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. 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.