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 intracellular proteases in fungi. These enzymes are characterized by conserved regions around the active site residues. Asp, His and Ser. Based on this amino acid (aa) sequence conservation, we have used degenerate primer PCR to isolate subtilisin-specific genomic probes from Aspergillus niger, and cloned a gene, pepD, by screening a λ. genomic library using a PCR probe. The pepD gene contains three putative introns, which 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 similarity to subtilisin-like proteases, in particular to fungal alkaline proteases. Signal sequence cleavage prediction indicates that the first 20 aa are probably removed upon transfer to the endoplasmic reticulum. The conservation of the pro-enzyme cleavage site in fungal alkaline proteases suggests that the mature protein is derived from this polypeptide via the removal of an additional 101 aa, resulting in a mature 30294-Da enzyme consisting of 295 aa.