A thermostable (thermophilic) extracellular metalloendopeptidase containing four calcium ions. Cofactors are zinc and calcium. Hydrolyzes protein bonds on the N-terminal side of hydrophobic amino acid residues. The pH optimum is 8.0 and the optimal temperature for activity is 70 °C. Considerably stable from pH 5 to 9.5. Thermolysin has a low cleavage specificity, therefore, it produces a number of short fragments that are suitable for sequencing. Preferential cleavage: X-cleavage-Y-Z where X=any amino acid; Y=Leu, Phe, Ile, Val, Met, Ala and Z is any amino acid other than Pro. Cleavage N-terminal to Leu is preferred over cleavage of N-terminal to Phe which is preferred over the others. Often used to do limited proteolysis for peptide mapping and studies of protein structure and conformational changes.