A 33 kDa serine proteinase from Scedosporium apiospermum.
An extracellular proteinase produced by the filamentous fungus Scedosporium apiospermum has been purified and characterized. Initially, in vitro conditions for enzyme synthesis were investigated. The highest yield of enzyme production was obtained when the fungus was cultivated in modified Czapek-Dox liquid medium supplemented with 0.1% bacteriological peptone and 1% (w/v) glucose as the nitrogen and carbon sources respectively. Purification to homogeneity of the proteinase was accomplished by (NH4)2SO4 precipitation, followed by gel filtration through Sephadex G-75 and finally affinity chromatography through immobilized phenylalanine. Analysis of the purified enzyme by SDS/PAGE revealed a single polypeptide chain with an apparent molecular mass of 33 kDa. Further investigation of its physical and biochemical properties disclosed numerous similarities with those of the previously described serine proteinase of Aspergillus fumigatus. The enzyme was not glycosylated and its pI was 9.3. Proteinase activity was optimum between 37 and 50 degrees C and at pH 9.0, but remained high within a large range of pH values between 7 and 11. The inhibition profile and N-terminal amino acid sequencing confirmed that this enzyme belongs to the subtilisin family of serine proteinases. In agreement with this, the specific synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide proved to be an excellent substrate for the proteinase with an estimated Km of 0.35 mM. Like the alkaline proteinase of A. fumigatus, this enzyme was able to degrade human fibrinogen, and thus may act as a mediator of the severe chronic bronchopulmonary inflammation from which cystic fibrosis patients suffer.
Larcher
G
G
Groupe d'Etude des Interactions, Hôte-Parasite, Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Angers, France.
Cimon
B
B
Symoens
F
F
Tronchin
G
G
Chabasse
D
D
Bouchara
J P
JP
D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011541:Pseudallescheria / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D005656:Fungal Proteins; D012697:Serine Endopeptidases