title: Isolation and characterization of a subtilisin-like serine proteinase secreted by the sap-staining fungus Ophiostoma piceae.
abstract: Identification of key enzymes of sapstain fungi which cause wood discoloration is necessary for targeted inhibition strategies. Therefore proteinases involved in the nitrogen pathway have been characterized. The sap-staining fungus Ophiostoma piceae strain 387N produced proteolytic enzymes when grown on wood and protein-supplemented media. Proteolytic activity in culture filtrates was inhibited by PMSF and EDTA. The major protein in culture filtrates was a proteinase with a pI of 5.6 and a molecular weight of 33 kDa. This was the major proteinase produced by O. piceae and it was purified from culture filtrates by hydrophobic interaction chromatography. The proteinase was susceptible to autolytic degradation during chromatographic separations when ammonium sulfate was not present. When azocoll was used as a substrate, the proteolytic activity of the purified proteinase was determined to be optimal at pH 7-9 and 40 degrees C. Similar pH and temperature optima were obtained using succinyl-ala-ala-pro-phe-p-nitroanilide as the substrate. The N-terminal sequence of the protein showed a high degree of homology with fungal alkaline serine proteinases classified as subtilisin class II enzymes. Agreement in inhibition patterns and electrophoretic and catalytic properties suggested the secretion of the same proteinase during growth on wood. Understanding the role of this proteinase during fungal colonization is an important step toward disrupting fungal growth on wood.
authors.affiliation: Department of Wood Science, Faculty of Forestry, University of British Columbia, Vancouver, Canada.
authors.forename: L D
authors.initials: LD
authors.lastname: Abraham
authors.forename: C
authors.initials: C
authors.lastname: Breuil
mesh_terms: D000595:Amino Acid Sequence; D002455:Cell Division; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000201:enzymology / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005658:Fungi / Q000201:enzymology*; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013381:Subtilisins / Q000145:classification / Q000235:genetics; D013696:Temperature; D014934:Wood
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D012697:Serine Endopeptidases; D013381:Subtilisins