High cleavage specificity of a subtilisin-like protease from a hyperthermophilic archaeon under extreme conditions

The cleavage specificity of Pernisine, a subtilisin-like protease from the hyperthermophilic archaeon Aeropyrum pernix, was established by mass spectrometry, analysing the peptides generated by digestion of oxidised bovine insulin B chain. The specificity was explored by changing several factors such as substrate/enzyme ratio, temperature and reaction media. Using a S/E ratio of 1000 (w/w) and a temperature of 60 °C, five primary cleavage sites in the insulin B chain were detected suggesting a broad specificity of Pernisine, which is different from that found for other bacterial subtilisin-like proteases. When the S/E ratio and/or temperature were increased, a higher selectivity of Pernisine was observed with a unique cleavage site occurring between Leu15 and Tyr16. In addition, the influence on the enzymatic hydrolysis of different organic solvent concentrations was investigated. The results demonstrated that Pernisine could specifically digest the peptide substrate even in the presence of 80% acetonitrile solution or 30% dimethyl sulfoxyde. Thereby the cleavage specificity of Pernisine can be opportunely modulated by controlling the in vitro digestion conditions, suggesting that this enzyme could be an attractive candidate to use in a variety of biotechnological applications.