CLONING AND CHARACTERIZATION OF THE PEPD GENE OF ASPERGILLUS-NIGER WHICH CODES FOR A SUBTILISIN-LIKE PROTEASE

Serine proteases constitute an important group of extra- and intracell ular proteases in fungi. These enzymes are characterized by conserved regions around the active site residues, Asp, His and Ser. Based on th is amino acid (aa) sequence conservation, we have used degenerate prim er PCR to isolate subtilisin-specific genomic probes from Aspergillus niger, and cloned a gene, pepD, by screening a lambda genomic library using a PCR probe. The pepD gene contains three putative introns, whic h are 51-, 47- and 55-bp long and has an open reading frame coding for a protein which consists of 416 aa. The deduced aa sequence shows sim ilarity to subtilisin-like proteases, in particular to fungal alkaline proteases. Signal sequence cleavage prediction indicates that the fir st 20 aa are probably removed upon transfer to the endoplasmic reticul um. The conservation of the pro-enzyme cleavage site in fungal alkalin e proteases suggests that the mature protein is derived from this poly peptide via the removal of an additional 101 aa, resulting in a mature 30 294-Da enzyme consisting of 295 aa.