We determined the specificity preferences of both recombinant shewasin D and shewasin A (pepsin-like aspartic proteases from S. denitrificans and S. amazonensis, respectively) using proteome-derived peptide libraries and observed remarkable similarities between both shewasins and eukaryotic pepsins, in particular with BACE-1 thereby confirming their phylogenetic proximity. We further observed subtle differences in sequence preferences at several positions, suggesting variations in their subsite binding pockets.