title: Purification and characterization of the extracellular aspartyl protease APSm1 from the phytopathogen fungus Stenocarpella maydis.
abstract: The extracellular protease APSm1 was purified to homogeneity from Stenocarpella maydis that was grown in acidic minimal media with glucose and ammonium sulfate. The purification procedure consisted of ion exchange chromatography coupled to an FPLC (Fast Protein Liquid Chromatography) system, resulting in a 15.3% recovery and a 2.3-fold increase in specific activity. The molecular weight of the purified enzyme was estimated to be 56.8 kDa by SDS-PAGE. Enzymatic activity toward hemoglobin was optimal at pH 2.0 and at 25 °C. The effects of six protease inhibitors on APSm1 activity were tested. Pepstatin A inhibited APSm1 activity, as the protein is in fact an aspartyl protease. The pure enzyme degraded hemoglobin, albumin and proteins obtained from corn germ at pH 3 but did not have any milk-clotting activities. The Km and Vmax values obtained were 0.514 mg/mL and 0.222 μmol/min, respectively, using hemoglobin as the substrate. This work is the first to report the purification of a secreted aspartyl protease from S. maydis.
authors.affiliation: Universidad Politécnica de Pachuca, Carretera Pachuca-Cd. Sahagún Km 20, Rancho Luna, Ex-Hacienda de Sta. Bárbara, Municipio de Zempoala, Hidalgo, Mexico.
authors.forename: Virginia
authors.initials: V
authors.lastname: Mandujano-González
authors.affiliation: Universidad Politécnica de Pachuca, Carretera Pachuca-Cd. Sahagún Km 20, Rancho Luna, Ex-Hacienda de Sta. Bárbara, Municipio de Zempoala, Hidalgo, Mexico.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Téllez-Jurado
authors.affiliation: Universidad Politécnica de Pachuca, Carretera Pachuca-Cd. Sahagún Km 20, Rancho Luna, Ex-Hacienda de Sta. Bárbara, Municipio de Zempoala, Hidalgo, Mexico.
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Anducho-Reyes
authors.affiliation: Universidad Politécnica de Pachuca, Carretera Pachuca-Cd. Sahagún Km 20, Rancho Luna, Ex-Hacienda de Sta. Bárbara, Municipio de Zempoala, Hidalgo, Mexico.
authors.forename: Ainhoa
authors.initials: A
authors.lastname: Arana-Cuenca
authors.affiliation: Universidad Politécnica de Pachuca, Carretera Pachuca-Cd. Sahagún Km 20, Rancho Luna, Ex-Hacienda de Sta. Bárbara, Municipio de Zempoala, Hidalgo, Mexico. Electronic address: yuridiamercado@upp.edu.mx.
authors.forename: Yuridia
authors.initials: Y
authors.lastname: Mercado-Flores
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D057055:Aspartic Acid Proteases / Q000737:chemistry* / Q000302:isolation & purification*; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D005656:Fungal Proteins; D057055:Aspartic Acid Proteases
keywords: Aspartyl protease; Phytopathogenic fungus; Stenocarpella maydis