Screening, purification, and characterization of an extracellular prolyl oligopeptidase from Coprinopsis clastophylla

Abstract

Culture filtrates of 22 mushrooms were screened for extracellular prolyl oligopeptidase activity. Four strains with relatively high enzyme activity were all from inky cap mushrooms. The production of Coprinopsis clastophylla prolyl oligopeptidase was associated with the growth of the fungus and the enzyme was not released by cell lysis. The enzyme was purified 285-fold to a specific activity of 52.05 U/mg. It was purified to a single band on a native polyacrylamide gel. However, the enzyme separated into three bands on a sodium dodecyl sulfate-polyacrylamide gel with mobility corresponding to molecular weights of approximately 84, 60, and 26 kDa. The results of tandem mass spectrometric analysis revealed that the 60 kDa protein was likely a degradation product of the 84 kDa protein. The isoelectric point of the purified enzyme was 5.2. The purified enzyme had an optimal pH and temperature of 8.0 and 37°C, respectively. Diisopropyl fluorophosphate (DFP), p-chloromercuribenzoaic acid (PCMB), Hg(2+), and Cu(2+) strongly inhibited C. clastophylla prolyl oligopeptidase. This enzyme is a serine peptidase and one or more cysteine residues of the enzyme are close to the active site.

Prolyl-specific peptidases for applications in food protein hydrolysis

“…Z-Gly-D-PropNA, Z-Gly-Pro-Ala, Z-Gly-Pro-D-Ala and Z-Ala-Ala-AlaPro-pNA (Yoshimoto et al 1988, Sattar et al 1990, Chen et al 2012. To differentiate prolyl oligopeptidases from amino-and carboxypeptidases, the substrates listed in Table 1 have been suggested.…”

“…Likewise, at least one amino acid residue N-terminal of proline was required for the hydrolysis of proline-containing peptides with an enzyme derived from the inky cap mushroom Coprinopsis clastophylla (Chen et al 2012). Comparable to the POP from A. bisporus and L. cinerascens, the POP of C. clastophylla had a pH optimum of 8.0 and showed the highest activity at 37°C.…”

The MSDIN family in amanitin-producing mushrooms and evolution of the prolyl oligopeptidase genes

“…Furthermore, abnormalities in POP activity are associated with diseases (Momeni et al 2005). The majority of reported POPs are intracellular enzymes, while mushrooms produce some extracellular POPs (Chen et al 2012) that can only be speculated to perform more specific peptide hydrolyzation roles compared to more general proteases.…”