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

Species of the Lacticaseibacillus casei group are GRAS organisms extensively used in dairy fermentations to release bioactive peptides (BPs) with health-promoting properties. However, the genetic basis of this proteolytic phenotype remains poorly investigated. In this study, we characterised the probiotic features, biosafety, and proteolytic system of the probiotic candidate Lacticaseibacillus sp. PRA205, a high producer of the anti-hypertensive 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 2977 predicted genes. Phylogenomics assigned PRA205, previously identified as Lcb. casei, to a lineage closely related yet distinct from Lacticaseibacillus parahuelsenbergensis. Genome annotation identified adhesion and stress-tolerance genes, along with three bacteriocin clusters, but no antibiotic resistance or virulence determinants. PRA205 exhibited a complete proteolytic system consistent with its strong proteolytic phenotype, including two pathways for the degradation of proline-rich caseins (PepX/PepQ and PepI/PepP). Notably, unlike other proteolytic genes such as prt, the single pepX gene found in PRA205 was only weakly repressed under peptide–rich nitrogen conditions, suggesting a regulatory mechanism partially independent of amino acid availability. The enzyme, a monomeric serine protease of ~ 80 kDa, was partially purified and biochemically characterised. PepX degraded BPs, including the tripeptides VPP and IPP, and retained activity at 4 °C and pH of 4.0, supporting a dual role in BPs production and breakdown during dairy fermentation and storage. Overall, these findings elucidate the genetic basis of the proteolytic activity of PRA205 and support its safety and potential use in the development of functional dairy foods enriched in BPs.