title: 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.
authors.affiliation: Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan.
authors.forename: Jen-Tao
authors.initials: JT
authors.lastname: Chen
authors.forename: Mei-Li
authors.initials: ML
authors.lastname: Chao
authors.forename: Chiou-Yen
authors.initials: CY
authors.lastname: Wen
authors.forename: Wen-Shen
authors.initials: WS
authors.lastname: Chu
mesh_terms: D000363:Agaricales / Q000201:enzymology* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008403:Mass Screening / Q000379:methods; D008970:Molecular Weight; D000085822:Prolyl Oligopeptidases; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D021122:Protein Subunits; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases