentation temperature of 42°C, PepX retained approximately 77% of its activity compared with the optimal temperature of 37°C. Similarly, the residual activity at 5°C was 47%. These data implicated that PepX is still active during the yoghurt production process and the low temperatures during the cold storage. The assays carried out with the partial purified enzyme in the presence of PMSF and EDTA confirmed the serine-peptidase nature of the enzyme (Fig. 9 ). PMSF was able to decrease the enzymatic activity of more than 40% at 1 mmol/L concentration, whereas the enzymatic activity was completely abolished at PMSF concentrations of 5 and 10 mmol/L. EDTA at 1 mmol/L had no effect on the enzymatic activity whereas a residual activity of 40% was detected at 10 mmol/L concentration, suggesting the requirement of metals for the catalytic activity of PepX. 3.10 Degradation of bioactive peptides by partially purified PepX Considering the cleavage specificity of PepX and the presence of proline residues in many dairy-derived BPs, the ability of PepX to hydrolyze selected BPs was evaluated. Selected BPs were well-known effective inhibitors of the enzymes ACE and di-peptidyl-peptidase-IV (DPP-IV). The lactotripeptides VPP and IPP (derived from the hydrolysis of β-casein) were potent inhibitors of ACE activity and have been effective in vivo in reducing blood pressure both in rats and human [ 8 ]. These two peptides have been identified in several fermented dairy products such as yogurt and cheeses [ 8 ]. The other peptides such as LPPT, APFPE, PPF, and IPPL have been identified as anti-diabetic peptides being potent DPP-IV-inhibitors [ 82 ]. As reported in Fig. 10 , all the tested peptides were cleaved by partially purified PepX although with different extents. The highest degradation rate was observed for PPF, VPP and APFPE whereas the lowest one for IPP. 4. Conclusions In summary, this present study provided three main findings. First, phylogenomic analyses indicated that strain PRA205 is closely related yet distinct from Lcb. parahuelsenbergenis . Owing to the limited availability of isolates and biochemical data, a conservative taxonomic position was adopted, leaving its species attribution unresolved. Furthermore, several LCG strains in public databases appear misclassified, underscoring the need for their taxonomic revision. Second, in silico biosafety assessment, performed according to EFSA guidelines [ 66 ], confirmed the GRAS status of PRA205, demonstrating the absence of transferable antibiotic-resistance genes and virulence factors. The genome harbors genes associated with probiotic traits, including bacteriocin production, acid stress tolerance, and cell-to-cell interactions. Strain PRA205 carries a complete proteolytic system, which underlies its strong casein-hydrolyzing capacity and the consequent release of BPs. Third, the gene expression profile and biochemical properties of PepX, a key enzyme within the proteolytic system, were established. Remarkably, the partial casitone-independent regulation of pepX_1 gene may contribute to the pronounced proteolytic aptitude of PRA205. The resulting PepX enzyme was identified as a serine-protease of approximately 80 kDa, with activity characterized across different temperatures and pH values. This enzyme retained activity under low temperature and low pH conditions, revealing a dual role both in the formation and degradation of BPs during milk fermentation and dairy product manufacture and storage. Declarations Funding The project was funded by the grant FAR2022 from the Department of Life Sciences (University of Modena and Reggio Emilia, Italy) (Title: Integration of Genomics and Metabolomics for the Characterization of the Proteolytic System Responsible for the Production of Bioactive Peptides in Lacticaseibacillus casei PRA205 - IGEM-PEP). The project was also partially funded under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4–Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union–NextGenerationEU, Award Number: Project Code CN_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUP E93C22001090001, Project Title “National Biodiversity Future Center–NBCF”. Author Contribution Conceptualization: DT and LS; methodology: DT and LS; resources: DT and LS; formal analysis: MC, AC, LS, LB, and DT; investigation: MC, AC, AB, GZ and LS; data curation: MC, AC, LB, and DT; writing—original draft preparation: LS and MC; writing—review and editing: MC, AC, AB, GZ, LB, DT, and LS; funding acquisition: DT and LS. All authors have read and agreed to the present version of the manuscript. Data Availability This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JBQQCW000000000. References Castellone V, Bancalari E, Rubert J, Gatti M, Neviani E, Bottari B (2021) Eating fermented: health benefits of LAB-fermented foods. Foods 10:2639. https://doi.org/10.3390/foods10112639 Leeuwendaal NK, Stanton C, O’Toole PW, Beresford TP (2022) Fermented foods, health and the gut microbiome. Nutrients 14:1527. https://doi.org/10.3390/nu14071527 Jahn LJ, Rekdal VM, Sommer MO (2023) Microbial foods for improving human and planetary health. Cell 186:469–478. https://doi.org/10.1016/j.cell.2022.12.002 Saleem GN, Gu R, Qu H, Bahar Khaskheli G, Rajput IR, Qasim M, Chen X (2024) Therapeutic potential of popular fermented dairy products and its benefits on human health. Front Nutr 11:1328620. https://doi.org/10.3389/fnut.2024.1328620 Gobbetti M, Stepaniak L, De Angelis M, Corsetti A, Di Cagno R (2002) Latent bioactive peptides in milk proteins: proteolytic activation and significance in dairy processing. Crit Rev Food Sci Nutr 42:223–239. https://doi.org/10.1080/10408690290825538 Korhonen H, Pihlanto A (2006) Bioactive peptides: production and functionality. Int Dairy J 16:945–960. https://doi.org/10.1016/j.idairyj.2005.10.012 Hafeez, Z., Cakir-Kiefer, C., Roux, E., Perrin, C., Miclo, L., & Dary-Mourot, A. (2014). Strategies of producing bioactive peptides from milk proteins to functionalize fermented milk products. Food Research International, 63, 71-80. https://doi.org/10.1016/j.foodres.2014.06.002 Tagliazucchi D, Martini S, Solieri L (2019) Bioprospecting for bioactive peptide production by lactic acid bacteria isolated from fermented dairy food. Fermentation 5:96. https://doi.org/10.3390/fermentation5040096 Ulug SK, Jahandideh F, Wu J (2021) Novel technologies for the production of bioactive peptides. Trends Food Sci Technol 108:27–39. https://doi.org/10.1016/j.tifs.2020.11.014 Kuhfeld RF, Eshpari H, Atamer Z, Dallas DC (2024) A comprehensive database of cheese-derived bitter peptides and correlation to their physical properties. Crit Rev Food Sci Nutr 64(27):10105–10119. https://doi.org/10.1080/10408398.2023.2220792 Kelly CD (1932) The influence of certain lactic acid streptococci on the chemical changes in cheddar cheese during ripening. N Y State Agric Exp Stn Bull 200:3–28. Smit G, Smit BA, Engels WJM (2005) Flavor formation by lactic acid bacteria and biochemical flavor profiling of cheese products. FEMS Microbiol Rev 29:592-610. https://doi.org/10.1016/j.fmrre.2005.04.002 McSweeney PLH, Sousa MJ (2000) Biochemical pathways for the production of flavour compounds in cheeses during ripening: a review. Lait 80(3): 293–324. https://doi.org/10.1051/lait:2000127 Anastasiou R, Kazou M, Georgalaki M, Aktypis A, Zoumpopoulou G, Tsakalidou E (2022) Omics approaches to assess flavor development in cheese. Foods 11(2):188. https://doi.org/10.3390/foods11020188. Griffiths MW, Tellez AM (2013) Lactobacillus helveticus : the proteolytic system. Front Microbiol 4:30. https://doi.org/10.3390/foods11020188 Teusink B, Molenaar D (2017) Beyond the laboratory: realistic growth conditions define the proteolytic demand of dairy lactic acid bacteria. Curr Opin Biotechnol 31:123–129. https://doi.org/10.1016/j.copbio.2014.11.010 Christensen JE, Dudley EG, Pederson JA, Steele JL (1999) Peptidases and amino acid catabolism in lactic acid bacteria. Antonie Van Leeuwenhoek 76(1):217–246. Liu M, Bayjanov JR, Renckens B, Nauta A, Siezen RJ (2010) The proteolytic system of lactic acid bacteria revisited: a genomic comparison. BMC Genomics 11:36. https://doi.org/10.1186/1471-2164-11-36 Raveschot C, Cudennec B, Coutte F, et al (2018) Production of bioactive peptides by Lactobacillus species: from gene to application. Front Microbiol 9:2354. https://doi.org/10.3389/fmicb.2018.02354 Solieri L, Bianchi A, Giudici P (2012) Inventory of non-starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture-dependent multiphasic approach. Syst Appl Microbiol 35:270–277. https://doi.org/10.1016/j.syapm.2012.04.005 Solieri L, Bianchi A, Mottolese G, Lemmetti F, Giudici P (2014) Tailoring the probiotic potential of non-starter Lactobacillus strains from ripened Parmigiano Reggiano cheese by in vitro screening and principal component analysis. Food Microbiol 38:240–249. Solieri L, Rutella GS, Tagliazucchi D (2015) Impact of non-starter lactobacilli on release of peptides with angiotensin-converting enzyme inhibitory and antioxidant activities during bovine milk fermentation. Food Microbiol 51:108–116. https://doi.org/10.1016/j.fm.2015.05.009 Solieri L, De Vero L, Tagliazucchi D (2018) Peptidomic study of casein proteolysis in bovine milk by Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331. Int Dairy J 85:237–246. https://doi.org/10.1016/j.idairyj.2018.06.011 Rutella GS, Tagliazucchi D, Solieri L (2016) Survival and bioactivities of selected probiotic lactobacilli in yogurt fermentation and cold storage: New insights for developing a bi-functional dairy food. Food Microbiol 60:54–61. https://doi.org/10.1016/j.fm.2016.06.001 Solieri L, Sola L, Vaccalluzzo A, Randazzo CL, Martini S, Tagliazucchi D (2022) Characterization of cell-envelope proteinases from two Lacticaseibacillus casei strains isolated from Parmigiano Reggiano cheese. Biology 11:139. https://doi.org/10.3390/biology11010139 Casey MG, Meyer J (1985) Presence of X-Prolyl-Dipeptidyl-Peptidase in Lactic Acid Bacteria. J Dairy Sci 68:3212–3215. Kiefer‑Partsch B, Bockelmann W, Geis A, Teuber M (1989) Purification of an X‑prolyl‑dipeptidyl aminopeptidase from the cell wall proteolytic system of Lactococcus lactis subsp. cremoris . Appl Microbiol Biotechnol 31:75–78. https://doi.org/10.1007/BF00252531 Rigolet P, Mechin I, Delage MM, Chich JF (2002) The structural basis for catalysis and specificity of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis . Structure 10:1383–1394. https://doi.org/10.1016/S0969-2126(02)00851-1 Stressler T, Eisele T, Schlayer M, Lutz-Wahl S, Fischer L (2013) Characterization of the recombinant exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 important for food protein hydrolysis. PLoS One 8:e70055. https://doi.org/10.1371/journal.pone.0070055 Yüksel G, Steele JL (1996) DNA sequence analysis, expression, distribution, and physiological role of the X-prolyl dipeptidyl aminopeptidase (PepX) gene from Lactobacillus helveticus CNRZ 32. Appl Microbiol Biotechnol 44:766–773. https://doi.org/10.1007/BF00178616 Wakai T, Yamamoto N (2012) Antihypertensive peptides specific to Lactobacillus helveticus fermented milk. In: Biotechnology - Molecular Studies and Novel Applications for Improved Quality of Human Life. pp 159–172 De Angelis M, Rizzello CG, Fasano A, Clemente MG, De Simone C, Silano M, De Vincenzi M, Losito I, Gobbetti M (2010) Mechanism of degradation of immunogenic gluten epitopes from Triticum turgidum by sourdough lactobacilli and fungal proteases. Appl Environ Microbiol 76:508–518. https://doi.org/10.1128/AEM.01630-09 Francavilla R, Piccolo M, Francavilla A, Polimeno L, Semeraro F, Cristofori F, Castellaneta S, Barone M, Indrio F, Gobbetti M, De Angelis M, Rizzello CG (2017) Selected probiotic lactobacilli hydrolyze gluten peptides during simulated digestion. Appl Environ Microbiol 83:e00376-17. https://doi.org/10.1128/AEM.00376-17 Brzozowski B, Bednarski W, Staniewski B, Walczak P, Bączek T, Michalski M, Błaszczyk U (2020) Reducing immunoreactivity of gliadins using peptidases from L. acidophilus . Catalysts 10:923. https://doi.org/10.3390/catal10080923 Parmar H, Hati S, Panchal G, Sakure AA (2020) Purification and production of novel angiotensin I-converting enzyme (ACE) inhibitory bioactive peptides derived from fermented goat milk. Int J Pept Res Ther 26:997–1011. https://doi.org/10.1007/ Matar C, Goulet J (1996) β-casomorphin-4 from milk fermented by a mutant of Lactobacillus helveticus . Int Dairy J 6(4):383–397. https://doi.org/10.1016/0958-6946(95)00015-1 Hafeez Z, Cakir-Kiefer C, Lecomte X, Miclo L, Dary-Mourot A (2019) The X-prolyl dipeptidyl-peptidase PepX of Streptococcus thermophilus initially described as intracellular is also responsible for peptidase extracellular activity. J Dairy Sci 102:113–123. https://doi.org/10.3168/jds.2018-14823 Otte J, Lenhard T, Flambard B, Sørensen KI (2011) Influence of fermentation temperature and autolysis on ACE-inhibitory activity and peptide profiles of milk fermented by selected strains of Lactobacillus helveticus and Lactococcus lactis . Int Dairy J 21:229–238. https://doi.org/10.1016/j.idairyj.2010.12.008 Kilpi E, Kahala M, Steele JL, Pihlanto A, Joutsjoki V (2007) Angiotensin I converting enzyme inhibitory activity in milk fermented by wild type and peptidase deletion derivatives of Lactobacillus helveticus CNRZ32. Int Dairy J 17:976–984. https://doi.org/10.1016/j.idairyj.2006.12.001 Grabner FM, Grabner HM, Schein H, Weidenholzer E, Busche T, Rückert-Reed C, Buchebner-Jance M (2023) Lacticaseibacillus huelsenbergensis sp. nov., isolated from grass silage and corn silage. Int J Syst Evol Microbiol 73:1–10. https://doi.org/10.1099/ijsem.0.006049 Grabner FM, Grabner HM, Schein H, Schrank A, Töglhofer M, Weidenholzer E, Rückert-Reed C, Busche T, Buchebner-Jance M (2024) Lacticaseibacillus parahuelsenbergensis sp. nov., Lacticaseibacillus styriensis sp. nov. and Lacticaseibacillus zeae subsp. silagei subsp. nov., isolated from different grass and corn silage. Int J Syst Evol Microbiol 74:006441. https://doi.org/10.1099/ijsem.0.006441 Agolino G, Cristofolini M, Vaccalluzzo A, Tagliazucchi D, Cattivelli A, Pino A, Randazzo CL (2025) Genome mining and characterization of two novel Lacticaseibacillus rhamnosus probiotic candidates with bile salt hydrolase activity. Biomolecules 15:86. https://doi.org/10.3390/biom15010086 Chen S (2023) Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta 2:e107. https://doi.org/10.1002/imt2.107 Blanco-Míguez A, Beghini F, Cumbo F, McIver LJ, Thompson KN, Zolfo M, Manghi P, Dubois L, Huang KD, Thomas AM, et al (2023) Extending and improving metagenomic taxonomic profiling with uncharacterized species using MetaPhlAn 4. Nat Biotechnol 41:1633–1644. https://doi.org/10.1038/s41587-023-01688-w Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A (2020) Using SPAdes De Novo Assembler. Curr Protoc Bioinform 70:e102. https://doi.org/10.1002/cpbi.102 Mikheenko A, Prjibelski A, Saveliev V, Antipov D, Gurevich A (2018) Versatile genome assembly evaluation with QUAST-LG. Bioinformatics 34:i142–i150. https://doi.org/10.1093/bioinformatics/bty266 Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. (2021) BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol. 38:4647–4654. https://doi.org/10.1093/molbev/msab199 Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153 Cantalapiedra CP, Hernández-Plaza A, Letunic I, Bork P, Huerta-Cepas J (2021) eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol 38:5825–5829. https://doi.org/10.1093/molbev/msab293 Grant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen CY, et al (2023) Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res 51:W484–W492. https://doi.org/10.1093/nar/gkad326 Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M (2016) KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res 44:D457–D462. https://doi.org/10.1093/nar/gkv1070 Richter M, Rosselló‑Móra R, Glöckner FO, Peplies J (2016) JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32:929–931. https://doi.org/10.1093/bioinformatics/btv681 Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S (2018) High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:4998. https://doi.org/10.1038/s41467-018-07641-9 Dieckmann MA, Beyvers S, Nkouamedjo‑Fankep RC, Hanel PHG, Jelonek L, Blom J, Goesmann A (2021) EDGAR3.0: comparative genomics and phylogenomics on a scalable infrastructure. Nucleic Acids Res 49:W185–W192. https://doi.org/10.1093/nar/gkab341 Qin QL, YZ A (2014) A proposed genus boundary for the prokaryotes based on genomic insights. J Bacteriol 196:2210–2215. https://doi.org/10.1128/jb.01688-14 Meier‑Kolthoff JP, Sardà Carbasse J, Peinado‑Olarte RL, Göker M (2022) TYGS and LPSN: a database tandem for fast and reliable genome‑based classification and nomenclature of prokaryotes. Nucleic Acids Res 50:D801–D807. https://doi.org/10.1093/nar/gkab938 Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. https://doi.org/10.1093/nar/gkh340 Asnicar F, Thomas AM, Beghini F, Mengoni C, Manara S, Manghi P, Zhu Q, Bolzan M, Cumbo F, May U, Sanders JG, Zolfo M, Kopylova E, Pasolli E, Knight R, Mirarab S, Huttenhower C, Segata N (2020) Precise phylogenetic analysis of microbial isolates and genomes from metagenomes using PhyloPhlAn 3.0. Nat Commun 11:2500. https://doi.org/10.1038/s41467-020-16366-7 Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312–1313. https://doi.org/10.1093/bioinformatics/btu033 Letunic I, Bork P (2024) Interactive Tree Of Life (iTOL) v6: an online tool for the display, annotation and management of phylogenetic and other trees. Nucleic Acids Res 52:W17–W24. https://doi.org/10.1093/nar/gkae268 Liu YY, Hsu CY, Yang YC, Huang CH, Chen CC (2023) ProbioMinServer: an integrated platform for assessing the safety and functional properties of potential probiotic strains. Bioinform Adv 3:vbad153. https://doi.org/10.1093/bioadv/vbad153 Blin K, Shaw S, Augustijn HE, Reitz ZL, Biermann F, Alanjary M, et al (2023) antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualization. Nucleic Acids Res 51:W46–W50. https://doi.org/10.1093/nar/gkad344 Carattoli A, Zankari E, García-Fernández A, Voldby Larsen M, Lund O, Villa L, et al (2014) In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 58:3895–3903. https://doi.org/10.1128/AAC.02412-14 Couvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, Néron B, et al (2018) CRISPRCasFinder, an update of CRISPRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res 46:W246–W251. https://doi.org/10.1093/nar/gky425 Brown CL, Mullet J, Hindi F, Stoll JE, Gupta S, Choi M, et al (2022) mobileOG-db: a manually curated database of protein families mediating the life cycle of bacterial mobile genetic elements. Appl Environ Microbiol 88:e00991-22. https://doi.org/10.1128/AEM.00991-22 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Rychen G, Aquilina G, et al (2018) Guidance on the characterisation of microorganisms used as feed additives or as production organisms. EFSA J 16:e05206. https://doi.org/10.2903/j.efsa.2018.5206 McArthur AG, Waglechner N, Nizam F, et al (2013) The Comprehensive Antibiotic Resistance Database. Antimicrob Agents Chemother 57:3348–3357. https://doi.org/ 10.1128/AAC.00419-13 Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M, Edalatmand A, Huynh W, Nguyen AV, Cheng AA, Liu S, Min SY, Miroshnichenko A, Tran HK, Werfalli RE, Nasir JA, Oloni M, Speicher DJ, Florescu A, Singh B, Faltyn M, Hernandez-Koutoucheva A, Sharma AN, Bordeleau E, Pawlowski AC, Zubyk HL, Dooley D, Griffiths E, Maguire F, Winsor GL, Beiko RG, Brinkman FSL, Hsiao WWL, Domselaar GV, McArthur AG (2020) CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res 48:D517–D525. https://doi.org/10.1093/nar/gkz935. Bortolaia V, Kaas RS, Ruppe E, Roberts MC, Schwarz S, Cattoir V, et al (2020) ResFinder 4.0 for predictions of phenotypic antimicrobial resistance and genotypic subtyping. J Antimicrob Chemother 75:3491–3500. https://doi.org/10.1093/jac/dkaa345 Liu B, Zheng D, Jin Q, Chen L, Yang J (2022) VFDB 2022: a general classification scheme for bacterial virulence factors. Nucleic Acids Res 50:D912–D917. https://doi.org/10.1093/nar/gkab1107 Malberg Tetzschner AM, Johnson JR, Johnston BD, et al (2020) In silico genotyping of Escherichia coli isolates for extraintestinal virulence genes by use of whole-genome sequencing data. J Clin Microbiol 58:e01269-20. https://doi.org/10.1128/JCM.01269-20 Bai Z, Zhang N, Jin Y, et al (2022) Comprehensive analysis of 84 Faecalibacterium prausnitzii strains uncovers their genetic diversity, functional characteristics, and potential risks. Front Cell Infect Microbiol 12:919701. https://doi.org/10.3389/fcimb.2022.919701 Bateman A, Martin MJ, Orchard S, Magrane M, Agivetova R, Ahmad S, et al (2021) UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 49:D480–D489. https://doi.org/10.1093/nar/gkaa1100 Pearson WR (2013) An introduction to sequence similarity (homology) searching. Curr Protoc Bioinform 42:e59. https://doi.org/10.1002/0471250953.bi0301s422 Eddy SR (2011) Accelerated profile HMM searches. PLoS Comput Biol 7:e1002195. https://doi.org/10.1371/journal.pcbi.1002195 Finn RD, Mistry J, Schuster‑Böckler B, Griffiths‑Jones S, Hollich V, Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R, Eddy SR, Sonnhammer ELL, Bateman A (2006) Pfam: clans, web tools and services. Nucleic Acids Res 34:D247–D251. https://doi.org/10.1093/nar/gkj149 Tagliazucchi D, Baldaccini A, Martini S, Bianchi A, Pizzamiglio V, Solieri L (2020) Cultivable non‑starter lactobacilli from ripened Parmigiano Reggiano cheeses with different salt content and their potential to release anti‑hypertensive peptides. Int J Food Microbiol 330:108688. https://doi.org/10.1016/j.ijfoodmicro.2020.108688 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25:402-408. Fernández de Palencia P, Pelaez C, Romero C, Martin-Hernandez MC (1997) Purification and characterization of the cell wall proteinase of Lactobacillus casei subsp. casei IFPL 731 isolated from raw goat's milk cheese. J Agric Food Chem 45(9):3401–3405. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. Donkor O, Henriksson A, Vasiljevic T, Shah N (2007) Proteolytic activity of dairy lactic acid bacteria and probiotics as determinant of growth and in vitro angiotensin‑converting enzyme inhibitory activity in fermented milk. Le Lait 87:21–38. https://doi.org/10.1051/lait:2006023 Martini S, Solieri L, Cattivelli A, Pizzamiglio V, Tagliazucchi D (2021) An integrated peptidomics and in silico approach to identify novel antidiabetic peptides in Parmigiano‑Reggiano cheese. Biology 10:563. https://doi.org/10.3390/biology10060563 Liu DD, Gu CT (2020) Proposal to reclassify Lactobacillus zhaodongensis , Lactobacillus zeae , Lactobacillus argentoratensis and Lactobacillus buchneri subsp. silagei as Lacticaseibacillus zhaodongensis comb. nov., Lacticaseibacillus zeae comb. nov., Lactiplantibacillus argentoratensis comb. nov. and Lentilactobacillus buchneri subsp. silagei comb. nov., respectively and Apilactobacillus kosoi as a later heterotypic synonym of Apilactobacillus micheneri . Int J Syst Evol Microbiol 70(12):6414-6417. Huang CH, Chen CC, Liou JS, Lee AY, Blom J, Lin YC, et al. (2020) Genome-based reclassification of Lactobacillus casei : emended classification and description of the species Lactobacillus zeae . Int J Syst Evol Microbiol 70:3755–3762. https://doi.org/10.1099/ijsem.0.003969 Koonin EV, Makarova KS, Wolf YI, Krupovic M (2020) Evolutionary entanglement of mobile genetic elements and host defense systems: guns for hire. Nat Rev Genet 21(2):119–131. https://doi.org/10.1038/s41576-019-0172-9 Hendrix RW, Casjens SR, Lavigne R (2012) Family - Siphoviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy. Elsevier, San Diego, pp 86–98. da Silva Barreira D, Lapaquette P, Novion Ducassou J, Couté Y, Guzzo J, Rieu A (2022) Spontaneous prophage induction contributes to the production of membrane vesicles by the Gram-positive bacterium Lacticaseibacillus casei BL23. MBio 13(5):e02375-22. https://doi.org/10.1128/mbio.02375-22 Chandran A, Beena AK, Vaiyapuri M, John L, Rajakumar SN, Babu S, James L, Rahila MP (2023) Detection of lytic phage infecting flavour-producing strain of Lacticaseibacillus paracasei in the dairy effluents of Kerala. J Dairy Res 90(2):178–181. https://doi.org/10.1017/S002202992300020X Rollie C, Chevallereau A, Watson BNJ, Chyou TY, Fradet O, McLeod I, Fineran PC, Brown CM, Gandon S, Westra ER (2020) Targeting of temperate phages drives loss of type I CRISPR‑Cas systems. Nature 578(7793):149–153. https://doi.org/10.1038/s41586-020-1936-2 Fernandes A, Jobby R (2022) Bacteriocins from lactic acid bacteria and their potential clinical applications. Appl Biochem Biotechnol 194(10):4377–4399. https://doi.org/10.1007/s12010-022-03870-3 Berlemont R, Martiny AC (2015) Genomic potential for polysaccharide deconstruction in bacteria. Appl Environ Microbiol 81(4):1513–1519. https://doi.org/10.1128/AEM.03718-14 Fukao M, Zendo T, Inoue T, Nakayama J, Suzuki S, Fukaya T, et al (2019) Plasmid-encoded glycosyltransferase operon is responsible for exopolysaccharide production, cell aggregation, and bile resistance in a probiotic strain, Lactobacillus brevis KB290. J Biosci Bioeng 128:391–397. doi:10.1016/j.jbiosc.2019.04.008 Kim E, Yang SM, Kim D, Kim HY (2022) Complete genome sequencing and comparative genomics of three potential probiotic strains, Lacticaseibacillus casei FBL6, Lacticaseibacillus chiayiensis FBL7, and Lacticaseibacillus zeae FBL8. Front Microbiol 12, 794315. https://doi.org/10.3389/fmicb.2021.794315 Leschonski KP, Mortensen MS, Hansen LB, Krogh KB, Kabel MA, Laursen MF (2024) Structure-dependent stimulation of gut bacteria by arabinoxylo-oligosaccharides (AXOS): a review. Gut Microbes 16(1):2430419. https://doi.org/10.1080/19490976.2024.2430419 Papadimitriou K, Alegría A, Bron PA, de Angelis M, Gobbetti M, Kleerebezem M, Lemos JA, Linares DM, Ross P, Stanton C, Turroni F, van Sinderen D, Varmanen P, Ventura M, Zúñiga M, Tsakalidou E, Kok J (2016) Stress physiology of lactic acid bacteria. Microbiol Mol Biol Rev 80:837–890. https://doi.org/10.1128/MMBR.00076-15 Romano A, Trip H, Lonvaud-Funel A, Lolkema JS, Lucas PM (2012) Evidence of two functionally distinct ornithine decarboxylation systems in lactic acid bacteria. Appl Environ Microbiol 78(6):1953–1961. https://doi.org/10.1128/AEM.07161-11 Vélez MP, De Keersmaecker SC, Vanderleyden J (2007) Adherence factors of Lactobacillus in the human gastrointestinal tract. FEMS Microbiol Lett 276(2):140–148. https://doi.org/10.1111/j.1574-6968.2007.00908.x Muscariello L, De Siena B, Marasco R (2020) Lactobacillus cell surface proteins involved in interaction with mucus and extracellular matrix components. Curr Microbiol 77(12):3831–3841. https://doi.org/10.1007/s00284-020-02243-5 Savijoki K, Ingmer H, Varmanen P (2006) Proteolytic systems of lactic acid bacteria. Appl Microbiol Biotechnol 71(4):394–406.http://doi10.1007/BF00395933. PMID: 8879407. Varmanen P, Savijoki K, Åvall S, Palva A, Tynkkynen S (2000) X-prolyl dipeptidyl aminopeptidase gene ( pepX ) is part of the glnRA operon in Lactobacillus rhamnosus . J Bacteriol 182:146–154. https://doi.org/10.1128/JB.182.1.146-154.2000 Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191 Guédon E, Sperandio B, Pons N, Ehrlich SD, Renault P (2001) Overall control of nitrogen metabolism in Lactococcus lactis by CodY and possible models for CodY regulation in Firmicutes. Microbiology 147(12):3895–3909. https://doi.org/10.1099/mic.0.28186-0 Petranovic D, Guédon E, Sperandio B, Delorme C, Ehrlich D, Renault P (2004) Intracellular effectors regulating the activity of the Lactococcus lactis CodY pleiotropic transcription regulator. Mol Microbiol 53(2):613–621. https://doi.org/10.1111/j.1365-2958.2004.04136.x Morel F, Frot Coutaz J, Aubel D, Portalier R, Atlan D (1999) Characterization of a prolidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 with an unusual regulation of biosynthesis. Microbiol 145:437–446. http://doi.10.1099/13500872-145-2-437 El Abboudi M, El Soda M, Pandian S, Simard RE, Olson NF (1992) Purification of X‑prolyl dipeptidyl aminopeptidase from Lactobacillus casei subspecies. Int J Food Microbiol 15(1‑2):87–98. https://doi.org/10.1016/0168-1605(92)90138-S Magboul AA, McSweeney PL (2000) Purification and characterization of an X-prolyl-dipeptidyl aminopeptidase from Lactobacillus curvatus DPC2024. Le Lait 80(4):385–396. https://doi.org/10.1051/lait:2000133 Stressler T, Eisele T, Kranz B, Fischer L (2014) PepX from Lactobacillus helveticus : automated multi‑step purification and determination of kinetic parameters with original tripeptide substrates. J Mol Catal B: Enzym 108:103–110. https://doi.org/10.1016/j.molcatb.2014.07.006. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTablecomplete.xlsx SupplementaryFigures.docx Cite Share Download PDF Status: Published Journal Publication published 23 Dec, 2025 Read the published version in Probiotics and Antimicrobial Proteins → Version 1 posted Editorial decision: Revision requested 25 Oct, 2025 Reviews received at journal 17 Oct, 2025 Reviews received at journal 11 Oct, 2025 Reviewers agreed at journal 23 Sep, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers invited by journal 21 Sep, 2025 Editor assigned by journal 08 Sep, 2025 Submission checks completed at journal 08 Sep, 2025 First submitted to journal 05 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":5083214,"visible":true,"origin":"","legend":"\u003cp\u003eCircular graphical representation of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. strain PRA205 contigs. The map contigs was generated using Proksee (https://proksee.ca; accessed on 13 February 2025). The black central circle shows the scale expressed in megabases. Starting from the outermost ring: Ring 1: VirSorter, Ring 2: PHASTEST, Ring 3: Bakta Annotation (+), Ring 4: Prokka Annotation (+), Ring 5: Phigaro, Ring 6: CARD RGI Results (+), Ring 7: CRISPRCasFinder Annotation (+), Ring 8: mobileOG-db Annotation (+), Ring 9: Features (+), Backbone (Contigs), Ring 11: Features (-), Ring 12: GC Content, Ring 13: GC Skew, Ring 14: mobileOG-db Annotation (-), Ring 15: CRISPRCasFinder Annotation (-), Ring 16: CARD RGI Results (-), Ring 17: Prokka Annotation (-), Ring 18: Bakta Annotation (-). Selected genes are labeled on the outer purple ring using Proksee’s default settings. The GC content is in dark green and the GC skew + and - in dark pink and dark green, respectively. The genomic order of the contigs is arbitrary.\u003c/p\u003e","description":"","filename":"Figure01map.png","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/8b803c5383e7b6f828df7b90.png"},{"id":92610597,"identity":"6f62f0fc-a6e5-4335-88de-dfbb84fbaab6","added_by":"auto","created_at":"2025-10-01 16:17:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1258624,"visible":true,"origin":"","legend":"\u003cp\u003eClustering and heatmap analyses of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. strain PRA205 and 7 close relatives based on similarity matrices of FastANI (a), AAI (b), and POCP (c) metrics, respectively.\u003c/p\u003e","description":"","filename":"Figure02ANI.png","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/ce5fbc46ae8a2749a1dd6d83.png"},{"id":92610636,"identity":"6f562644-c9b6-4e46-9d05-029e6c1aa58d","added_by":"auto","created_at":"2025-10-01 16:18:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2542467,"visible":true,"origin":"","legend":"\u003cp\u003eCore genome phylogenetic analyses of \u003cem\u003eLacticaseibacillus \u003c/em\u003esp. PRA205. (a) Core genome phylogenetic tree depicting the relationships of strain PRA205 with 7 publicly available \u003cem\u003eLacticaseibacillus\u003c/em\u003e spp. genomes, as computed in EDGAR3.0 [54]. Reciprocal best BLAST hits against all other genomes were checked with \u003cem\u003eLacticaseibacillus \u003c/em\u003esp. strain PRA205 as the reference genome. Alignments of each core gene set were generated with MUSCLE and concatenated [57]. A phylogenetic tree was constructed with approximately-maximum-likelihood using FastTree software (http://www.microbesonline.org/fasttree/). (b) Core genome phylogenetic tree depicting the relationships of strain PRA205 with 25 publicly available \u003cem\u003eLacticaseibacillus\u003c/em\u003e spp. genomes obtained with PhyloPhlAn 3 in the fast mode [58].The final phylogenetic tree was inferred using the maximum-likelihood method with RAxML v.8.2.12 [59]. All trees were visualized using iTOL [60].\u003c/p\u003e","description":"","filename":"Figure03tree.png","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/2b34d12cd12a9a4c1500ead7.png"},{"id":92610599,"identity":"b58f5191-3b42-4458-808b-5251c942d717","added_by":"auto","created_at":"2025-10-01 16:17:58","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1842885,"visible":true,"origin":"","legend":"\u003cp\u003eComparative genomics analysis across 6 \u003cem\u003eLacticaseibacillus\u003c/em\u003e spp. strains, including \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205, \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei \u003c/em\u003eDSM 116376\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eLcb. huelsenbergensis \u003c/em\u003eDSM 115425\u003csup\u003eT\u003c/sup\u003e, and \u003cem\u003eLcb. styriensis\u003c/em\u003e DSM 116297\u003csup\u003eT\u003c/sup\u003e.(a) Distribution of core-genome and accessory genome. (b) Upset plot showing the distribution and intersection of orthogroups across the six genomes. (c) KEGG classification of unique genes. (d) Distribution of Genetic Mobile Elements (MGEs). Abbreviations: T, transposase; STD; stability/transfer/defense; P, phage-related genes; RRR, replication/recombination/repair; IE, integration/excision; Lh, \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e; Lph, \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e; Ls, \u003cem\u003eLcb. styriensis\u003c/em\u003e; Lzs, \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e; Lz, \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure04DEF.png","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/f47de1c99286a30c84b621ef.png"},{"id":92610600,"identity":"1b04d6db-9916-4671-b07f-7cf3c29dcd57","added_by":"auto","created_at":"2025-10-01 16:17:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":259021,"visible":true,"origin":"","legend":"\u003cp\u003eBAGEL5 graphical output for four putative bacteriocin gene clusters identified in \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205.\u003c/p\u003e","description":"","filename":"Figure05BAGEL5.png","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/5a2b64b0432e00cf6f6c82e8.png"},{"id":92611268,"identity":"7d80b312-b23c-4e07-960d-e2324755aba8","added_by":"auto","created_at":"2025-10-01 16:25:58","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":880356,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of PepX_1 and PePX_2 proteins. (a) Protein alignment of PepX_1 and Pep_X2 performed with Muscle [57] and visualized with Jalview [101]. (b) Domain structures of the PepX candidate proteins. The codes denote the PFAM (PF) domain identifiers.\u003c/p\u003e","description":"","filename":"Figure06aln.png","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/04889b40859b53c5697f4b96.png"},{"id":92611265,"identity":"1b602ecd-be9f-431c-a14f-3f2a1f640032","added_by":"auto","created_at":"2025-10-01 16:25:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1100672,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression analysis of \u003cem\u003epepX_1\u003c/em\u003e gene in \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205. (A) RT-PCR analysis of \u003cem\u003epepX_1\u003c/em\u003e expression in PRA205 cells grown on milk and MRS medium. The figure shows amplified cDNA products from three biological replicates; gDNA amplification was used as a positive PCR control. RT+ and RT- indicate reaction with and without reverse-transcription, respectively. Abbreviations: M, DNA ladder used as molecular size marker; NTC, no template control. (B) \u003cem\u003epepx_1\u003c/em\u003e expression level assessed by RT-qPCR assay in PRA205 grown on milk and MRS medium. Data represent the mean ± standard deviation of at least three biological replicates. Statistical differences were evaluated using Student’s t-test (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05); *** indicate \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure07RT.png","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/888751ee779f454cef62f602.png"},{"id":92610605,"identity":"4c9d71b6-3739-4899-9b22-e5e4bb8e3905","added_by":"auto","created_at":"2025-10-01 16:17:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":766247,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of different pH values (a) and temperature (b) on PepX activity. PepX activity (expressed as a percentage) was quantified using the substrate Gly-Pro-pNA. Residual activity values were calculated based on 100% activity obtained at pH 7 and 37 °C. Different letters means that the values are significantly different (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure08pHT.png","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/8ef3cf925329ac7312f8a8ed.png"},{"id":92611267,"identity":"61240a60-4ac8-4134-97ec-23107f7c519e","added_by":"auto","created_at":"2025-10-01 16:25:58","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":693201,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of PMSF and EDTA on PepX activity. PepX activity (expressed as a percentage) was quantified using the substrate Gly-Pro-\u003cem\u003ep\u003c/em\u003eNA. Residual activity (expressed in % and calculated with respect to the control reaction without inhibitors) was determined at increasing concentrations (mM) of the inhibitors EDTA and PMSF. Different letters indicate significantly different values (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure09EDTAPMFS.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/3853400dd5474bfef266679a.jpg"},{"id":92611269,"identity":"14ea8c62-d528-477e-936d-f124e0b659bd","added_by":"auto","created_at":"2025-10-01 16:25:58","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":744498,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation of the bioactive peptides IPP, VPP, LPPT, APFPE, PPF, and IPPL by PepX. Data are expressed as residual percentage of the peptide calculated by comparing the area under the peak (AUP) obtained from peptides incubated for 24h with PepX and peptides incubated in the same condition but without PepX (control reaction). AUP was calculated by integrating the area under the peak, measured from the extracted ion chromatograms (EIC) obtained for each peptide (tolerance ± 5 ppm). Different letters indicate significantly different values (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/d3eb274016f086d08aab0e5c.jpg"},{"id":99172817,"identity":"69b95aed-40e6-4536-a2ed-3e95c800cdf7","added_by":"auto","created_at":"2025-12-29 16:11:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16722307,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/73ec1bbe-5003-45a5-8481-4e50c96a4832.pdf"},{"id":92611264,"identity":"ae500e73-1126-4ef0-a0e1-b4edf0f3ce36","added_by":"auto","created_at":"2025-10-01 16:25:58","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":128320,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablecomplete.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/c64a5ec11e699909a005ce95.xlsx"},{"id":92610606,"identity":"a2e540dc-12a3-4fa5-80bc-58ef554020c1","added_by":"auto","created_at":"2025-10-01 16:17:58","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":402917,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7544664/v1/3bb226a28ee259790c11018f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic and Proteolytic Profiling of Lacticaseibacillus sp. PRA205: Insights into PepX- Mediated Bioactive Peptide Metabolism","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn the dairy industry, microbial fermentation is one of the oldest and most effective methods to develop functional food [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In this context, lactic acid bacteria (LAB) serve as cell factories to produce biofunctional compounds, including milk protein-derived bioactive peptides (BPs) [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBPs are encrypted sequences within milk proteins that, once released, exhibit various biological activities such as antimicrobial, hypocholesterolemic, antihypertensive, antioxidant, anticancer, antithrombotic, osteogenic, immunomodulatory, and mineral binding activity effects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These peptides typically range from 2 to 40 amino acids in length, with their activity influenced by sequence and composition [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Many are multifunctional, acting through multiple mechanisms and exerting more than one physiological effect [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn addition to their biological activity, short peptides released by LAB also affect cheese maturation, influencing texture and flavor of the final products [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The link between cheese bitterness and specific peptide fractions was recognized as early as 1932 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Conversely, other peptides enhance salty and umami notes of cheese by interacting with compounds like reducing sugars and unsaturated fatty acids and forming flavor molecules such as sulfur compounds, methyl ketones, and aldehydes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Key amino acids contributing to dairy flavor include branched-chain (valine, leucine, isoleucine), aromatic (tyrosine, tryptophan, phenylalanine), and sulfur-containing (methionine, cysteine) amino acids [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn LAB, a well-coordinated proteolytic system hydrolyzes milk proteins into peptides, including BPs, and essential amino acids. This system complements the autotrophies of LAB for several amino acids, enabling growth by utilizing external proteins as a nitrogen source [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This system includes cell surface proteinases (CEPs), responsible for the initial cleavage of caseins into oligopeptides (up to 17 amino acid residues); specific transport systems for the peptide internalization, such as oligopeptide permease (Opp), dipeptide permeases (DtpT, Dpp), and di/tri-peptide transporters; and a broad range of intracellular peptidases, including specific endopeptidases, aminopeptidases, tri- and dipeptidases, as well as peptidases involved in proline metabolism, which further degrade peptides into di/tri-peptides and/or free amino acids [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The considerable variability in proteolytic systems among LAB strains results in marked phenotypic differences in their ability to hydrolyze milk proteins and release BPs both at species and strain level [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. strain PRA205, isolated from Parmigiano Reggiano PDO cheese and previously identified as \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e PRA205 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], is a mesophilic, non-starter lactic acid bacterium with probiotic tolerance to environmental cues such as acidity, salinity, bile salts, and digestive enzymes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] (Solieri et al. 2014). During milk fermentation it produced significant concentrations of anti-hypertensive tripeptides Valine-Proline-Proline (VPP) (32.88 mg/L) and Isoleucine-Proline-Proline (IPP) (7.52 mg/L) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Consequently, fermented milk prepared with this strain exerts strong angiotensin-converting enzyme (ACE) inhibitory activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Moreover, when used as adjunct culture during yogurt fermentation with \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003ebulgaricus\u003c/em\u003e and \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e, strain PRA205 maintained a viable cell count higher than 10⁸ CFU/g in yogurt at 4\u0026deg;C for 28 days, exceeding the minimum threshold required to confer probiotic health benefits [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn a previous work, we have characterized the cell envelope proteinase (CEP)-encoding \u003cem\u003eprt\u003c/em\u003e gene in \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205, demonstrating that the enzyme PrtR1 is responsible for the release of several BPs through αs1- and β-casein hydrolysis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, despite the probiotic potential of strain PRA205, the entire proteolytic system is still unknown, and the intracellular peptidases involved in subsequent peptide processing remain uncharacterized.\u003c/p\u003e\u003cp\u003eAmong these, X-prolyl dipeptidyl aminopeptidase (PepX, EC 3.4.14.11) is a serine protease belonging to the MEROPS peptidase family S15 clan SC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ebi.ac.uk/interpro/entry/IPR008252\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/interpro/entry/IPR008252\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), which cleaves Xaa-Pro releasing N-terminal dipeptide from polypeptides [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the model organism \u003cem\u003eLactococcus lactis\u003c/em\u003e, this aminopeptidase has a narrow specificity for proline (or alanine or glycine with, respectively, 10- and 100-fold lower efficiency) in the P1 position. In the positions P2 and P\u0026prime;1, PepX accepts any residue except proline [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. While the positive role of PepX in cheese peptide debittering is well-known, its contribution to the release of BPs remains ambiguous. Several studies have linked PepX activity to notable biofunctions. For instance, PepX from \u003cem\u003eLactobacillus helveticus\u003c/em\u003e has been shown to hydrolyze caseins [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and, together with PepC and PepO, it contributes to release VPP and IPP from a 28-amino-acid β-casein precursor peptide [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The PepX enzyme from \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e has also been reported to hydrolyze highly proline rich gliadins and coeliac-toxic peptides that contain a high percentage of proline residues [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, PepX enzymes from five LAB species have been shown to generate ACE-inhibitory peptides from goat milk [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Deletion of the \u003cem\u003epepX\u003c/em\u003e gene resulted in the loss of hydrolytic activity on the typical substrate β-casomorphin-7 (β-CN 7) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eConversely, other studies have negatively correlated the PepX activity with BPs release. In milk fermented by \u003cem\u003eLactococcus lactis\u003c/em\u003e and \u003cem\u003eL. helveticus\u003c/em\u003e, high PepX activity did not enhance ACE-inhibitory capacity, suggesting that ACE-inhibitory peptides were primarily generated by early-stage proteases [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Similarly, in \u003cem\u003eL. helveticus\u003c/em\u003e CNRZ32, deletion of \u003cem\u003epepX\u003c/em\u003e and \u003cem\u003epepN\u003c/em\u003e was associated with increased ACE-inhibitory activity, likely due to reduced degradation of proline-rich BPs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe aim of the present study was to explore the genome of the probiotic candidate \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205 and to characterize its proteolytic system, with a focus on the X-prolyl dipeptidyl aminopeptidase PepX. Given the recent taxonomic revisions within the genus \u003cem\u003eLacticaseibacillus\u003c/em\u003e, including the introduction of four novel species [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], a comparative genomic approach was employed to accurately determine the phylogenetic position of strain PRA205, as well as to investigate its safety and probiotic traits.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Reagents and cultivation media\u003c/h2\u003e\u003cp\u003eUnless otherwise indicated, all media and anaerobic systems used in this study were purchased from Oxoid (Oxoid, Basingstoke, Hampshire, UK), while the chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), except for chemicals and solvents for mass spectrometry analysis that were supplied by Carlo Erba (Milan, Italy). The primers were provided by BMR Genomics (Padova, Italy). The molecular biology reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Amicon Ultra-4 centrifugal filter units (regenerated cellulose, nominal cut 30 kDa) were supplied by Millipore (Milan, Italy).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Bacteria Strain and Culture Conditions\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. strain PRA205 used in this study was isolated from Parmigiano Reggiano PDO cheese [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and was deposited in the Culture Collection of Department of Life Sciences (University of Modena and Reggio Emilia, Italy). The strain was cryo-preserved at -80\u0026deg;C in the Man, Rogosa and Sharpe (MRS) medium (pH 6.5) containing 25% (v/v) of glycerol. To activate the strain from its glycerol storage, it was propagated in 5 mL of MRS medium, supplemented with 1.5% (w/v) agar when required, and incubated at 37\u0026deg;C for 24 h, under anaerobic conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003e2.3 Reference genomes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, genome sequences of \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e group (LCG) species, including \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e, \u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e, \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e, \u003cem\u003eLacticaseibacillus zeae\u003c/em\u003e, and the four recently proposed species such as \u003cem\u003eLacticaseibacillus huelsenbergensis\u003c/em\u003e, \u003cem\u003eLacticaseibacillus zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e, \u003cem\u003eLacticaseibacillus parahuelsenbergensis\u003c/em\u003e, and \u003cem\u003eLacticaseibacillus styriensis\u003c/em\u003e, were used for comparative purposes. In addition, two \u003cem\u003eLactobacillus\u003c/em\u003e species with a well-characterized proteolytic system, namely \u003cem\u003eLactobacillus helveticus\u003c/em\u003e and \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003elactis\u003c/em\u003e were used as reference genomes for the reconstruction of proteolytic system. All the genomes were obtained from the National Center for Biotechnology Information (NCBI, Bethesda, Rockville, ML, USA) genome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/genomes/lproks.cgi\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, last accessed 12 March 2025). The genomes and the associated information are listed in Supplementary \u003cb\u003eTable S1\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Genomic Sequencing and annotation\u003c/h2\u003e\u003cp\u003eThe genomic DNA was extracted carried out as previously reported [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. BMR Genomics (Padova, Italy) performed both the library preparation and genome sequencing. Briefly, the purified DNA was processed using the Illumina DNAprep kit following the manufacturer\u0026rsquo;s instructions, and the resulting shotgun library was sequenced on the Illumina MiSeq Sequencing System, using the proprietary V3 reagent kit, producing 2 x 300 bp paired end reads. Reads pre-processing was carried out with Fastp v0.23.2 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] to remove residual adapter sequences, short reads (length\u0026thinsp;\u0026lt;\u0026thinsp;150) and low quality data (base quality\u0026thinsp;\u0026lt;\u0026thinsp;20, average read quality\u0026thinsp;\u0026lt;\u0026thinsp;25, read complexity threshold\u0026thinsp;\u0026lt;\u0026thinsp;30). Possible contaminants were assessed by MetaPhlan v4.0.1 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSPAdes v3.15.5 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] with \u003cem\u003ecareful\u003c/em\u003e option was applied to perform \u003cem\u003ede novo\u003c/em\u003e genome assembly. QUAST v5.0.2 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] and BUSCO v5.4.3 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], run on \u003cem\u003elactobacillales_odb10\u003c/em\u003e (v2020-03-06) lineage dataset, were used for assessing assembly metrics and genomic completeness, respectively. The gene prediction and gene annotation were performed with Prokka v.1.14.6 [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] and eggNOG-mapper v2.1.7 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCustom graphic map of the genome was obtained via Proksee v1.1.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://proksee.ca\u003c/span\u003e\u003cspan address=\"https://proksee.ca\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed 13 February 2025) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] using the GenBank annotation file (gbk). BlastKOALA was used to calculate the relative abundances of genes in the KEGG categories as percentages of the genes assigned to each respective KEGG category versus the total genes number [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Species identification and phylogenomics\u003c/h2\u003e\u003cp\u003eDraft genome sequence of PRA205 and seven \u003cem\u003eLacticaseibacillus\u003c/em\u003e complete genomes were uploaded to the JspeciesWS server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ribocon.com/jspeciesws.html\u003c/span\u003e\u003cspan address=\"https://www.ribocon.com/jspeciesws.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 14 Mar 2025) to calculate ANI values with BLAST algorithm (ANIb) and MUMmer (Maximal Unique Match) alignment tool (ANIm), respectively [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. FastANI, which is a \u003cem\u003ek\u003c/em\u003e-mer and alignment-free method of ANI calculation [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], was implemented in EDGAR3.0 webtool [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] to cross-validate the ANI values. The calculations of average amino acid identity (AAI) and of the percentage of conserved proteins (POCP) between two genomes as proposed by [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] were also implemented in EDGAR3.0 pipeline [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. For POCP analysis the following cutoff values were used: an e-value threshold of 1\u003cem\u003ee\u003c/em\u003e\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, a minimum sequence identity\u0026thinsp;\u0026gt;\u0026thinsp;50%, and an alignment coverage of the query protein\u0026thinsp;\u0026gt;\u0026thinsp;50%.\u003c/p\u003e\u003cp\u003eGGDC 3.0 available in Type (Strain) Genome Server (TYGS) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://tygs.dsmz.de/\u003c/span\u003e\u003cspan address=\"https://tygs.dsmz.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to calculate digital DNA-DNA hybridization (dDDH) values [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Phylogenomics were carried out with two approaches. Firstly, the core genes of 8 genomes were computed in EDGAR3.0 pipeline [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The alignments of each core gene set are generated using MUSCLE [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], and the alignments are concatenated to one huge alignment. This alignment was the input for the FastTree software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.microbesonline.org/fasttree/\u003c/span\u003e\u003cspan address=\"http://www.microbesonline.org/fasttree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to generate approximately-maximum-likelihood (ML) phylogenetic tree. The values at the branches of FastTree tree were local support values computed by FastTree using the Shimodaira-Hasegawa (SH) test.\u003c/p\u003e\u003cp\u003eIn the second phylogenetic analysis the PRA205 assembled genome was compared with 25 \u003cem\u003eLacticaseibacillus\u003c/em\u003e genomes (Supplementary \u003cb\u003eTable S1\u003c/b\u003e) downloaded from National Center for Biotechnology Information (NCBI, Bethesda, Rockville, ML, USA). To perform such procedure, PhyloPhlAn v3.1.68 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] was applied in fast mode against a customized PhyloPhlAn database containing only \u003cem\u003eLactobacillus\u003c/em\u003e associated markers (n\u0026thinsp;=\u0026thinsp;339453). In addition, the following parameters were used to generate high-resolution strain-level phylogeny: --diversity low --trim greedy --min_num_entries 9 (~\u0026thinsp;34.6% of the total number of genomes in order to keep more informative signals for distinguishing between very closely related organisms; 75481 markers were selected) --remove_fragmentary_entries --fast --force_nucleotides. The Maximum Likelihood (ML) tree was reconstructed from the PhyloPhlAn output using RAxML version 8.2.12 [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The GTRCAT model was applied to account for nucleotide substitution and rate heterogeneity. A rapid bootstrap analysis (-f a) with 1000 replicates was performed to assess branch support. To guarantee the result\u0026rsquo;s reproducibility, the parsimony random seed (-p) and the rapid bootstrap random seed (-x) were set to 1989 and 42, respectively.\u003c/p\u003e\u003cp\u003eAll the resulting phylogenetic trees were further refined and visually enhanced using the iTOL tool [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Genome analyses\u003c/h2\u003e\u003cp\u003eThe annotated genome (Genbank format) of PRA205 and 5 \u003cem\u003eLacticaseibacillus\u003c/em\u003e reference genomes (Supplementary \u003cb\u003eTable S1\u003c/b\u003e) were submitted to EDGAR3.0 web-based tool to investigate core, dispensable, and accessory genes, as well as to identify KEGG Orthologs (KOs) and clusters Clusters of Orthologous Groups of proteins (COGs) functions across the different genomes [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCarbohydrate-active enzyme (CAZy) database implemented in the ProbioMinServer web-platform was used for functional assignment of CAZy enzymes [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe biosynthetic gene clusters (BGCs) encoding secondary metabolites were predicted using antiSMASH 4.0 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://antismash.secondarymetabolites.org/#!/about\u003c/span\u003e\u003cspan address=\"https://antismash.secondarymetabolites.org/#!/about\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 20 June 2025).\u003c/p\u003e\u003cp\u003eBAGEL5 web-server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bagel5.molgenrug.nl\u003c/span\u003e\u003cspan address=\"http://bagel5.molgenrug.nl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 20 May 2025) was used to identify potential bacteriocin gene clusters, as well as other bacterial ribosomally synthesized and post-translationally modified peptides, with all parameters set to default.\u003c/p\u003e\u003cp\u003ePutative plasmids were identified using the PlasmidFinder v2.1 database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.food.dtu.dk/services/PlasmidFinder/\u003c/span\u003e\u003cspan address=\"https://cge.food.dtu.dk/services/PlasmidFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) according to the following screening criteria: 95% identity threshold and 60% minimum coverage [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences and Cas protein encoding genes were identified using CRISPRCasFinder v2.2 [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The presence of genes of mobile elements was examined using BLASTX searches compared to the full mobileOG-db v1.1.3 database [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], with an identity\u0026thinsp;\u0026gt;\u0026thinsp;90% and coverage\u0026thinsp;\u0026gt;\u0026thinsp;90%.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003ein silico\u003c/em\u003e analyses for microbial safety assessments were implemented in ProbioMinServer [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], following the guidelines of the European Food Safety Authority (EFSA) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Specifically, antibiotic resistance genes (ARGs) were identified using the Comprehensive Antibiotic Resistance Database (CARD) Variants v4.0.0 [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], ARMFinder [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], and ResFinder v4.3.2 [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. BLASTN v2.8.1\u0026thinsp;+\u0026thinsp;was used to detect virulence factors (VFs) by searching against the set B database from the Virulence Factor Database (VFDB) [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] and VirulenceFinder v2.0.3 [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. BLASTP v2.8.1\u0026thinsp;+\u0026thinsp;search was performed against the Pathogen Host Interaction v4.14 database to identify the probable pathogenic genes (PGs) [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e] (Liu et al. 2022). The probiotic potential risk score (PPRS) was computed as defined by [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] to evaluate the risks associated with PRA205 probiotic strain. The score was classified as low-risk (\u0026le;\u0026thinsp;4), medium-risk (4\u0026ndash;6), and high-risk (\u0026ge;\u0026thinsp;6).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Identification of orthologs of the proteolytic system\u003c/h2\u003e\u003cp\u003eProtein sequences of experimentally verified proteolytic system members, i.e. CEPs and various peptidases, were derived from the non-redundant protein database Uniprot (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"http://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; accessed on 17 May 2025; [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e] (Bateman et al. 2021) (Supplementary \u003cb\u003eTable S2\u003c/b\u003e). The amino acid sequences were used as query to perform a TBLASTN search against the nucleotide sequences of the selected species (Supplementary \u003cb\u003eTable S1\u003c/b\u003e). Two sequences were considered homologous if their alignment had a minimum sequence identity of 30% and a query coverage of at least 70% [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] (Pearson 2013). The peptidase/protease candidates which were not present in genome annotation were subjected to HMMER v.3.2.1 using the hmmsearch function from HMMER v.3.4 [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. The corresponding Hidden Markov Models (HMMs) of each protein family were obtained from the Pfam database [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Milk fermentation\u003c/h2\u003e\u003cp\u003eStrain PRA205 was pre-cultured in 50 mL of MRS broth for 72 h at 37\u0026deg;C under anaerobic conditions. After centrifugation at 10,000\u0026times; rpm for 20 min at 4\u0026deg;C, cells were washed with physiological solution (0.9% NaCl) and then inoculated in triplicates in scraw-cap flasks containing 45 mL UHT skimmed milk at the final concentration of 10\u003csup\u003e10\u003c/sup\u003e CFU/mL. After 72 h of incubation at 37\u0026deg;C under shaking conditions (10 rpm), fermented milk samples were left to settle for 10 min. The liquid phase was separated from the coagulated proteins through Whatman paper (grade 4). After centrifugation at 6,000x rpm for 20 min at 4\u0026deg;C, cells were resuspended in 6 mL of phosphate buffer pH 7.0 50 mM a final concentration of 10\u003csup\u003e10\u003c/sup\u003e CFU/mL and divided into two aliquots for RNA extraction and biochemical characterization of pepX enzyme, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Gene expression analysis\u003c/h2\u003e\u003cp\u003eRNA was extracted approximately from 2 x 10\u003csup\u003e8\u003c/sup\u003e cells out using the Zymo Direct-zol RNA MiniPrep kit (Cat. No. R2071, Zymo Research, Irvine, CA, USA) and applying few modifications to the manufacturer\u0026rsquo;s instructions. Briefly, after adding up to 700 \u0026micro;L of the Tri reagent, the mechanical lysis of cells was achieved using a Vortex Genie 2 instrument (Mo Bio Laboratories Carlsbad, CA, USA) by performing two rounds of 20 min at the highest speed alternating with 3 min on ice. The quantity of total RNA was measured spectrophotometrically using a Nanodrop Nd 1000 system (Nanodrop Technologies, Wilmington, DE, USA) and only samples with A260/280 absorbance ratio between 1.8 and 2.2 were considered for further analyses. The integrity of the total RNA was evaluated by denaturing gel electrophoresis on a 0.9% (w/v) agarose gel with formaldehyde (10 mL of 10\u0026times; 3-morpholinepropane sulfonic acid [MOPS] running buffer) and 18 mL of 37% formaldehyde (12 mol/L) in pH 7.0 1\u0026times; MOPS running buffer (0.4 mol/L MOPS, 1 mol/L sodium acetate, and 0.01 mol/L EDTA) after the RNA treatment at 65\u0026deg;C for 10 min. To remove any contamination of gDNA, 1 \u0026micro;g of the RNA sample was treated with dsDNase (Cat. No. EN0771, Thermo Fisher Scientific) (final volume 40 \u0026micro;L) and, thereafter, RNA was reverse transcribed to cDNA at 42\u0026deg;C for 60 min with random hexamers (Cat. No. SO142; Thermo Fisher Scientific) and oligo (dT)18 primers (Cat. No. SO131; Thermo Fisher Scientific) using the RevertAid RT Kit (Cat. No. EP0441; Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003eThe end-point RT-PCR amplification of \u003cem\u003epepX\u003c/em\u003e gene was carried out with a Dream Taq DNA polymerase (Cat. No. EP0712; Thermo Fisher Scientific). RT-PCR of the 16S rRNA gene was used as positive control and carried out as previously reported [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. All RT-qPCR reactions were done in a 96-well plate using the PowerUp SYBR Green Master Mix (Cat. No. A25742; Thermo Fisher Scientific) on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). 16S rRNA was selected as housekeeping gene. Each reaction was prepared in a 20 \u0026micro;L mixture containing 10 \u0026micro;L of the Power SYBR Green master mix, 0.3 \u0026micro;M of each primer, and 5 \u0026micro;L of properly diluted cDNA (5 \u0026micro;g/\u0026micro;L). The thermal conditions were as follows: 50\u0026deg;C for 2 min, 95\u0026deg;C for 2 min, 40 cycles at 95\u0026deg;C for 15 s, and then at 60\u0026deg;C for 1 min with fluorescence measurement, and the melt curve stage including 95\u0026deg;C for 15 s, 60\u0026deg;C for 1 min, and increasing the temperature step to 95\u0026deg;C at a rate of 0.15\u0026deg;C/s.\u003c/p\u003e\u003cp\u003eAll the primers used in this study are listed in Supplementary \u003cb\u003eTable S3\u003c/b\u003e. The measurement of gene expression was tested in triplicate, and the mean of values for the target gene was analyzed and normalized to that of the 16S rRNA gene using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Preparation of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205 cytoplasmic extract\u003c/h2\u003e\u003cp\u003eInduced cells obtained from milk fermentation were harvested by centrifugation at 8000 rpm for 10 min at 4\u0026deg;C and washed 3 times with 100 mmol/L sodium phosphate buffer, pH 7.0. At the end of the washing procedure, the precipitate was re-suspended in 20 mmol/L Tris-Cl buffer pH 7.5 (1 mL of buffer per 10\u003csup\u003e10\u003c/sup\u003e total cells). Cell lysate was obtained by adding acid-washed glass beads (\u0026lt;\u0026thinsp;106 \u0026micro;m) (Cat. No. G4649; Sigma Aldrich) to the cell suspension in the proportion of 1:1 w/v and samples were vortexed at the maximum power with a Vortex-Genie 2 (Scientific Industries, Inc., Bohemia, NY, USA) for 4 min at 4\u0026deg;C. The extraction step was repeated four times with 2 min of resting in ice after each vortexing cycle. At the end of the last cycle, cell debris and glass beads were removed by centrifugation for 40 min at 10,000 rpm at 4\u0026deg;C. Finally, the supernatant, representing the cytoplasmic extract, was withdrawn, aliquoted and stored at -80\u0026deg;C until further analyses [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eProteins in the cytoplasmic extract were quantified by the Bradford method using bovine serum albumin (BSA) as standard [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. The results were expressed in mg/L of BSA equivalents.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Determination of PepX activity in the cytoplasmic extract of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205\u003c/h2\u003e\u003cp\u003eIn order to verify the presence of the X-propyl-dipeptidyl-aminopeptidase (PepX) in the \u003cem\u003eLcb. casei\u003c/em\u003e PRA205 cytoplasmic extract, the hydrolytic reaction was set up in a 96-well plate using the specific substrate glycyl-prolyl-\u003cem\u003ep\u003c/em\u003e-nitroanilide (Gly-Pro-\u003cem\u003ep\u003c/em\u003eNA) in the absence and presence of inhibitors [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. The reaction mixture containing 235 \u0026micro;L of Tris-Cl buffer 50 mmol/L pH 7 and 5 \u0026micro;L of Gly-Pro-\u003cem\u003ep\u003c/em\u003eNA substrate (previously dissolved in the same buffer in a concentration of 6.4 mmol/L) was pre-incubated at 37\u0026deg;C for 5 min, before the addition of 10 \u0026micro;L of cytoplasmic extract. The same reactions were also carried out in the presence of EDTA and PMSF at increasing concentrations of 0.1, 0.5, 1, 5 and 10 mmol/L. The corresponding control reactions were prepared by replacing the cytoplasmic extract with Tris-Cl buffer 50 mmol/L pH 7. The reaction was carried out for 2 h at 37\u0026deg;C and finally blocked by the addition of 50 \u0026micro;L of acetic acid (30%). At the end of the incubation, the amount of \u003cem\u003ep\u003c/em\u003eNA released following the enzymatic hydrolysis was determined by spectrophotometric reading at 405 nm.\u003c/p\u003e\u003cp\u003eThe enzyme activity, defined as the amount of enzyme needed to release 1 \u0026micro;mol p-NA per minute (U\u0026thinsp;=\u0026thinsp;\u0026micro;mol/min x mL), was calculated with the following \u003cb\u003eEq.\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEquation 1\u003c/b\u003e:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:U=\\left[\\:\\frac{\\left(\\left(\\frac{\\varDelta\\:\\text{A}\\text{b}\\text{s}}{\\text{t}}\\right)*Vf\\right)}{\\left({\\epsilon\\:}\\:\\text{*}\\text{V}\\text{c}\\right)}\\:\\right]$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhere: ΔAbs is the absorbance change at 410 nm; t is the incubation time in minutes; Vf is the final reaction volume in mL; ε is the molar extinction coefficient of p-NA, 0,00945; and Vc is the sample volume in mL. Finally, the specific enzyme activity was calculated by relating the units of enzyme activity to the protein content expressed in mg/mL, using the formula U/mg\u0026thinsp;=\u0026thinsp;\u0026micro;mol/min x mg.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.12 Partial purification of PepX from the cytoplasmic extract of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205\u003c/h2\u003e\u003cp\u003ePepX was partially purified by using a dimensional exclusion chromatographic column. During all stages of the purification process, both the protein content and the specific activity of PepX were monitored using the methods described above. First, a column for molecular exclusion chromatography packed with Sephadex G-100 resin, with a fractionation range between 5 and 150 kDa, was prepared and then conditioned with a Tris-HCl 50 mM buffer at pH 7.5 containing NaCl 0.1 M. The column was previously calibrated by eluting 4 standard components of known molecular weight (bromophenol blue, ferritin, bovine serum albumin and chymotrypsin). The cytoplasmic extract was first concentrated about 4 times by ultrafiltration with 30 kDa filters, centrifuging at 8360 rpm for 10 min at 4\u0026deg;C to obtain an optimum separation into small volumes. Elution was carried out under isocratic conditions using the same conditioning buffer, namely Tris-HCl 50 mM at pH 7,5 containing NaCl 0.1 M. All eluted fractions were subjected to spectrophotometric reading at 280 nm and 410 nm for the presence of proteins and specific reaction catalyzed by PepX using Gly-Pro-pNA as substrate. Subsequently, the fractions positive for enzyme activity were collected, joined and subjected to an ultrafiltration process as described above, to concentrate the sample and remove the sodium chloride contained in the elution buffer which would interfere with the subsequent analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Effects of Temperature, pH, and Inhibitors on PepX Activity\u003c/h2\u003e\u003cp\u003eThe effect of temperature on the PepX activity was determined as described above, modifying the incubation temperature while maintaining a constant pH of 7. The assay was carried out at four temperatures: 5\u0026deg;C, 35\u0026deg;C, 40\u0026deg;C, and 45\u0026deg;C. The effect of pH on enzyme activity was evaluated at the constant temperature at 37\u0026deg;C by modifying the pH of the reaction buffer. Tris-HCL buffer (50 mM) was utilized for reactions at pH 4\u0026ndash;9, whereas sodium Tris-HCL buffer (50 mM) was used for the assay at pH 7. The effect of protease inhibitors was tested by supplementing the reaction mix with EDTA or PMSF at final concentrations of 0, 1, 2, 5, or 10 mM under standard conditions (pH 7, 37\u0026deg;C).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.14 Peptide degradation by partial-purified PepX\u003c/h2\u003e\u003cp\u003eTo evaluate the proteolytic activity of the partially purified PepX on various peptides, enzymatic reactions were carried out using the following well-known bioactive substrates: IPP, VPP, LPPT, APFPE, IPPL, and PPF.\u003c/p\u003e\u003cp\u003eIn each reaction mixture, 10 \u0026micro;L of partially purified PepX enzyme was incubated with 90 \u0026micro;L of peptide substrate, previously dissolved in 50 mM Tris-HCl buffer (pH 7), resulting in a final peptide concentration of 0.5 mM. Control reactions were also prepared by replacing the enzyme with 10 \u0026micro;L of potassium phosphate buffer (pH 7.5) containing 0.2 M NaCl, to assess potential spontaneous degradation of the peptides.\u003c/p\u003e\u003cp\u003eThe reaction mixtures were incubated at 37\u0026deg;C for 24 h. At the end of incubation, degradation products were analyzed using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, San Jose, CA, USA) mass spectrometer coupled to UHPLC system (UHPLC Ultimate 3000 separation module, Thermo Scientific, San Jose, CA, USA) equipped with a C18 column (Acquity UPLC HSS C18 reversed phase, 2.1 \u0026times; 100 mm, 1.8 \u0026micro;m particle size, Waters, Milan, Italy). Chromatographic conditions and the mass spectrometer parameters were fully described in [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe relative quantities of the peptides IPP, VPP, LPPT, APFPE, IPPL, and PPF were estimated by integrating the area under the corresponding peaks in the extracted ion chromatograms.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Genome Sequencing\u003c/h2\u003e\u003cp\u003eStrain PRA205 was characterized at the genome level. The assembly of 1,124,896 reads resulted in 38 contigs, corresponding to a total of 3,195,478 bp and 3032 features. The GC content was 47.82% and a clear definition of the positive and negative strands was obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The genome assembly metrics of strain PRA205 are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The genome size of strain PRA205 was slightly larger than those of \u003cem\u003eLcb. casei\u003c/em\u003e and \u003cem\u003eLcb. rhamnosus\u003c/em\u003e and was more comparable to those of \u003cem\u003eLcb. zeae\u003c/em\u003e and four novel species recently isolated from silage, including \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e, \u003cem\u003eLcb. styriensis\u003c/em\u003e, and \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAssembly and annotation of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA 205 genome.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAssembly statistics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFeatures\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAnnotation statistics\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFeatures\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eContigs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStrand +\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1323\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eContigs (\u0026gt;\u0026thinsp;1000 bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStrand -\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1709\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eContigs (\u0026gt;\u0026thinsp;10.000 bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCDS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2977\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLargest contig\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e684.487\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal length\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.195.478\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003erRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e279.481\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003etmRNA (ssrA)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eN90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43.054\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003encRNA regions\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eoriC/oriV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eL90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGC (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e47.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCoverage (10X)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e99.97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAvg. coverage depth\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e104\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Species identification and phylogenomic analysis\u003c/h2\u003e\u003cp\u003eStrain PRA205 was previously identified as \u003cem\u003eLcb. casei\u003c/em\u003e (formerly \u003cem\u003eLactobacillus casei\u003c/em\u003e) based on 16S rRNA gene sequencing [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. \u003cem\u003eLcb. casei\u003c/em\u003e is closely related to other \u003cem\u003eLacticaseibacillus\u003c/em\u003e species, including \u003cem\u003eLcb. rhamnosus\u003c/em\u003e, \u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e, and the new described species \u003cem\u003eLcb. zeae\u003c/em\u003e [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e], \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e, \u003cem\u003eLcb. styriensis\u003c/em\u003e, and \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, 16S rRNA barcoding is insufficient to distinguish phylogenetically related species sharing more than 99.7% sequence identity.\u003c/p\u003e\u003cp\u003eTo clarify the classification of PRA205, we computed ANIb and ANIm values of PRA205 genome with 11 phylogenetically related strains (Supplementary \u003cb\u003eTables S4\u003c/b\u003e and \u003cb\u003eS5\u003c/b\u003e). In two of these comparisons, ANI values exceed the 95% species threshold, making it difficult to assign PRA205 unambiguously to either \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e (ANIb/ANIm of 97.72%/98.07%) or \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e (ANIb/ANIm of 94.85%/95.33%). Similarly, dDDH values were 93.3% (confidence interval: 90.6\u0026ndash;95.3%) with \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e; 82% (confidence interval: 78.2\u0026ndash;85.1%) with \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e; and 69.0% (confidence interval: 65.1\u0026ndash;72.7%) with \u003cem\u003eLcb. casei\u003c/em\u003e DSM 20011\u003csup\u003eT\u003c/sup\u003e, respectively (Supplementary \u003cb\u003eTable S6\u003c/b\u003e). Furthermore, FastANI and AAI analyses showed that strain PRA205 clustered with \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), whereas POCP metrics supported the clustering of strain PRA205 with \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Given that the proposed species boundaries are defined by an ANI value of 95\u0026ndash;96% and a dDDH value of 70%, we can confidently exclude \u003cem\u003eLcb. casei\u003c/em\u003e as the species designation for strain PRA205. However, the data does not allow for a conclusive assignment of PRA205 to either \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e or \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo resolve the taxonomic position of strain PRA205, two different whole genome phylogeny construction methods were used. In the core genome based phylogenetic analysis performed with the EDGAR3.0 pipeline, a dataset of eight \u003cem\u003eLacticaseibacillus\u003c/em\u003e genomes was used, where strain PRA205 formed a separate branch close to \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e and \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e, suggesting close but distinct relatedness (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eAnalysis with PhyloPhlAn 3.0 included a larger dataset of 25 \u003cem\u003eLacticaseibacillus\u003c/em\u003e genomes. The resulting topology of the ML tree identified three clusters. The first cluster, referred to as \u003cem\u003eLcb. styriensis\u003c/em\u003e, consisted of \u003cem\u003eLcb. styriensis\u003c/em\u003e DSM 116297\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eLcb. styriensis\u003c/em\u003e MRD1975, and \u003cem\u003eLcb. casei\u003c/em\u003e strain LC5 which should be re-attributed to \u003cem\u003eLcb. styriensis\u003c/em\u003e. The second cluster, referred to as \u003cem\u003eLcb. casei\u003c/em\u003e, included the only two genomes present in GenBank (last accessed March 2025), namely those of \u003cem\u003eLcb. casei\u003c/em\u003e strains DSM 20011\u003csup\u003eT\u003c/sup\u003e and MGB0470. The third cluster, referred to as \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e, including \u003cem\u003eLcb. casei\u003c/em\u003e strains N and FBL6, in addition to the two strains previously described as \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e such as DSM 115425\u003csup\u003eT\u003c/sup\u003e and DSM 115424\u003csup\u003eT\u003c/sup\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Also in this case, strains FBL6 and N, previously annotated as \u003cem\u003eLcb. casei\u003c/em\u003e, could be re-attributed to the novel species \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e. According to [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e branched alone. Finally, strain PRA205 was closely related but distinct from \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e, \u003cem\u003eLcb. styriensis\u003c/em\u003e, \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e, \u003cem\u003eLcb. zeae\u003c/em\u003e, and \u003cem\u003eLcb. casei\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eBased on these findings, strain PRA205 was strongly related to \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e but cannot be univocally attributed to this species.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Functional analysis and genome comparison\u003c/h2\u003e\u003cp\u003eGenome annotation of PRA205 predicted 2977 CDS, 53 tRNA, 6 rRNA, and 1 tmRNA (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The tRNAs, covering all 20 amino acids, were scattered across the contigs, with two clusters on contig 1 (772 bp, 8 tRNA) and contig 21 (\u0026sim;2,293 bp,14 tRNA). The CDS counts in PRA205 and in \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e were higher than in other related species (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Out of 2977 CDS, 1544 (51.8%) were assigned to 23 different KEGG pathways. The most represented KOs were carbohydrate metabolism (282, 18.35%), protein families: genetic information processing (13.66%), and protein families: signaling and cellular processes (11.06%), respectively (Supplementary \u003cb\u003eTable S7\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eOrthology relationships among PRA205 and five related strains were assessed using the EDGAR3.0 pipeline. The pan-genome included 3,930 CDS: 2,136 core genes (54.4%), 789 strain-specific genes (20.1%), and 1,005 dispensable genes (25.6%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The large core genome suggests a strong conservation of essential functions across all strains despite their attribution to different species. COG category distribution across demonstrated that most orthogroups were non annotated, followed by orthogroups classified as carbohydrate transport and metabolism in core, dispensable, and singletons genomes (Supplementary \u003cb\u003eTable S8\u003c/b\u003e). \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e had the highest number of unique genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), while PRA205 displayed 108 singletons, 105 of which are unclassified. The reaming three singletons are involved in unclassified \u0026ndash; genetic information processing (1), environmental information processing (1), and glycan biosynthesis and metabolism (1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eMobile genetic elements (MGEs) contribute to genome plasticity and horizontal gene transfer (HGT) [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Analysis with MobileOG-db revealed that PRA205 displayed a similar MGE profile to \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e, and a greater MGEs number than \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e DSM 115425\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eLcb. styriensis\u003c/em\u003e DSM 116297\u003csup\u003eT\u003c/sup\u003e, and \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e DSM 116376\u003csup\u003eT\u003c/sup\u003e. Most of 147 MGE-associated regions in the PRA205 genome were involved in integration/excision, replication/recombination/repair, and phage-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Biosafety, prophage, and CRISPR-Cas assessments\u003c/h2\u003e\u003cp\u003eAccording to EFSA [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], the genome of the PRA205 strain was checked for the presence of AMR genes. All the results of \u003cem\u003ein silico\u003c/em\u003e biosafety assessments were summarized in Supplementary \u003cb\u003eTable S9\u003c/b\u003e. Both AMRFinder and ResFinder analyses did not reveal AMR genes, indicating that strain PRA205 can be considered safe in relation to the potential dissemination of AMR genes. Neither virulence nor pathogenic genes were detected. PathogenFinder showed a probability of being a human pathogen of 0.092 (above 1), while the probiotic potential risk score (PPRS) was 2.00. These results are in accordance with the QPS status of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. strain PRA205.\u003c/p\u003e\u003cp\u003eNo plasmid replication initiation proteins were detected in the genome of strain PRA205 using PlasmidFinder v2.1, suggesting the absence of plasmids. Similarly, no plasmid sequences were found in \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e, in \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e, or in the closely relative species \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e DSM 115425\u003csup\u003eT\u003c/sup\u003e, \u003cem\u003eLcb. styriensis\u003c/em\u003e DSM 116297\u003csup\u003eT\u003c/sup\u003e, and \u003cem\u003eLacticaseibacillus zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e DSM 116376\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAt least one prophage sequence was detected in each of analyzed genomes (Supplementary \u003cb\u003eTable S10\u003c/b\u003e). All these prophages belonged to the \u003cem\u003eSiphoviridae\u003c/em\u003e family [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e], which is the most prevalent prophage family infecting \u003cem\u003eLacticaseibacillus\u003c/em\u003e strains [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. Specifically, strain PRA205 harbored three prophage regions, including one transposable element, whereas \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e had only one.\u003c/p\u003e\u003cp\u003eAnalysis of CRISPR-Cas systems showed that PRA205 genome displayed two putative \u003cem\u003ecas\u003c/em\u003e clusters (13.2 kb and 3.5 kb) but none CRISPR arrays (Supplementary \u003cb\u003eTables S12\u003c/b\u003e and \u003cb\u003eS13\u003c/b\u003e). The absence of complete CRISPR-Cas systems may be due to assembly limitations from short-read Illumina sequencing, which struggles to resolve repetitive regions. Conversely, \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e and \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e DSM 115425\u003csup\u003eT\u003c/sup\u003e had one CRISPR array each, but no \u003cem\u003ecas\u003c/em\u003e genes. \u003cem\u003eLcb. styriensis\u003c/em\u003e lacked both CRISPR elements and \u003cem\u003ecas\u003c/em\u003e genes. Complete CRISPR-Cas systems were found only in \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e and \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Supplementary \u003cb\u003eTables S11\u003c/b\u003e and \u003cb\u003eS12\u003c/b\u003e). CRISPR-Cas systems can be lost under phage infection pressure [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. The high abundance of phage-related MGEs in PRA205 could be linked to the absence of a functional CRISPR-Cas defense, supporting the hypothesis that MGEs play a dominant role in shaping its genome evolution.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOverview of genomic features, Glycosil transferase (GT) genes, and CRISPR-Cas systems in the genomes of strain PRA205 and its close relatives. Abbreviation s: Lh, \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e DSM 115425\u003csup\u003eT\u003c/sup\u003e; Lph, \u003cem\u003eLcb. parahelsenbergenesis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e; Ls, \u003cem\u003eLcb. styriensis\u003c/em\u003e DSM 116297\u003csup\u003eT\u003c/sup\u003e; Lzs, \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e DSM 116376\u003csup\u003eT\u003c/sup\u003e; Lzz, \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e; GT: Glycosyl transferase; na, not applicable.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFeatures\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePRA 205\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLzz\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLh\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLph\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLzs\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSize (Mb)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.95\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.91\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.91\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGC (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e47.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e47.93\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e47.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e48.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e48.03\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCDS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2977\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2961\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2906\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2798\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2792\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2777\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003erRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003etRNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e59\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT35\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGT8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGH38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003earaA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCRISPR region\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN\u0026deg;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ecas\u003c/em\u003e gene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN\u0026deg;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eType\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eboth CAS putative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCAS-TypeIC,\u003c/p\u003e\u003cp\u003eCAS putative,\u003c/p\u003e\u003cp\u003eCAS putative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ena\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNa\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ena\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eCAS-TypeIC,\u003c/p\u003e\u003cp\u003eCAS-TypeIIA_1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Search for functional traits-associated genes\u003c/h2\u003e\u003cp\u003eTo identify genes potentially linked to probiotic traits, we first analyzed the genome of strain PRA205 for biosynthetic gene clusters (BGCs), putative bacteriocins, and carbohydrate-active enzymes (CAZymes).\u003c/p\u003e\u003cp\u003eantiSMASH 8.0 predicted five BGCs in PRA205 genome, namely three ribosomally encoded and post-translationally modified peptides (RiPP)-like gene clusters, one linear azol(in)e-containing peptides gene clusters, and one terpene precursor (Supplementary \u003cb\u003eTable S12\u003c/b\u003e). The three RiPP-like clusters resembled the \u003cem\u003eEscherichia coli\u003c/em\u003e BGC encoding microcin L (similarity 0.44), the \u003cem\u003eLactobacillus gasseri\u003c/em\u003e BGC encoding gassericin T/E (similarity 0.53), and the \u003cem\u003eEnterococcus faecium\u003c/em\u003e BGC encoding enterocin A (0.56 similarity), respectively. Among closely related strains, \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e exhibited the most similar BGCs profile to PRA205 (Supplementary \u003cb\u003eTable S12\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eBAGEL5 analysis identified four areas of interest (AOIs), three of which overlapped with those predicted by antiSMASH results. The fourth AOI (PFCGCLEC_1.10.AOI_01) contained several genes involved in class IIc bacteriocin production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), supporting a potential antimicrobial role of PRA205. In particular, the cluster included two bacteriocins: the bacteriocin at orf00033 was identified as a ComC/BlpC family leader-containing pheromone/ bacteriocin (WP_138130143.1) from \u003cem\u003eLcb. zeae\u003c/em\u003e, and the adjacent downstream ORF encoded Enterocin X beta chain, identified as a bacteriocin leader domain-containing protein commonly reported in many \u003cem\u003eLacticaseibacillus\u003c/em\u003e strains [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe search for CAZymes revealed that 54.40% of the carbohydrate metabolism-related genes were annotated as glycosyltransferases (GTs) and 41.33% as glycoside hydrolases (GHs) in PRA205 genome (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A similar distribution was found in \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e (GT: 52.11%, GH: 42.25%), \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e (GT: 54.17%, GH: 40.28%), and \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e (GT: 54.29%, GH: 41.43%). In contrast, \u003cem\u003eLcb. styriensis\u003c/em\u003e and \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e showed slightly lower GT levels (48.61% and 47.89%, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGHs have a critical role in the metabolism of complex carbohydrates as they are mainly used to hydrolyze glycosidic bonds or carbohydrates and glycosidic parts between carbohydrates [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Among GHs, glycoside hydrolases of family 38 are Class II α-mannosidases involved in the hydrolysis of terminal, non-reducing α-D-mannose residues in α-D-mannosides. \u003cem\u003eLcb. styriensis\u003c/em\u003e was the only \u003cem\u003eLacticaseibacillus\u003c/em\u003e species possessing a gene encoding α-mannosidase and therefore positive for α-mannosidase activity [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This gene was missing in strain PRA205 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGTs catalyze glycosidic bond formation between phospho-activated sugars and various acceptors. These enzymes are involved in stress response, biofilm formation, and the biosynthesis of exopolysaccharides (EPS), which contribute to probiotic traits such as colonization and persistence [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. PRA205 contained 41 GT genes from 9 families, 19 of which belonged to family 4 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). GT families 2 and 4 were most prevalent across all strains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eNotably, the GT8 gene, proposed as a molecular marker to distinguish \u003cem\u003eLcb. casei\u003c/em\u003e from \u003cem\u003eLcb. zeae\u003c/em\u003e [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e], was present in PRA205 (96.44% identity), \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e, \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e, \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e, and \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e but absent in \u003cem\u003eLcb. styriensis\u003c/em\u003e and \u003cem\u003eLcb. casei\u003c/em\u003e DSM 20011\u003csup\u003eT\u003c/sup\u003e. Analysis of pairwise sequence similarity revealed that GT8 nucleotide sequences are poorly conserved among these strains (Supplementary \u003cb\u003eTable\u0026nbsp;13\u003c/b\u003e), suggesting that this gene may serve as a target for PRA205-specific primer design.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003earaA\u003c/em\u003e gene, which encodes L-arabinose isomerase, was detected in PRA205 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This gene is also present in \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e, \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e, and \u003cem\u003eLcb. styriensis\u003c/em\u003e but absent in \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Its presence suggests that PRA205 may metabolize arabinose, a potentially valuable probiotic trait [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong the probiotic-associated traits, acid tolerance and adhesion capacity are of relevance. In our previous study, strain PRA205 was shown to withstand low pH conditions, exhibiting strong auto-aggregation and high cell surface hydrophobicity, two properties which are predictive of adhesion capability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Consistent with these phenotypes, PRA205 genome contained multiple genes involved in acid stress response, including those encoding the F-type proton pump (\u003cem\u003eatpA\u003c/em\u003e\u0026ndash;\u003cem\u003eatpH\u003c/em\u003e), Na+:H\u0026thinsp;+\u0026thinsp;antiporter (\u003cem\u003enpaA\u003c/em\u003e), and ornithine decarboxylase (\u003cem\u003eodcI\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These systems contribute to intracellular pH homeostasis by coupling ATP hydrolysis to proton extrusion [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNotably, PRA205 lacks genes of the arginine deiminase (ADI) pathway as well as tyrosine-, lysine-, and histidine-decarboxylases. This indicates that, although genetically adapted to cope with acidic stress, the strain does not produce harmful biogenic amines, with the possible exception of putrescine (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The latter is generated via ornithine decarboxylation, a process generally coupled with amino acid transport by antiporter proteins [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]. This pathway contributes to cytosolic alkalinization and proton motive force generation, which can be exploited for stress resistance and ATP production. Consistently, PRA205 harbors both the ornithine decarboxylase encoding gene \u003cem\u003eodcI\u003c/em\u003e and \u003cem\u003epotE\u003c/em\u003e, encoding a substrate/product exchanger (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) [\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough PRA205 also tolerates bile acids [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], another stress encountered in gastrointestinal transit, its genome lacks the cholylglycine hydrolase gene (\u003cem\u003ebsh\u003c/em\u003e). This finding supports the view that bile salt tolerance is a polygenic trait determined by genes other than \u003cem\u003ebsh\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eLastly, the adhesive phenotype of PRA205 is supported by several genes implicated in binding to intestinal epithelial cells, mucin, and fibronectin (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eProbiotic-related genes found in \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhenotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGene description\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePosition (Contig; coordinates)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAcid tolerance\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02899\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF-type H+-transporting ATPase epsilon chain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_19; 5940..6371\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02900\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpD\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF-type H+-transporting ATPase subunit beta\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_19; 6386..7852\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02901\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpG\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF-type H+-transporting ATPase gamma chain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_19; 8028..8951\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02902\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF-type H+-transporting ATPase subunit alpha\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_19; 8963..10492\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02903\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpH\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF-type H+-transporting ATPase subunit delta\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_19; 10516..11061\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02904\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpF\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF-type H+-transporting ATPase subunit b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_19; 11048..11536\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02905\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpE\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF-type H+-transporting ATPase subunit c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_19; 11571..11783\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02906\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eatpB\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eF-type H+-transporting ATPase subunit a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_19; 11806..12516\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02705\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003enapA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNa(+)/H(+) antiporter (CPA2 family)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_14; 30646..31800\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02301\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eodcI\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eInducible ornithine decarboxylase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_09; 39301..41391\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02602\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003epotE\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOrnithine-Putrescine antiporter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_12; 23228..24583\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAdhesion\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02966\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eltasS\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLipoteichoic acid synthase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_21; 12038..14134\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_01247\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003etuf\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eElongation factor Tu\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_03; 244598..245788\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02369\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003etsf\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eElongation factor Ts\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_09; 106024..106905\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_00024\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003efusA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eElongation factor G\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_01; 28760..30862\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02853\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003edltA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eD-alanine\u0026ndash;D-alanyl carrier protein ligase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_17; 33208..34728\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02962\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003ecpoA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAlpha-galactosylglucosyldiacylglycerol synthase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_21; 7736..8764\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_01140\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003efbpA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFibronectin-binding protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_03; 126400..128100\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_00218\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHypothetical protein with\u003c/p\u003e\u003cp\u003eMucBP domain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_01; 221391..222581\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_00997\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHypothetical protein with\u003c/p\u003e\u003cp\u003eMucBP domain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_02; 377598..379850\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_02256\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHypothetical protein with\u003c/p\u003e\u003cp\u003eMucBP domain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_08; 142726..143214\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_00943\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eoppA\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOligopeptide-binding protein OppA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_02; 332806..334416\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePFCGCLEC_00350\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003egroL\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003echaperonin GroEL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eContig_01; 356658..358295\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Reconstruction of the proteolytic system\u003c/h2\u003e\u003cp\u003eAmong the functional traits of strain PRA205, the ability to release antihypertensive peptides from caseins has been well documented [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To elucidate the genetic basis of this phenotype, we analyzed the distribution protease and peptidase-encoding genes in PRA205 and 12 related \u003cem\u003eLacticaseibacillus\u003c/em\u003e species (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDistribution of proteinase and peptidases in the proteolytic system of \u003cem\u003eLacticaseibacillus\u003c/em\u003e species. The number of identified genes is indicated. MEROPS families are indicated. Color shading shows absence of a gene (white), a single gene (yellow) or multiple genes (green). The Locus_Tag codes of the genes can be found in Supplementary \u003cb\u003eTable S14\u003c/b\u003e. Abbreviations: Lca: \u003cem\u003eLcb. casei\u003c/em\u003e DSM 20011\u003csup\u003eT\u003c/sup\u003e; Lpa: \u003cem\u003eLcb. paracasei\u003c/em\u003e BL23; Lc: \u003cem\u003eLcb. chiayiensis\u003c/em\u003e FBL7; LrT: \u003cem\u003eLcb. rhamnosus\u003c/em\u003e DSM 20021\u003csup\u003eT\u003c/sup\u003e; Lr GG: \u003cem\u003eLcb. rhamnosus\u003c/em\u003e GG; Lzz: \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. zeae DSM 20178\u003csup\u003eT\u003c/sup\u003e; PRA, \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. strain PRA205; Lzs: \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e 116376T; Lh: \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e DSM 115425\u003csup\u003eT\u003c/sup\u003e; Lph; \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e DSM 116105\u003csup\u003eT\u003c/sup\u003e; Ls: \u003cem\u003eLcb. styriensis\u003c/em\u003e DSM 116297\u003csup\u003eT\u003c/sup\u003e; Lhel: \u003cem\u003eL. helveticus\u003c/em\u003e DSM 20075\u003csup\u003eT\u003c/sup\u003e and Ld: \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003elactis\u003c/em\u003e DSM 20072\u003csup\u003eT\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"17\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c16\" colnum=\"16\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c17\" colnum=\"17\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePeptidase\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFamily\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSubstrate / Annotation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLca\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLpa\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLc\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eLrT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eLrGG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eLzz\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003ePRA\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003eLzs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003eLh\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003eLph\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c15\"\u003e\u003cp\u003eLs\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c16\"\u003e\u003cp\u003eLhel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c17\"\u003e\u003cp\u003eLd\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProteinase\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eCell-wall bound proteinase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePrtP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eS8-A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePrtR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePrtH\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePrtB\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePrtM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePeptidases\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c15\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c16\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c17\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eEndopeptidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepE/PepG\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC1-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(X)m|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepF\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM3-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(X)m|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepO\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e(X)m|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eAminopeptidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepA\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGlu/Asp|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepC\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC1-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepM\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM24-A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMet|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepN\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepS\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eDipeptidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepD\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eC69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX|X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepV\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM20-A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX|X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTripeptidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM20-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX|X-X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eProline peptidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepI\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePro|X-(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePro|X\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepL\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLeu|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepP\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM24-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX|Pro-(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepQ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eM24-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX|Pro\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePepX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eS15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX-Pro|(X)n\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c16\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c17\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eConsistent with previous findings in \u003cem\u003eL. helveticus\u003c/em\u003e and \u003cem\u003eL. delbrueckii\u003c/em\u003e subsp. \u003cem\u003elactis\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], protease and peptidase genes were conserved across all genomes analyzed. Core peptidases\u0026mdash;PepC, PepN, PepM, and proline-specific peptidases PepR, PepI, PepX, and PepQ\u0026mdash;were present in single copies in all species. Gene encoding endopeptidases (PepO, PepF) and dipeptidases (PepV, PepD) were found in multiple copies in some genomes.\u003c/p\u003e\u003cp\u003eStrain PRA205 exhibited a complete and enriched proteolytic profile, with two copies of \u003cem\u003ePepO\u003c/em\u003e, \u003cem\u003ePepF\u003c/em\u003e, and \u003cem\u003ePepV\u003c/em\u003e and four of \u003cem\u003ePepD\u003c/em\u003e. A similar profile was found in \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003ezeae\u003c/em\u003e DSM 20178\u003csup\u003eT\u003c/sup\u003e. In contrast, \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e, \u003cem\u003eLcb. huelsenbergensis\u003c/em\u003e, \u003cem\u003eL. styriensis\u003c/em\u003e, and \u003cem\u003eLcb. zeae\u003c/em\u003e subsp. \u003cem\u003esilagei\u003c/em\u003e had only one PepV-encoding gene.\u003c/p\u003e\u003cp\u003eLike the thermophilic species \u003cem\u003eL. helveticus, Lacticaseibacillus\u003c/em\u003e sp. PRA205 was well-equipped to degrade proline-rich proteins like caseins as two catabolic pathways were identified in its genome: (i) PepX/PepQ, in which PepX cleaves X-Pro dipeptides, followed by PepQ-mediated hydrolysis; and (ii) PepI/PepN, where PepP releases Pro-Y-Z tripeptides, and PepI cleaves the N-terminal proline [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]. In contrast, mesophilic LAB such as \u003cem\u003eL. lactis\u003c/em\u003e lack PepI and rely solely on the PepX/PepQ route [\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn PRA205 genome, two candidate genes encoding for a X-prolyl dipeptidyl aminopeptidase (EC 3.4.14.11) were identified: \u003cem\u003epepX_1\u003c/em\u003e (PFCGCLEC_02304; contig 9) and \u003cem\u003epepX_2\u003c/em\u003e (PFCGCLEC_02630; contig 12). Alignment of deduced amino acid sequences with the prototype \u003cem\u003eL. lactis\u003c/em\u003e PepX (A0A0V8AJV2) revealed that pepX_1 contained all domains characteristic of the S15 family, namely the PepX N-terminal domain (PF09168), the catalytic S15 domain (PF02129), and the C-terminal non-catalytic domain (PF08530) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, pepX_2 lacked the N-terminal domain and resembled a CocE/Serine esterase (Q45289), indicating a misannotation. Furthermore, synteny analysis identified \u003cem\u003eglnR\u003c/em\u003e and \u003cem\u003eglnA\u003c/em\u003e genes 5\u0026rsquo;-downstream of \u003cem\u003epepX_1\u003c/em\u003e gene according to the operon structure found in the close relative \u003cem\u003eLcb. rhamnosus\u003c/em\u003e [\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese results confirm that PRA205 harbors a functional PepX-encoding gene (\u003cem\u003epepX_1\u003c/em\u003e), supporting its proteolytic activity and the functional ability to degrade caseins, releasing BPs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Expression analysis of the \u003cem\u003epepX\u003c/em\u003e gene\u003c/h2\u003e\u003cp\u003ePeptide-rich media downregulate proteolytic genes in \u003cem\u003eLacticaseibacillus\u003c/em\u003e spp., mediated mainly by the CodY regulator, which responds to intracellular BCAA levels by binding conserved motifs in promoters of nitrogen metabolism genes (e.g., \u003cem\u003eprt, pepN, pepC, opp-pepO1\u003c/em\u003e) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e, \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e]. In \u003cem\u003eL. lactis\u003c/em\u003e, CodY represses \u003cem\u003epepX\u003c/em\u003e in the presence of peptides, while in \u003cem\u003eL. delbrueckii subsp. bulgaricus\u003c/em\u003e its regulation is CodY-independent [\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e, \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn strain PRA205, \u003cem\u003eprt\u003c/em\u003e was completely silenced in MRS medium but expressed in a milk-like medium [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To investigate the regulation of \u003cem\u003epepX\u003c/em\u003e expression under these two conditions, both RT-PCR and RT-qPCR were performed. RT-PCR showed \u003cem\u003epepX\u003c/em\u003e transcription in both media, with stronger signals observed in the milk-like medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Consistently, RT-qPCR confirmed that \u003cem\u003epepX_1\u003c/em\u003e was constitutively expressed in MRS medium but significantly upregulated in the milk-like medium, indicating induction under amino acid-limited conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003eTaken together, these results suggest that, unlike \u003cem\u003eprt\u003c/em\u003e, \u003cem\u003epepX\u003c/em\u003e in PRA205 is not fully repressed in peptide-rich environments, pointing to a regulatory mechanism that is at least partially independent of CodY regulator.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.8 PepX partial purification\u003c/h2\u003e\u003cp\u003eTo link genomic data to enzymatic function, the prolyl-dipeptidyl aminopeptidase activity in \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205 was assessed on cytoplasmic extract obtained from cells grown on milk by using the specific substrate Gly-Pro-pNA. The specific prolyl-dipeptidyl aminopeptidase activity was found to be 2.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3 U/mg of proteins. Large variation has been found for the prolyl-dipeptidyl aminopeptidase activity of \u003cem\u003eLacticaseibacilluss\u003c/em\u003e spp. strains. Depending on the strain, the prolyl-dipeptidyl aminopeptidase activity ranged between 2.4 to 50 U/mg of proteins [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePurification of PepX from the cytoplasmic extract of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205 was carried out as reported in [\u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Gel filtration chromatography (Supplementary \u003cb\u003eFig. S2\u003c/b\u003e) revealed that PepX from strain PRA205 exists predominantly as a monomer with an estimated molecular mass of approximately 80 kDa. In contrast, PepX had been previously purified as a dimer of about 170\u0026ndash;200 kDa in \u003cem\u003eL. acidophilus\u003c/em\u003e, \u003cem\u003eL. delbrueckii\u003c/em\u003e, \u003cem\u003eL. curvatus\u003c/em\u003e, \u003cem\u003eL. helveticus\u003c/em\u003e, and \u003cem\u003eL. lactis\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e, \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. However, in several strains of \u003cem\u003eL. helveticus\u003c/em\u003e, \u003cem\u003eL. delbrueckii\u003c/em\u003e as well as \u003cem\u003eLcb. casei\u003c/em\u003e PepX was found active in a monomeric form of about 70\u0026ndash;90 kDa [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The purification details of PepX from \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205 are summarized in the Supplementary \u003cb\u003eTable S15\u003c/b\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.9 PepX characterization\u003c/h2\u003e\u003cp\u003eTo define the biochemical characteristics of the purified enzyme, the effect of pH, temperature, and inhibitors was assessed by using the specific substrate Gly-Pro-pNA.\u003c/p\u003e\u003cp\u003eThe enzyme showed high activity over a wide pH range, from 6.0 to 8.0 with optimum activity at pH 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Values of pH optima for PepX between 6.0 and 8.0 have been already published [\u003cspan additionalcitationids=\"CR106\" citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. A significant reduction of about 64% of PepX activity was observed at pH value of 9. Interesting, at the pH value of 4.5 (as observed in fermented dairy products) the enzyme retained about the 40% of their original activity.\u003c/p\u003e\u003cp\u003eThe temperature dependence of \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205 PepX activity was assessed at 5 and 42\u0026deg;C, which are the typical temperature of yoghurt fermentation and fermented dairy food storage, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Data showed that, at the typical yoghurt fermentation temperature of 42\u0026deg;C, PepX retained approximately 77% of its activity compared with the optimal temperature of 37\u0026deg;C. Similarly, the residual activity at 5\u0026deg;C was 47%. These data implicated that PepX is still active during the yoghurt production process and the low temperatures during the cold storage.\u003c/p\u003e\u003cp\u003eThe assays carried out with the partial purified enzyme in the presence of PMSF and EDTA confirmed the serine-peptidase nature of the enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). PMSF was able to decrease the enzymatic activity of more than 40% at 1 mmol/L concentration, whereas the enzymatic activity was completely abolished at PMSF concentrations of 5 and 10 mmol/L. EDTA at 1 mmol/L had no effect on the enzymatic activity whereas a residual activity of 40% was detected at 10 mmol/L concentration, suggesting the requirement of metals for the catalytic activity of PepX.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.10 Degradation of bioactive peptides by partially purified PepX\u003c/h2\u003e\u003cp\u003eConsidering the cleavage specificity of PepX and the presence of proline residues in many dairy-derived BPs, the ability of PepX to hydrolyze selected BPs was evaluated. Selected BPs were well-known effective inhibitors of the enzymes ACE and di-peptidyl-peptidase-IV (DPP-IV). The lactotripeptides VPP and IPP (derived from the hydrolysis of β-casein) were potent inhibitors of ACE activity and have been effective \u003cem\u003ein vivo\u003c/em\u003e in reducing blood pressure both in rats and human [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These two peptides have been identified in several fermented dairy products such as yogurt and cheeses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The other peptides such as LPPT, APFPE, PPF, and IPPL have been identified as anti-diabetic peptides being potent DPP-IV-inhibitors [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs reported in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, all the tested peptides were cleaved by partially purified PepX although with different extents. The highest degradation rate was observed for PPF, VPP and APFPE whereas the lowest one for IPP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, this present study provided three main findings. First, phylogenomic analyses indicated that strain PRA205 is closely related yet distinct from \u003cem\u003eLcb. parahuelsenbergenis\u003c/em\u003e. Owing to the limited availability of isolates and biochemical data, a conservative taxonomic position was adopted, leaving its species attribution unresolved. Furthermore, several LCG strains in public databases appear misclassified, underscoring the need for their taxonomic revision.\u003c/p\u003e\u003cp\u003eSecond, \u003cem\u003ein silico\u003c/em\u003e biosafety assessment, performed according to EFSA guidelines [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], confirmed the GRAS status of PRA205, demonstrating the absence of transferable antibiotic-resistance genes and virulence factors. The genome harbors genes associated with probiotic traits, including bacteriocin production, acid stress tolerance, and cell-to-cell interactions. Strain PRA205 carries a complete proteolytic system, which underlies its strong casein-hydrolyzing capacity and the consequent release of BPs.\u003c/p\u003e\u003cp\u003eThird, the gene expression profile and biochemical properties of PepX, a key enzyme within the proteolytic system, were established. Remarkably, the partial casitone-independent regulation of \u003cem\u003epepX_1\u003c/em\u003e gene may contribute to the pronounced proteolytic aptitude of PRA205. The resulting PepX enzyme was identified as a serine-protease of approximately 80 kDa, with activity characterized across different temperatures and pH values. This enzyme retained activity under low temperature and low pH conditions, revealing a dual role both in the formation and degradation of BPs during milk fermentation and dairy product manufacture and storage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe project was funded by the grant FAR2022 from the Department of Life Sciences (University of Modena and Reggio Emilia, Italy) (Title: Integration of Genomics and Metabolomics for the Characterization of the Proteolytic System Responsible for the Production of Bioactive Peptides in \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e PRA205 - IGEM-PEP). The project was also partially funded under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4\u0026ndash;Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union\u0026ndash;NextGenerationEU, Award Number: Project Code CN_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUP E93C22001090001, Project Title \u0026ldquo;National Biodiversity Future Center\u0026ndash;NBCF\u0026rdquo;.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: DT and LS; methodology: DT and LS; resources: DT and LS; formal analysis: MC, AC, LS, LB, and DT; investigation: MC, AC, AB, GZ and LS; data curation: MC, AC, LB, and DT; writing\u0026mdash;original draft preparation: LS and MC; writing\u0026mdash;review and editing: MC, AC, AB, GZ, LB, DT, and LS; funding acquisition: DT and LS. All authors have read and agreed to the present version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThis Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JBQQCW000000000.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCastellone V, Bancalari E, Rubert J, Gatti M, Neviani E, Bottari B (2021) Eating fermented: health benefits of LAB-fermented foods. Foods 10:2639. https://doi.org/10.3390/foods10112639\u003c/li\u003e\n\u003cli\u003eLeeuwendaal NK, Stanton C, O\u0026rsquo;Toole PW, Beresford TP (2022) Fermented foods, health and the gut microbiome. Nutrients 14:1527. https://doi.org/10.3390/nu14071527\u003c/li\u003e\n\u003cli\u003eJahn LJ, Rekdal VM, Sommer MO (2023) Microbial foods for improving human and planetary health. Cell 186:469\u0026ndash;478. https://doi.org/10.1016/j.cell.2022.12.002\u003c/li\u003e\n\u003cli\u003eSaleem GN, Gu R, Qu H, Bahar Khaskheli G, Rajput IR, Qasim M, Chen X (2024) Therapeutic potential of popular fermented dairy products and its benefits on human health. Front Nutr 11:1328620. https://doi.org/10.3389/fnut.2024.1328620\u003c/li\u003e\n\u003cli\u003eGobbetti M, Stepaniak L, De Angelis M, Corsetti A, Di Cagno R (2002) Latent bioactive peptides in milk proteins: proteolytic activation and significance in dairy processing. Crit Rev Food Sci Nutr 42:223\u0026ndash;239. https://doi.org/10.1080/10408690290825538 \u003c/li\u003e\n\u003cli\u003eKorhonen H, Pihlanto A (2006) Bioactive peptides: production and functionality. Int Dairy J 16:945\u0026ndash;960. https://doi.org/10.1016/j.idairyj.2005.10.012\u003c/li\u003e\n\u003cli\u003eHafeez, Z., Cakir-Kiefer, C., Roux, E., Perrin, C., Miclo, L., \u0026amp; Dary-Mourot, A. (2014). Strategies of producing bioactive peptides from milk proteins to functionalize fermented milk products. Food Research International, 63, 71-80. https://doi.org/10.1016/j.foodres.2014.06.002\u003c/li\u003e\n\u003cli\u003eTagliazucchi D, Martini S, Solieri L (2019) Bioprospecting for bioactive peptide production by lactic acid bacteria isolated from fermented dairy food. Fermentation 5:96. https://doi.org/10.3390/fermentation5040096\u003c/li\u003e\n\u003cli\u003eUlug SK, Jahandideh F, Wu J (2021) Novel technologies for the production of bioactive peptides. Trends Food Sci Technol 108:27\u0026ndash;39. https://doi.org/10.1016/j.tifs.2020.11.014\u003c/li\u003e\n\u003cli\u003eKuhfeld RF, Eshpari H, Atamer Z, Dallas DC (2024) A comprehensive database of cheese-derived bitter peptides and correlation to their physical properties. Crit Rev Food Sci Nutr 64(27):10105\u0026ndash;10119. https://doi.org/10.1080/10408398.2023.2220792\u003c/li\u003e\n\u003cli\u003eKelly CD (1932) The influence of certain lactic acid streptococci on the chemical changes in cheddar cheese during ripening. N Y State Agric Exp Stn Bull 200:3\u0026ndash;28.\u003c/li\u003e\n\u003cli\u003eSmit G, Smit BA, Engels WJM (2005) Flavor formation by lactic acid bacteria and biochemical flavor profiling of cheese products. FEMS Microbiol Rev 29:592-610. https://doi.org/10.1016/j.fmrre.2005.04.002\u003c/li\u003e\n\u003cli\u003eMcSweeney PLH, Sousa MJ (2000) Biochemical pathways for the production of flavour compounds in cheeses during ripening: a review. Lait 80(3): 293\u0026ndash;324. https://doi.org/10.1051/lait:2000127\u003c/li\u003e\n\u003cli\u003eAnastasiou R, Kazou M, Georgalaki M, Aktypis A, Zoumpopoulou G, Tsakalidou E (2022) Omics approaches to assess flavor development in cheese. Foods 11(2):188. https://doi.org/10.3390/foods11020188. \u003c/li\u003e\n\u003cli\u003eGriffiths MW, Tellez AM (2013) \u003cem\u003eLactobacillus helveticus\u003c/em\u003e: the proteolytic system. Front Microbiol 4:30. https://doi.org/10.3390/foods11020188\u003c/li\u003e\n\u003cli\u003eTeusink B, Molenaar D (2017) Beyond the laboratory: realistic growth conditions define the proteolytic demand of dairy lactic acid bacteria. Curr Opin Biotechnol 31:123\u0026ndash;129. https://doi.org/10.1016/j.copbio.2014.11.010\u003c/li\u003e\n\u003cli\u003eChristensen JE, Dudley EG, Pederson JA, Steele JL (1999) Peptidases and amino acid catabolism in lactic acid bacteria. Antonie Van Leeuwenhoek 76(1):217\u0026ndash;246. \u003c/li\u003e\n\u003cli\u003eLiu M, Bayjanov JR, Renckens B, Nauta A, Siezen RJ (2010) The proteolytic system of lactic acid bacteria revisited: a genomic comparison. BMC Genomics 11:36. https://doi.org/10.1186/1471-2164-11-36\u003c/li\u003e\n\u003cli\u003eRaveschot C, Cudennec B, Coutte F, et al (2018) Production of bioactive peptides by Lactobacillus species: from gene to application. Front Microbiol 9:2354. https://doi.org/10.3389/fmicb.2018.02354\u003c/li\u003e\n\u003cli\u003eSolieri L, Bianchi A, Giudici P (2012) Inventory of non-starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture-dependent multiphasic approach. Syst Appl Microbiol 35:270\u0026ndash;277. https://doi.org/10.1016/j.syapm.2012.04.005\u003c/li\u003e\n\u003cli\u003eSolieri L, Bianchi A, Mottolese G, Lemmetti F, Giudici P (2014) Tailoring the probiotic potential of non-starter \u003cem\u003eLactobacillus \u003c/em\u003estrains from ripened Parmigiano Reggiano cheese by in vitro screening and principal component analysis. Food Microbiol 38:240\u0026ndash;249.\u003c/li\u003e\n\u003cli\u003eSolieri L, Rutella GS, Tagliazucchi D (2015) Impact of non-starter lactobacilli on release of peptides with angiotensin-converting enzyme inhibitory and antioxidant activities during bovine milk fermentation. Food Microbiol 51:108\u0026ndash;116. https://doi.org/10.1016/j.fm.2015.05.009\u003c/li\u003e\n\u003cli\u003eSolieri L, De Vero L, Tagliazucchi D (2018) Peptidomic study of casein proteolysis in bovine milk by \u003cem\u003eLactobacillus casei\u003c/em\u003e PRA205 and \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e PRA331. Int Dairy J 85:237\u0026ndash;246. https://doi.org/10.1016/j.idairyj.2018.06.011\u003c/li\u003e\n\u003cli\u003eRutella GS, Tagliazucchi D, Solieri L (2016) Survival and bioactivities of selected probiotic lactobacilli in yogurt fermentation and cold storage: New insights for developing a bi-functional dairy food. Food Microbiol 60:54\u0026ndash;61. https://doi.org/10.1016/j.fm.2016.06.001\u003c/li\u003e\n\u003cli\u003eSolieri L, Sola L, Vaccalluzzo A, Randazzo CL, Martini S, Tagliazucchi D (2022) Characterization of cell-envelope proteinases from two \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e strains isolated from Parmigiano Reggiano cheese. Biology 11:139. https://doi.org/10.3390/biology11010139\u003c/li\u003e\n\u003cli\u003eCasey MG, Meyer J (1985) Presence of X-Prolyl-Dipeptidyl-Peptidase in Lactic Acid Bacteria. J Dairy Sci 68:3212\u0026ndash;3215.\u003c/li\u003e\n\u003cli\u003eKiefer‑Partsch B, Bockelmann W, Geis A, Teuber M (1989) Purification of an X‑prolyl‑dipeptidyl aminopeptidase from the cell wall proteolytic system of \u003cem\u003eLactococcus lactis\u003c/em\u003e subsp. \u003cem\u003ecremoris\u003c/em\u003e. Appl Microbiol Biotechnol 31:75\u0026ndash;78. https://doi.org/10.1007/BF00252531\u003c/li\u003e\n\u003cli\u003eRigolet P, Mechin I, Delage MM, Chich JF (2002) The structural basis for catalysis and specificity of the X-prolyl dipeptidyl aminopeptidase from \u003cem\u003eLactococcus lactis\u003c/em\u003e. Structure 10:1383\u0026ndash;1394. https://doi.org/10.1016/S0969-2126(02)00851-1\u003c/li\u003e\n\u003cli\u003eStressler T, Eisele T, Schlayer M, Lutz-Wahl S, Fischer L (2013) Characterization of the recombinant exopeptidases PepX and PepN from \u003cem\u003eLactobacillus helveticus\u003c/em\u003e ATCC 12046 important for food protein hydrolysis. PLoS One 8:e70055. https://doi.org/10.1371/journal.pone.0070055\u003c/li\u003e\n\u003cli\u003eY\u0026uuml;ksel G, Steele JL (1996) DNA sequence analysis, expression, distribution, and physiological role of the X-prolyl dipeptidyl aminopeptidase (PepX) gene \u003cem\u003efrom Lactobacillus helveticus\u003c/em\u003e CNRZ 32. Appl Microbiol Biotechnol 44:766\u0026ndash;773. https://doi.org/10.1007/BF00178616\u003c/li\u003e\n\u003cli\u003eWakai T, Yamamoto N (2012) Antihypertensive peptides specific to \u003cem\u003eLactobacillus helveticus\u003c/em\u003e fermented milk. In: Biotechnology - Molecular Studies and Novel Applications for Improved Quality of Human Life. pp 159\u0026ndash;172\u003c/li\u003e\n\u003cli\u003eDe Angelis M, Rizzello CG, Fasano A, Clemente MG, De Simone C, Silano M, De Vincenzi M, Losito I, Gobbetti M (2010) Mechanism of degradation of immunogenic gluten epitopes from Triticum turgidum by sourdough lactobacilli and fungal proteases. Appl Environ Microbiol 76:508\u0026ndash;518. https://doi.org/10.1128/AEM.01630-09\u003c/li\u003e\n\u003cli\u003eFrancavilla R, Piccolo M, Francavilla A, Polimeno L, Semeraro F, Cristofori F, Castellaneta S, Barone M, Indrio F, Gobbetti M, De Angelis M, Rizzello CG (2017) Selected probiotic lactobacilli hydrolyze gluten peptides during simulated digestion. Appl Environ Microbiol 83:e00376-17. https://doi.org/10.1128/AEM.00376-17\u003c/li\u003e\n\u003cli\u003eBrzozowski B, Bednarski W, Staniewski B, Walczak P, Bączek T, Michalski M, Błaszczyk U (2020) Reducing immunoreactivity of gliadins using peptidases from \u003cem\u003eL. acidophilus\u003c/em\u003e. Catalysts 10:923. https://doi.org/10.3390/catal10080923\u003c/li\u003e\n\u003cli\u003eParmar H, Hati S, Panchal G, Sakure AA (2020) Purification and production of novel angiotensin I-converting enzyme (ACE) inhibitory bioactive peptides derived from fermented goat milk. Int J Pept Res Ther 26:997\u0026ndash;1011. https://doi.org/10.1007/\u003c/li\u003e\n\u003cli\u003eMatar C, Goulet J (1996) \u0026beta;-casomorphin-4 from milk fermented by a mutant of \u003cem\u003eLactobacillus helveticus\u003c/em\u003e. \u003cem\u003eInt Dairy J\u003c/em\u003e6(4):383\u0026ndash;397. https://doi.org/10.1016/0958-6946(95)00015-1\u003c/li\u003e\n\u003cli\u003eHafeez Z, Cakir-Kiefer C, Lecomte X, Miclo L, Dary-Mourot A (2019) The X-prolyl dipeptidyl-peptidase PepX of \u003cem\u003eStreptococcus thermophilus\u003c/em\u003e initially described as intracellular is also responsible for peptidase extracellular activity. J Dairy Sci 102:113\u0026ndash;123. https://doi.org/10.3168/jds.2018-14823\u003c/li\u003e\n\u003cli\u003eOtte J, Lenhard T, Flambard B, S\u0026oslash;rensen KI (2011) Influence of fermentation temperature and autolysis on ACE-inhibitory activity and peptide profiles of milk fermented by selected strains of \u003cem\u003eLactobacillus helveticus\u003c/em\u003e and \u003cem\u003eLactococcus lactis\u003c/em\u003e. Int Dairy J 21:229\u0026ndash;238. https://doi.org/10.1016/j.idairyj.2010.12.008\u003c/li\u003e\n\u003cli\u003eKilpi E, Kahala M, Steele JL, Pihlanto A, Joutsjoki V (2007) Angiotensin I converting enzyme inhibitory activity in milk fermented by wild type and peptidase deletion derivatives of \u003cem\u003eLactobacillus helveticus\u003c/em\u003e CNRZ32. Int Dairy J 17:976\u0026ndash;984. https://doi.org/10.1016/j.idairyj.2006.12.001\u003c/li\u003e\n\u003cli\u003eGrabner FM, Grabner HM, Schein H, Weidenholzer E, Busche T, R\u0026uuml;ckert-Reed C, Buchebner-Jance M (2023) \u003cem\u003eLacticaseibacillus huelsenbergensis\u003c/em\u003e sp. nov., isolated from grass silage and corn silage. Int J Syst Evol Microbiol 73:1\u0026ndash;10. https://doi.org/10.1099/ijsem.0.006049\u003c/li\u003e\n\u003cli\u003eGrabner FM, Grabner HM, Schein H, Schrank A, T\u0026ouml;glhofer M, Weidenholzer E, R\u0026uuml;ckert-Reed C, Busche T, Buchebner-Jance M (2024) \u003cem\u003eLacticaseibacillus parahuelsenbergensis\u003c/em\u003e sp. nov., \u003cem\u003eLacticaseibacillus styriensis\u003c/em\u003e sp. nov. and \u003cem\u003eLacticaseibacillus zeae\u003c/em\u003e subsp. \u003cem\u003esilagei \u003c/em\u003esubsp. nov., isolated from different grass and corn silage. Int J Syst Evol Microbiol 74:006441. https://doi.org/10.1099/ijsem.0.006441\u003c/li\u003e\n\u003cli\u003eAgolino G, Cristofolini M, Vaccalluzzo A, Tagliazucchi D, Cattivelli A, Pino A, Randazzo CL (2025) Genome mining and characterization of two novel \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e probiotic candidates with bile salt hydrolase activity. Biomolecules 15:86. https://doi.org/10.3390/biom15010086\u003c/li\u003e\n\u003cli\u003eChen S (2023) Ultrafast one-pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta 2:e107. https://doi.org/10.1002/imt2.107\u003c/li\u003e\n\u003cli\u003eBlanco-M\u0026iacute;guez A, Beghini F, Cumbo F, McIver LJ, Thompson KN, Zolfo M, Manghi P, Dubois L, Huang KD, Thomas AM, et al (2023) Extending and improving metagenomic taxonomic profiling with uncharacterized species using MetaPhlAn 4. Nat Biotechnol 41:1633\u0026ndash;1644. https://doi.org/10.1038/s41587-023-01688-w\u003c/li\u003e\n\u003cli\u003ePrjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A (2020) Using SPAdes De Novo Assembler. Curr Protoc Bioinform 70:e102. https://doi.org/10.1002/cpbi.102\u003c/li\u003e\n\u003cli\u003eMikheenko A, Prjibelski A, Saveliev V, Antipov D, Gurevich A (2018) Versatile genome assembly evaluation with QUAST-LG. Bioinformatics 34:i142\u0026ndash;i150. https://doi.org/10.1093/bioinformatics/bty266\u003c/li\u003e\n\u003cli\u003eManni M, Berkeley MR, Seppey M, Sim\u0026atilde;o FA, Zdobnov EM. (2021) BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol. 38:4647\u0026ndash;4654. https://doi.org/10.1093/molbev/msab199\u003c/li\u003e\n\u003cli\u003eSeemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30:2068\u0026ndash;2069. https://doi.org/10.1093/bioinformatics/btu153\u003c/li\u003e\n\u003cli\u003eCantalapiedra CP, Hern\u0026aacute;ndez-Plaza A, Letunic I, Bork P, Huerta-Cepas J (2021) eggNOG-mapper v2: functional annotation, orthology assignments, and domain prediction at the metagenomic scale. Mol Biol Evol 38:5825\u0026ndash;5829. https://doi.org/10.1093/molbev/msab293\u003c/li\u003e\n\u003cli\u003eGrant JR, Enns E, Marinier E, Mandal A, Herman EK, Chen CY, et al (2023) Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res 51:W484\u0026ndash;W492. https://doi.org/10.1093/nar/gkad326\u003c/li\u003e\n\u003cli\u003eKanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M (2016) KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res 44:D457\u0026ndash;D462. https://doi.org/10.1093/nar/gkv1070\u003c/li\u003e\n\u003cli\u003eRichter M, Rossell\u0026oacute;‑M\u0026oacute;ra R, Gl\u0026ouml;ckner FO, Peplies J (2016) JSpeciesWS: a web server for prokaryotic species circumscription based on pairwise genome comparison. Bioinformatics 32:929\u0026ndash;931. https://doi.org/10.1093/bioinformatics/btv681\u003c/li\u003e\n\u003cli\u003eJain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S (2018) High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 9:4998. https://doi.org/10.1038/s41467-018-07641-9 \u003c/li\u003e\n\u003cli\u003eDieckmann MA, Beyvers S, Nkouamedjo‑Fankep RC, Hanel PHG, Jelonek L, Blom J, Goesmann A (2021) EDGAR3.0: comparative genomics and phylogenomics on a scalable infrastructure. Nucleic Acids Res 49:W185\u0026ndash;W192. https://doi.org/10.1093/nar/gkab341\u003c/li\u003e\n\u003cli\u003eQin QL, YZ A (2014) A proposed genus boundary for the prokaryotes based on genomic insights. J Bacteriol 196:2210\u0026ndash;2215. https://doi.org/10.1128/jb.01688-14\u003c/li\u003e\n\u003cli\u003eMeier‑Kolthoff JP, Sard\u0026agrave; Carbasse J, Peinado‑Olarte RL, G\u0026ouml;ker M (2022) TYGS and LPSN: a database tandem for fast and reliable genome‑based classification and nomenclature of prokaryotes. Nucleic Acids Res 50:D801\u0026ndash;D807. https://doi.org/10.1093/nar/gkab938\u003c/li\u003e\n\u003cli\u003eEdgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792\u0026ndash;1797. https://doi.org/10.1093/nar/gkh340\u003c/li\u003e\n\u003cli\u003eAsnicar F, Thomas AM, Beghini F, Mengoni C, Manara S, Manghi P, Zhu Q, Bolzan M, Cumbo F, May U, Sanders JG, Zolfo M, Kopylova E, Pasolli E, Knight R, Mirarab S, Huttenhower C, Segata N (2020) Precise phylogenetic analysis of microbial isolates and genomes from metagenomes using PhyloPhlAn 3.0. Nat Commun 11:2500. https://doi.org/10.1038/s41467-020-16366-7\u003c/li\u003e\n\u003cli\u003eStamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30:1312\u0026ndash;1313. https://doi.org/10.1093/bioinformatics/btu033 \u003c/li\u003e\n\u003cli\u003eLetunic I, Bork P (2024) Interactive Tree Of Life (iTOL) v6: an online tool for the display, annotation and management of phylogenetic and other trees. Nucleic Acids Res 52:W17\u0026ndash;W24. https://doi.org/10.1093/nar/gkae268\u003c/li\u003e\n\u003cli\u003eLiu YY, Hsu CY, Yang YC, Huang CH, Chen CC (2023) ProbioMinServer: an integrated platform for assessing the safety and functional properties of potential probiotic strains. Bioinform Adv 3:vbad153. https://doi.org/10.1093/bioadv/vbad153\u003c/li\u003e\n\u003cli\u003eBlin K, Shaw S, Augustijn HE, Reitz ZL, Biermann F, Alanjary M, et al (2023) antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualization. Nucleic Acids Res 51:W46\u0026ndash;W50. https://doi.org/10.1093/nar/gkad344\u003c/li\u003e\n\u003cli\u003eCarattoli A, Zankari E, Garc\u0026iacute;a-Fern\u0026aacute;ndez A, Voldby Larsen M, Lund O, Villa L, et al (2014) In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 58:3895\u0026ndash;3903. https://doi.org/10.1128/AAC.02412-14\u003c/li\u003e\n\u003cli\u003eCouvin D, Bernheim A, Toffano-Nioche C, Touchon M, Michalik J, N\u0026eacute;ron B, et al (2018) CRISPRCasFinder, an update of CRISPRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins. Nucleic Acids Res 46:W246\u0026ndash;W251. https://doi.org/10.1093/nar/gky425\u003c/li\u003e\n\u003cli\u003eBrown CL, Mullet J, Hindi F, Stoll JE, Gupta S, Choi M, et al (2022) mobileOG-db: a manually curated database of protein families mediating the life cycle of bacterial mobile genetic elements. Appl Environ Microbiol 88:e00991-22. https://doi.org/10.1128/AEM.00991-22 \u003c/li\u003e\n\u003cli\u003eEFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Rychen G, Aquilina G, et al (2018) Guidance on the characterisation of microorganisms used as feed additives or as production organisms. EFSA J 16:e05206. https://doi.org/10.2903/j.efsa.2018.5206 \u003c/li\u003e\n\u003cli\u003eMcArthur AG, Waglechner N, Nizam F, et al (2013) The Comprehensive Antibiotic Resistance Database. Antimicrob Agents Chemother 57:3348\u0026ndash;3357. https://doi.org/ 10.1128/AAC.00419-13\u003c/li\u003e\n\u003cli\u003eAlcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M, Edalatmand A, Huynh W, Nguyen AV, Cheng AA, Liu S, Min SY, Miroshnichenko A, Tran HK, Werfalli RE, Nasir JA, Oloni M, Speicher DJ, Florescu A, Singh B, Faltyn M, Hernandez-Koutoucheva A, Sharma AN, Bordeleau E, Pawlowski AC, Zubyk HL, Dooley D, Griffiths E, Maguire F, Winsor GL, Beiko RG, Brinkman FSL, Hsiao WWL, Domselaar GV, McArthur AG (2020) CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res 48:D517\u0026ndash;D525. https://doi.org/10.1093/nar/gkz935.\u003c/li\u003e\n\u003cli\u003eBortolaia V, Kaas RS, Ruppe E, Roberts MC, Schwarz S, Cattoir V, et al (2020) ResFinder 4.0 for predictions of phenotypic antimicrobial resistance and genotypic subtyping. J Antimicrob Chemother 75:3491\u0026ndash;3500. https://doi.org/10.1093/jac/dkaa345 \u003c/li\u003e\n\u003cli\u003eLiu B, Zheng D, Jin Q, Chen L, Yang J (2022) VFDB 2022: a general classification scheme for bacterial virulence factors. Nucleic Acids Res 50:D912\u0026ndash;D917. https://doi.org/10.1093/nar/gkab1107\u003c/li\u003e\n\u003cli\u003eMalberg Tetzschner AM, Johnson JR, Johnston BD, et al (2020) In silico genotyping of \u003cem\u003eEscherichia coli\u003c/em\u003e isolates for extraintestinal virulence genes by use of whole-genome sequencing data. J Clin Microbiol 58:e01269-20. https://doi.org/10.1128/JCM.01269-20\u003c/li\u003e\n\u003cli\u003eBai Z, Zhang N, Jin Y, et al (2022) Comprehensive analysis of 84 \u003cem\u003eFaecalibacterium prausnitzii \u003c/em\u003estrains uncovers their genetic diversity, functional characteristics, and potential risks. Front Cell Infect Microbiol 12:919701. https://doi.org/10.3389/fcimb.2022.919701\u003c/li\u003e\n\u003cli\u003eBateman A, Martin MJ, Orchard S, Magrane M, Agivetova R, Ahmad S, et al (2021) UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 49:D480\u0026ndash;D489. https://doi.org/10.1093/nar/gkaa1100\u003c/li\u003e\n\u003cli\u003ePearson WR (2013) An introduction to sequence similarity (homology) searching. Curr Protoc Bioinform 42:e59. https://doi.org/10.1002/0471250953.bi0301s422\u003c/li\u003e\n\u003cli\u003eEddy SR (2011) Accelerated profile HMM searches. PLoS Comput Biol 7:e1002195. https://doi.org/10.1371/journal.pcbi.1002195\u003c/li\u003e\n\u003cli\u003eFinn RD, Mistry J, Schuster‑B\u0026ouml;ckler B, Griffiths‑Jones S, Hollich V, Lassmann T, Moxon S, Marshall M, Khanna A, Durbin R, Eddy SR, Sonnhammer ELL, Bateman A (2006) Pfam: clans, web tools and services. Nucleic Acids Res 34:D247\u0026ndash;D251. https://doi.org/10.1093/nar/gkj149\u003c/li\u003e\n\u003cli\u003eTagliazucchi D, Baldaccini A, Martini S, Bianchi A, Pizzamiglio V, Solieri L (2020) Cultivable non‑starter lactobacilli from ripened Parmigiano Reggiano cheeses with different salt content and their potential to release anti‑hypertensive peptides. Int J Food Microbiol 330:108688. https://doi.org/10.1016/j.ijfoodmicro.2020.108688\u003c/li\u003e\n\u003cli\u003eLivak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2\u0026minus; \u0026Delta;\u0026Delta;CT method. Methods 25:402-408.\u003c/li\u003e\n\u003cli\u003eFern\u0026aacute;ndez de Palencia P, Pelaez C, Romero C, Martin-Hernandez MC (1997) Purification and characterization of the cell wall proteinase of \u003cem\u003eLactobacillus casei\u003c/em\u003e subsp. \u003cem\u003ecasei\u003c/em\u003e IFPL 731 isolated from raw goat\u0026apos;s milk cheese. J Agric Food Chem 45(9):3401\u0026ndash;3405. \u003c/li\u003e\n\u003cli\u003eBradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248\u0026ndash;254.\u003c/li\u003e\n\u003cli\u003eDonkor O, Henriksson A, Vasiljevic T, Shah N (2007) Proteolytic activity of dairy lactic acid bacteria and probiotics as determinant of growth and in vitro angiotensin‑converting enzyme inhibitory activity in fermented milk. Le Lait 87:21\u0026ndash;38. https://doi.org/10.1051/lait:2006023\u003c/li\u003e\n\u003cli\u003eMartini S, Solieri L, Cattivelli A, Pizzamiglio V, Tagliazucchi D (2021) An integrated peptidomics and in silico approach to identify novel antidiabetic peptides in Parmigiano‑Reggiano cheese. Biology 10:563. https://doi.org/10.3390/biology10060563\u003c/li\u003e\n\u003cli\u003eLiu DD, Gu CT (2020) Proposal to reclassify \u003cem\u003eLactobacillus zhaodongensis\u003c/em\u003e, \u003cem\u003eLactobacillus zeae\u003c/em\u003e, \u003cem\u003eLactobacillus argentoratensis\u003c/em\u003e and \u003cem\u003eLactobacillus buchneri\u003c/em\u003e subsp. \u003cem\u003esilagei \u003c/em\u003eas \u003cem\u003eLacticaseibacillus zhaodongensis\u003c/em\u003e comb. nov., \u003cem\u003eLacticaseibacillus zeae\u003c/em\u003e comb. nov., \u003cem\u003eLactiplantibacillus argentoratensis\u003c/em\u003e comb. nov. and \u003cem\u003eLentilactobacillus buchneri\u003c/em\u003e subsp. \u003cem\u003esilagei \u003c/em\u003ecomb. nov., respectively and \u003cem\u003eApilactobacillus kosoi\u003c/em\u003e as a later heterotypic synonym of \u003cem\u003eApilactobacillus micheneri\u003c/em\u003e. Int J Syst Evol Microbiol 70(12):6414-6417.\u003c/li\u003e\n\u003cli\u003eHuang CH, Chen CC, Liou JS, Lee AY, Blom J, Lin YC, et al. (2020) Genome-based reclassification of \u003cem\u003eLactobacillus casei\u003c/em\u003e: emended classification and description of the species \u003cem\u003eLactobacillus zeae\u003c/em\u003e. Int J Syst Evol Microbiol 70:3755\u0026ndash;3762. https://doi.org/10.1099/ijsem.0.003969\u003c/li\u003e\n\u003cli\u003eKoonin EV, Makarova KS, Wolf YI, Krupovic M (2020) Evolutionary entanglement of mobile genetic elements and host defense systems: guns for hire. Nat Rev Genet 21(2):119\u0026ndash;131. https://doi.org/10.1038/s41576-019-0172-9\u003c/li\u003e\n\u003cli\u003eHendrix RW, Casjens SR, Lavigne R (2012) Family - Siphoviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy. Elsevier, San Diego, pp 86\u0026ndash;98.\u003c/li\u003e\n\u003cli\u003eda Silva Barreira D, Lapaquette P, Novion Ducassou J, Cout\u0026eacute; Y, Guzzo J, Rieu A (2022) Spontaneous prophage induction contributes to the production of membrane vesicles by the Gram-positive bacterium \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e BL23. MBio 13(5):e02375-22. https://doi.org/10.1128/mbio.02375-22\u003c/li\u003e\n\u003cli\u003eChandran A, Beena AK, Vaiyapuri M, John L, Rajakumar SN, Babu S, James L, Rahila MP (2023) Detection of lytic phage infecting flavour-producing strain of \u003cem\u003eLacticaseibacillus paracasei\u003c/em\u003e in the dairy effluents of Kerala. J Dairy Res 90(2):178\u0026ndash;181. https://doi.org/10.1017/S002202992300020X\u003c/li\u003e\n\u003cli\u003eRollie C, Chevallereau A, Watson BNJ, Chyou TY, Fradet O, McLeod I, Fineran PC, Brown CM, Gandon S, Westra ER (2020) Targeting of temperate phages drives loss of type I CRISPR‑Cas systems. Nature 578(7793):149\u0026ndash;153. https://doi.org/10.1038/s41586-020-1936-2\u003c/li\u003e\n\u003cli\u003eFernandes A, Jobby R (2022) Bacteriocins from lactic acid bacteria and their potential clinical applications. Appl Biochem Biotechnol 194(10):4377\u0026ndash;4399. https://doi.org/10.1007/s12010-022-03870-3\u003c/li\u003e\n\u003cli\u003eBerlemont R, Martiny AC (2015) Genomic potential for polysaccharide deconstruction in bacteria. Appl Environ Microbiol 81(4):1513\u0026ndash;1519. https://doi.org/10.1128/AEM.03718-14\u003c/li\u003e\n\u003cli\u003eFukao M, Zendo T, Inoue T, Nakayama J, Suzuki S, Fukaya T, et al (2019) Plasmid-encoded glycosyltransferase operon is responsible for exopolysaccharide production, cell aggregation, and bile resistance in a probiotic strain, \u003cem\u003eLactobacillus brevis\u003c/em\u003e KB290. J Biosci Bioeng 128:391\u0026ndash;397. doi:10.1016/j.jbiosc.2019.04.008\u003c/li\u003e\n\u003cli\u003eKim E, Yang SM, Kim D, Kim HY (2022) Complete genome sequencing and comparative genomics of three potential probiotic strains, \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e FBL6, \u003cem\u003eLacticaseibacillus chiayiensis\u003c/em\u003e FBL7, and \u003cem\u003eLacticaseibacillus zeae\u003c/em\u003e FBL8. Front Microbiol 12, 794315. https://doi.org/10.3389/fmicb.2021.794315\u003c/li\u003e\n\u003cli\u003eLeschonski KP, Mortensen MS, Hansen LB, Krogh KB, Kabel MA, Laursen MF (2024) Structure-dependent stimulation of gut bacteria by arabinoxylo-oligosaccharides (AXOS): a review. Gut Microbes 16(1):2430419. https://doi.org/10.1080/19490976.2024.2430419\u003c/li\u003e\n\u003cli\u003ePapadimitriou K, Alegr\u0026iacute;a A, Bron PA, de Angelis M, Gobbetti M, Kleerebezem M, Lemos JA, Linares DM, Ross P, Stanton C, Turroni F, van Sinderen D, Varmanen P, Ventura M, Z\u0026uacute;\u0026ntilde;iga M, Tsakalidou E, Kok J (2016) Stress physiology of lactic acid bacteria. Microbiol Mol Biol Rev 80:837\u0026ndash;890. https://doi.org/10.1128/MMBR.00076-15\u003c/li\u003e\n\u003cli\u003eRomano A, Trip H, Lonvaud-Funel A, Lolkema JS, Lucas PM (2012) Evidence of two functionally distinct ornithine decarboxylation systems in lactic acid bacteria. Appl Environ Microbiol 78(6):1953\u0026ndash;1961. https://doi.org/10.1128/AEM.07161-11\u003c/li\u003e\n\u003cli\u003eV\u0026eacute;lez MP, De Keersmaecker SC, Vanderleyden J (2007) Adherence factors of \u003cem\u003eLactobacillus\u003c/em\u003e in the human gastrointestinal tract. FEMS Microbiol Lett 276(2):140\u0026ndash;148. https://doi.org/10.1111/j.1574-6968.2007.00908.x\u003c/li\u003e\n\u003cli\u003eMuscariello L, De Siena B, Marasco R (2020) \u003cem\u003eLactobacillus\u003c/em\u003e cell surface proteins involved in interaction with mucus and extracellular matrix components. Curr Microbiol 77(12):3831\u0026ndash;3841. https://doi.org/10.1007/s00284-020-02243-5\u003c/li\u003e\n\u003cli\u003eSavijoki K, Ingmer H, Varmanen P (2006) Proteolytic systems of lactic acid bacteria. Appl Microbiol Biotechnol 71(4):394\u0026ndash;406.http://doi10.1007/BF00395933. PMID: 8879407.\u003c/li\u003e\n\u003cli\u003eVarmanen P, Savijoki K, \u0026Aring;vall S, Palva A, Tynkkynen S (2000) X-prolyl dipeptidyl aminopeptidase gene (\u003cem\u003epepX\u003c/em\u003e) is part of the \u003cem\u003eglnRA\u003c/em\u003e operon in \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e. J Bacteriol 182:146\u0026ndash;154. https://doi.org/10.1128/JB.182.1.146-154.2000\u003c/li\u003e\n\u003cli\u003eWaterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ (2009) Jalview Version 2\u0026mdash;a multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189\u0026ndash;1191\u003c/li\u003e\n\u003cli\u003eGu\u0026eacute;don E, Sperandio B, Pons N, Ehrlich SD, Renault P (2001) Overall control of nitrogen metabolism in \u003cem\u003eLactococcus lactis\u003c/em\u003e by CodY and possible models for CodY regulation in Firmicutes. Microbiology 147(12):3895\u0026ndash;3909. https://doi.org/10.1099/mic.0.28186-0\u003c/li\u003e\n\u003cli\u003ePetranovic D, Gu\u0026eacute;don E, Sperandio B, Delorme C, Ehrlich D, Renault P (2004) Intracellular effectors regulating the activity of the \u003cem\u003eLactococcus lactis\u003c/em\u003e CodY pleiotropic transcription regulator. Mol Microbiol 53(2):613\u0026ndash;621. https://doi.org/10.1111/j.1365-2958.2004.04136.x\u003c/li\u003e\n\u003cli\u003eMorel F, Frot Coutaz J, Aubel D, Portalier R, Atlan D (1999) Characterization of a prolidase from \u003cem\u003eLactobacillus delbrueckii\u003c/em\u003e subsp. \u003cem\u003ebulgaricus\u003c/em\u003e CNRZ 397 with an unusual regulation of biosynthesis. Microbiol 145:437\u0026ndash;446. http://doi.10.1099/13500872-145-2-437\u003c/li\u003e\n\u003cli\u003eEl Abboudi M, El Soda M, Pandian S, Simard RE, Olson NF (1992) Purification of X‑prolyl dipeptidyl aminopeptidase from \u003cem\u003eLactobacillus casei\u003c/em\u003e subspecies. Int J Food Microbiol 15(1‑2):87\u0026ndash;98. https://doi.org/10.1016/0168-1605(92)90138-S\u003c/li\u003e\n\u003cli\u003eMagboul AA, McSweeney PL (2000) Purification and characterization of an X-prolyl-dipeptidyl aminopeptidase from \u003cem\u003eLactobacillus curvatus\u003c/em\u003e DPC2024. Le Lait 80(4):385\u0026ndash;396. https://doi.org/10.1051/lait:2000133\u003c/li\u003e\n\u003cli\u003eStressler T, Eisele T, Kranz B, Fischer L (2014) PepX from \u003cem\u003eLactobacillus helveticus\u003c/em\u003e: automated multi‑step purification and determination of kinetic parameters with original tripeptide substrates. J Mol Catal B: Enzym 108:103\u0026ndash;110. https://doi.org/10.1016/j.molcatb.2014.07.006.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"probiotics-and-antimicrobial-proteins","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"paap","sideBox":"Learn more about [Probiotics and Antimicrobial Proteins](http://link.springer.com/journal/12601)","snPcode":"12602","submissionUrl":"https://submission.nature.com/new-submission/12602/3","title":"Probiotics and Antimicrobial Proteins","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Proteolytic system, X-prolyl dipeptidyl aminopeptidase, pepX gene, Lacticaseibacillus, probiotics, bioactive peptides","lastPublishedDoi":"10.21203/rs.3.rs-7544664/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7544664/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpecies of the \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e group are GRAS organisms extensively used in dairy fermentations to release bioactive peptides (BPs) with health-promoting properties. This study characterized the probiotic features, biosafety, and proteolytic system of the probiotic candidate \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205, a high producer of the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), originally isolated from Parmigiano Reggiano cheese, with a focus on the PepX enzyme. Whole-genome sequencing revealed a 3.2 Mb genome encoding 2979 predicted genes. Phylogenomics assigned PRA205, previously identified as \u003cem\u003eLacticaseibacillus casei\u003c/em\u003e, to a lineage closely related yet distinct from \u003cem\u003eLcb. parahuelsenbergensis\u003c/em\u003e. Genome annotation identified adhesion and stress-tolerance genes, along with three bacteriocin clusters, but no antibiotic resistance or virulence factors. \u003cem\u003eLacticaseibacillus\u003c/em\u003e sp. PRA205 exhibited a complete proteolytic system, consistent with its strong proteolytic phenotype. Notably, a single \u003cem\u003epepX\u003c/em\u003e gene, only weakly repressed under amino acid\u0026ndash;rich conditions, encoded a serine protease of ~\u0026thinsp;80 kDa that was partially purified and biochemically characterized. PepX degraded BPs, including the tripeptides VPP and IPP, and retained activity at low temperature and acidic pH, suggesting a dual role in BPs production and breakdown during dairy fermentation and storage. Overall, these findings elucidate the genetic basis of PRA205 proteolytic activity and support its safety and potential as probiotic culture for developing functional dairy foods enriched in BPs.\u003c/p\u003e","manuscriptTitle":"Genomic and Proteolytic Profiling of Lacticaseibacillus sp. 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