Identification and characterization of a new proline-specific endopeptidase, PvPSE1, from Paecilomycesvariotii

Fungal proline-specific endopeptidases (PSEs) show strong potential in both therapeutic and biotechnological applications, while most known members are acidic proteases from Aspergillus species. Here, we report the identification and characterization of a novel neutral PSE from the ascomycete Paecilomyces variotii (PvPSE1). Recombinant PvPSE1 (rPvPSE1) was expressed extracellularly using Trichoderma reesei RUT-C30 strain, and purified by Ni-NTA affinity chromatography. Biochemical characterization identified rPvPSE1 as a serine protease with an optimum temperature of 45 °C and a neutral pH optimum of 6.0, maintaining >85% activity across pH 5.0-7.0. Kinetic analysis with the substrate AAAP-pNA yielded a K_m of 0.46 mM and a specific activity of 5.17 U/mg. The N-glycosylated enzyme functions as a monomer and exhibits good stability against pepsin and trypsin. Interestingly, data suggest that rPvPSE1 is heterogeneously processed in T. reesei, resulting in a fraction of non-covalently associated subunits. Application trials demonstrated that rPvPSE1 efficiently degrades residual gluten in a 10% (w/w) commercial gluten-containing oat suspension at its native pH. These findings position rPvPSE1 as a robust candidate for industrial food processing and potential therapeutic adjuncts where neutral pH activity and specific protease resistance are required.