A prolyl endopeptidase, FvpP, was identified in the culture supernatant of the basidiomycete Flatranulina velutipes and functionally expressed for gene verification in Aspergillus otyzae. The wild-type FvpP with a molecular mass of 50 kDa under denaturing conditions, exhibited optima at pH 5, and 45 degrees C. and an isoelectric point of 3.8. Five mM Fe2+ and Fe3+ activated the prolyl endopeptidase, while it was not affected by 10 mM CaCl2, EDTA, and Pepstatin A. Five mM Cu2+, Mn2+, Zn2+, Ni2+, Co2+ and low Z-Pro-prolinal concentrations decreased the enzyme activity. Concluded from comparative RP-HPLC and high-resolution QTOF-MS/MS analyses of the hydrolytic cleavage products of gliadin, casein and gelatin, FvpP possessed a Pl-P1'-substrate specificity similar to the prolyl endopeptidase from Aspergillus niger (An-Pep), although the sequence similarity was only 39%. FvpP and An-Pep (S28.004; MEROPS) are unique in the serine peptidase family S28, because they share more sequence similarity with Pro-X carboxypeptidase (S28.001; MEROPS) and dipeptidyl peptidase II (S28.002; MEROPS) than with prolyl oligopeptidases of the serine peptidase family S09. As a potential degrader of the antigenic epitopes of alpha-gliadin, FvpP might provide an efficient tool for the processing of wheat and other gluten containing cereals to better digestible foods for celiac patients.