ISOLATION, CHARACTERIZATION, AND CLONING OF CDNA AND THE GENE FOR AN ELASTINOLYTIC SERINE PROTEINASE FROM ASPERGILLUS-FLAVUS

An elastinolytic serine proteinase produced by Aspergillus flavus 28 t hat was isolated from a patient who died of aspergillosis has been pur ified and characterized. The enzyme was inhibited by the serine protei nase inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluoroph osphate. The metal-chelating agents EDTA and EGTA [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] did not severely i nhibit the enzyme. A cDNA and a 2.95-kb segment of genomic DNA contain ing the proteinase gene were sequenced. The open reading frame that wo uld code for a protein containing 403 amino acids was interrupted by t hree introns. The mature protein lacks 121 N-terminal amino acids incl uding a putative 21-amino-acid signal peptide. The purified mature pro tein showed a molecular mass of 36 kDa by sodium dodecyl sulfate-polya crylamide gel electrophoresis, whereas that calculated from the deduce d protein sequence was 30 kDa. This elastinolytic serine proteinase of A. flavus has 83 and 82% sequence homology to the similar proteinases from A. fumigatus and A. oryzae. The catalytic properties and the seq uence homology around the putative catalytic amino acids suggest that this enzyme belongs to the serine proteinases of the subtilisin family .