A 33-KDA SERINE PROTEINASE FROM SCEDOSPORIUM-APIOSPERMUM

An extracellular proteinase produced by the filamentous fungus Scedosp orium apiospermum has been purified and characterized. Initially, in v itro conditions for enzyme synthesis were investigated. The highest yi eld of enzyme production was obtained when the fungus was cultivated i n modified Czapek-Dox liquid medium supplemented with 0.1%, bacteriolo gical peptone and 1% (w/v) glucose as the nitrogen and carbon sources respectively. Purification to homogeneity of the proteinase was accomp lished by (NH4)(2)SO4 precipitation, followed by gel filtration throug h Sephadex G-75 and finally affinity chromatography through immobilize d 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 discl osed numerous similarities with those of the previously described seri ne proteinase of Aspergillus fumigatus. The enzyme was not glycosylate d and its pi was 9.3. Proteinase activity was optimum between 37 and 5 0 degrees C and at pH 9.0, but remained high within a large range of p H 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 syn thetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide proved to b e an excellent substrate for the proteinase, with an estimated K-m 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 t he severe chronic bronchopulmonary inflammation from which cystic fibr osis patients suffer.