Abstract: The invention relates to protease enzymes (that can be thermostable in some embodiments) with unique cleavage specificity as well as their production, isolation, activation and applications. These enzymes can be engineered for quick production and purification. The peptides produced by the action of these protease enzymes have unique properties for biochemical determination of protein sequence. In particular, using the steps of protease digestion, ionization of the specifically produced peptides and fragmentation of these peptides in a mass spectrometer, the amino acid sequence of the peptide may be read as a ladder from the N-terminus.